METHODS AND COMPOSITIONS FOR MODULATING THYMIC FUNCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/361,067, filed on July 12, 2016, the contents of which is hereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The thymus is a specialized primary lymphoid organ of the immune system, responsible for the development, selection and maturation of T cells. The thymus provides an inductive environment for development of T cells from hematopoietic progenitor cells. In addition, thymic stromal cells allow for the selection of a functional and self-tolerant T cell repertoire. The thymus is largest and most active during the neonatal and pre-adolescent periods. By the early teens, the thymus begins to atrophy and thymic stroma is mostly replaced by adipose tissue.

SUMMARY OF THE INVENTION

The invention features methods for modulating (e.g., in vivo) thymic function, e.g., for decreasing or reversing thymic involution, improving thymic function, treating thymic damage, or treating conditions or diseases related to thymic function, in a subject (e.g., a human or an agricultural animal). The invention also features methods for treating physiological senescence and age associated diseases in a subject. Also featured herein are methods of treating a subject who has a heterologous thymic capability, e.g., who has, will have or has had a thymic implant or thymus cell implant.

In one aspect, the invention features methods of decreasing or reversing thymic involution (e.g., decreasing the rate of thymic involution) in a subject in need thereof. The method includes administering to the subject an agent that modulates, e.g., increases or decreases, thymic function (also referred to herein as a thymic function modulator), thereby decreasing or reversing thymic involution (e.g., decreasing the rate of thymic involution) in the subject. In some embodiments, the thymic function modulator increases or decreases the expression or activity of one or more thymic function factors described herein.

Thymic Function Factors

In some embodiments of any of the methods described herein, the thymic function modulator increases or decreases the expression or activity of a thymic function factor described herein.

In some embodiments of any of the methods described herein, the thymic function factor can be characterized by one or more, e.g., one, two, three, four, five, or all, of the following:

1) increases the proliferation or cell count of stromal cells or non-adipocyte cells (e.g., as assessed by proliferation assays, e.g., Ki67 staining), e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more.

2) changes the stromal/non- stromal cellular balance in the thymus (e.g., as assessed by ultrasound, histological analysis, or FDG avidity via PET imaging), e.g., an increase or decrease in the ratio of stromal to non-stromal cells, e.g., an increase of decrease in the ratio of thymic epithelial cells to thymocytes, or an increase or decrease in the ratio of adipocytes to thymocytes;

3) increases thymic hormonal production and/or levels; wherein the thymic hormone is associated with thymic size, e.g., thymulin (e.g., as assessed by rosette inhibition assay, e.g., as described in Consolini et al.), e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more.

4) changes in peripheral T cell phenotype, e.g., measuring changes in thymic emigrant profile (e.g., as assessed by comparing levels of different T cell populations, e.g., comparing percentages of T cells and clonal populations of T cells with unique TCRs that are FoxP3 positive, e.g., Tregs)

5) polyclonality of B-cell response or emergence of a particular antibody (e.g., as

assessed by changes in B-cell isotype distribution or polyclonal antibody titers to an antigen of interest, or particular set of thymus-dependent antigens); and/or

6) changes in thymocyte flux (e.g., as assessed by determining TRECs per number of cells, e.g., over time). In some embodiments of any of the methods described herein, the thymic function factor comprises a T cell growth factor, a T cell growth factor receptor, a T cell proliferation factor, a T cell migration factor, a T cell activity factor, a thymic epithelial cell (TEC) proliferation factor, a thymic epithelial cell (TEC) growth factor, a thymic epithelial cell (TEC) growth factor receptor, a thymic epithelial cell (TEC) function factor, or a thymus homeostasis factor. In some embodiments, the thymic function modulator increases or decreases the expression or activity of one or more thymic function factors described herein, e.g., listed in Table 1.

In one embodiment, the T cell growth factor is selected from BMP4, Bmprla, CD70, Cldn4, DM, Egrl, Egr2, Egr3, EphB2, Flt3, Flt3L, Foxol, FoxP3, Gata2, Gata3, Gfil, Ghrl (ghrelin), GnRH, Icaml, Id3, Ifnarl, IL-2, IL-6, IL-7, IL-12b (IL12 p40 subunit), IL- 15, IL-18, IL-21, IFNy, K , Klf3, Lefl, leptin, Lif, Nfat5, Nfatcl, Notch 1, Prolactin, Ragl, Rag2, Runxl, Satbl, SCF (K ), Shh (T-cell), Tcfl, TCR (e.g., a TCR comprising an alpha and a beta chain, a TCR comprising a gamma chain and a delta chain, a TCR comprising a CD3 chain, a TCR comprising a zeta chain, a TCR comprising a complementarity determining region, a TCR comprising a T cell co-receptor (e.g., CD4 or CD8), a TCR that binds to an epitope presented on an MHCI molecule, a TCR that binds to an epitope presented on an MHCII molecule, or a TCR that binds to an epitope presented on an MHCIII molecule), Tshb (thyrotropin beta chain), Tslp, Vcaml, Wnt3a, Wnt4, Zfp3611, or Zfp3612.

In one embodiment, the T cell growth factor receptor is selected from CD 127 (non- soluble IL-7R), CD27, Crlf2 (TSLP-R), Fzdl, FzdlO, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Ihh, Ptchl, and Ptch2, or IL-7R alpha. In one embodiment, the T cell growth factor is selected from at least one of Flt3, Flt3L, Foxol, FoxP3, Gfil, Ghrl (ghrelin), GnRH, Icaml, Id3, Ifnarl, IL-2, IL-6, IL-7, IL-12b (IL12 p40 subunit), IL-15, IL-18, IL-21, KM, Klf3, Lefl, leptin, Lif, and SCF (Kitl). In one embodiment, the T cell growth factor is selected from at least one of IL-7, IL-21, Delta-like 4 (DLL4), Flt3L, SCF (Kitl), and miR-29a. In one embodiment, the T cell growth factor may also include at least one selected from IL-15, IL-2, IL-12, IL-18, and IFNy.

In one embodiment, the T cell migration factor is selected from CC119, Ccl21, CCL25, Ccr7, Ccr9, Cxcll2, Cxcr4, S 1PR1 (S 1P1), Sele (E-selectin, CD62E, ELAM-1, or LECAM2), Sell (CD62L), Selp (P-selectin), CCL20, CCR6, CXCLl-3, CXCR2, IL8, CXCR1, CCL2, CCL4, CCL5, CCL22, CXCL8, or CXCL10. In one embodiment, the T cell activity factor is E2F2.

In one embodiment, the TEC proliferation factor is selected from Cyr61, E2F2 (TEC), IGF-1, IL-22, IL-23, or KGF (Fgf7).

In one embodiment, the TEC growth factor is selected from Atf3, BMP4 (TEC), Cbx4, Cdh5 (VE cadherin), E2F3, E2F4, Foxnl, Fspl, Fstll, Isll, Kl (Klotho), Ltbr, NFkB l,

PPARgamma, Pten, RANK, RANKL, Shh (TEC), Sin/Polr3e, Stat3, Tbata, Tbxl, Tgfbr2, Tnfrsfl la /RANK, Tnfrsf 1 lb, Traf6, Wnt3a (TEC), or Wnt4 (TEC). In one embodiment, the TEC growth factor enhances thymus function by modulating, e.g., increasing, e.g., enhancing, proliferation, survival and/or generation of thymic epithelial cells. In one embodiment, the TEC growth factor is selected from at least one of FGF21, FGF7 (KGF), FGF8, FGF10, IL-22, Wnt4, Bmp4, RANKL, LTa, CL40L, Foxnl, leptin, IGF-1, GH, (Ghrl) ghrelin, GnRH, and NGF.

In one embodiment, the TEC growth factor receptor is selected from Bmpr2, Fgfr2, or bmprla (TEC).

In one embodiment, the thymic function factor is selected from AIRE, beta(5t)/Psmbl 1, Fezf2, HLA (e.g., an HLA molecule described herein, e.g., HLA-A, HLA-B, HLA-B27, HLA- B47, HLA-C, HLA-E, HLA-F, HLA-G, p2-microglobulin, HLA-DM (e.g., HLA-DMA1 and/or HLA-DMB 1), HLA-DO (e.g., HLA-DOA1 and/or HLA-DOB 1), HLA-DP (e.g., HLA-DPA1 and/or HLA-DPB l), HLA-DQ (e.g., HLA-DQAl and/or HLA-DQB l), HLA-DQ2, HLA-DQ8, HLA-DR (e.g., HLA-DRA, HLA-DRB 1, HLA-DRB3, HLA-DRB4, and/or HLA-DRB5), HLA- DR2, HLA-DR3, HLA-DR4, and/or an HLA encoding a component of the complement system), a Major Histocompatibility Complex (MHC) molecule (e.g., Class I MHC molecule (MHC I), e.g., comprising one or more polypeptides encoded by a HLA-A, HLA-B, HLA-C, HLA-G, and/or HLA-E gene; Class II MHC molecule (MHC II), e.g., comprising one or more

polypeptides encoded by a HLA-DP, HLA-DQ, and/or HLA-DR gene; or Class III MHC molecule (MHC III), e.g., comprising a polypeptide involved in inflammation, e.g., a component of the complement system (e.g., C2, C4, or factor B), tumor necrosis factor (TNF)-a,

lymphotoxin-a, lymphotoxin-β, or a heat shock protein), or Prssl6.

In one embodiment, the thymus homeostasis factor is selected from 1 lb-HSD2, AR, ASC, Axin, Fgf21, IL10, IL2 (stroma), Leptin (TEC), Meisl, or NLRP3.

In embodiments, the thymic function factor comprises Cd44, gpl30, hGH (GH1 and GH2), Nfkb2, Ptma (thymosin alpha- 1), Smad4, Smad6, or Tmpo (thymopeotin). In embodiments, the thymic function factor comprises a chemokine (e.g., secreted chemokine), cytoplasmic protein, enzyme (e.g., phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound ligand, non-secreted ligand, or secreted ligand), matrix protein, transcription factor, or receptor (e.g., cell surface receptor or nuclear receptor).

In embodiments, the chemokine is selected from Ccl21, CCL25, or Cxcll2. In embodiments, the secreted chemokine is Cxcll2.

In embodiments, the cytoplasmic protein is selected from ASC, Axin, or Fspl.

In embodiments, the enzyme is selected from Cbx4, Kl (Klotho), Ragl, Rag2, Sin/Polr3e, Tmpo (thymopoetin), Traf6, Pten, Prssl6, or beta(5t)/Psmbl l. In embodiments, the enzyme comprises a phosphatase, e.g., Pten. In embodiments, the enzyme comprises a protease, e.g., Prssl6. In embodiments, the enzyme comprises a proteinase, e.g., beta(5t)/Psmbl l.

In embodiments, the hormone, e.g., secreted hormone, is selected from GnRH, hGH (GH1 and GH2), leptin, leptin (TEC), prolactin, Ptma (thymosin alpha-1), Tshb (thyrotropin (beta chain)), or Ghrl (ghrelin). In embodiments, the secreted hormone encodes a preprotein, e.g., a ghrelin-obestatin preprotein.

In embodiments, the ligand is a secreted ligand. In embodiments, the ligand, e.g., secreted ligand, is selected from SCF (Kitl), BMP4, BMP4 (TEC), Ccll9, Cyr61, D114, Fstll, Fgf21, IGF-1, Ihh, IL10, IL-12b (IL12 p40 subunit), IL-15, IL18, IL2, IL2 (stroma), IL21, IL-22, IL23, IL6, IL-7, KGF (Fgf7), Lif, Shh (T-cell), Shh (TEC), Tslp, Wnt3a, Wnt3a (TEC), Wnt4, Wn4 (TEC), Kitl, or Flt3L. In embodiments, the ligand is a non-secreted ligand, e.g., membrane bound ligand. In embodiments, the ligand, e.g., non-secreted ligand, e.g., membrane bound ligand, is selected from 1 lb-HSD2, CD70, Icaml, RANKL, Tbata, Vcaml, SCF (Kitl), Kitl, or Flt3L.

In embodiments, the matrix protein is Satbl.

In embodiments, the receptor is selected from TCR (e.g., a TCR comprising an alpha and a beta chain, a TCR comprising a gamma chain and a delta chain, a TCR comprising a CD3 chain, a TCR comprising a zeta chain, a TCR comprising a complementarity determining region, and a TCR comprising a T cell co-receptor (e.g., CD4 or CD8)), AR, Bmprla, Bmprla (TEC), Bmpr2, Ccr7, Ccr9, CD127 (non-soluble IL-7R), CD27, Cd44, Cdh5 (VE cadherin), Cldn4, Crlf2 (TSLP-R), Cxcr4, EphB2, Fgfr2, Flt3, Fzdl, FzdlO, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, gpl30, HLA (e.g., HLA-A, HLA-B, HLA-B27, HLA-B47, HLA-C, HLA-E, HLA- F, HLA-G, p2-microglobulin, HLA-DM (e.g., HLA-DMA1 and/or HLA-DMB 1), HLA-DO (e.g., HLA-DO A 1 and/or HLA-DOB 1), HLA-DP (e.g, HLA-DPA1 and/or HLA-DPB 1), HLA- DQ (e.g., HLA-DQA1 and/or HLA-DQB 1), HLA-DQ2, HLA-DQ8, HLA-DR (e.g., HLA- DRA, HLA-DRB 1, HLA-DRB3, HLA-DRB4, and/or HLA-DRB5), HLA-DR2, HLA-DR3, HLA-DR4, and/or an HLA encoding a component of the complement system), Ifnarl,

IL7Ralpha, Ltbr, Major Histocompatibility Complexes (e.g., Class I MHC molecule (MHC I), e.g., comprising one or more polypeptides encoded by a HLA-A, HLA-B, HLA-C, HLA-G, and/or HLA-E gene; Class II MHC molecule (MHC II), e.g., comprising one or more

polypeptides encoded by a HLA-DP, HLA-DQ, and/or HLA-DR gene; or Class III MHC molecule (MHC III), e.g., comprising a polypeptide involved in inflammation, e.g., a component of the complement system (e.g., C2, C4, or factor B), tumor necrosis factor (TNF)-a,

lymphotoxin-a, lymphotoxin-β, or a heat shock protein), NLRP3, Notchl, Ptchl, Ptch2, RANK, S 1PR1 (S 1P1), Sele (E-selectin, CD62E, ELAM-1, or LECAM2), Sell (CD62L), Selp (P- selectin), Tgfbr2, Tnfrsfl la/RANK, Tnfrsf 1 lb, or PPARgamma. In embodiments, the receptor is a nuclear receptor, e.g PPARgamma. In embodiments, a TCR includes a TCR that binds to an epitope presented on an MHCI molecule, an epitope presented on an MHCII molecule, or an epitope presented on an MHCIII molecule).

In embodiments, the transcription factor is PPARgamma, AIRE, Atf3, E2F2, E2F2 (TEC), E2F3, E2F4, Egrl, Egr2, Egr3, Fezf2, Foxnl, Foxol, FoxP3, Gata2, Gata3, Gfil, Id3, l, Klf3, Lefl, Meisl, Nfat5, Nfatcl, NFkB l, Runxl, Smad4, Smad6, Stat3, Tbxl, Tcfl, Zfp3611, Zfp3612, or Nfkb2.

In embodiments, the thymic function factor is a positive regulator of a thymic function, e.g., the thymic function factor is selected from Shh (T-cell), E2F2, CD70, Cldn4, D114, Egrl, Egr2, Egr3, EphB2, Flt3, Flt3L, Foxol, FoxP3, Gata3, Gfil, Ghrl (ghrelin), GnRH, Icaml, Id3, Ifnarl, IL-12b (IL12 p40 subunit), IL-15, IL18, IL2, IL21, IL-7, K , Lefl, leptin, Nfat5, Nfatcl, Notchl, prolactin, Ragl, Rag2, Runxl, Satbl, SCF (KM), Tcfl, Tshb (thyrotropin beta chain), Tslp, Vcaml, Wnt3a, Wnt4, Zfp3611, Zfp3612, CD127 (non-soluble IL-7R), CD27, Crlf2 (TSLP- R), Fzdl, FzdlO, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, IL-7Ralpha, Ptchl, Ptch2, Ccll9, Ccl21, CCL25, Ccr7, Ccr9, Cxcll2, Cxcr4, S 1PR1 (S 1P1), Sele (E-selectin, CD62E, ELAM-1, or LECAM2), Sell (CD62L), Selp (P-selectin), AIRE, beta(5t)/Psmbl l, Fezf2, Prssl6, BMP4 (TEC), Cbx4, Cdh5 (VE cadherin), E2F3, Foxnl, Fspl, Kl (Klotho), Ltbr, NFkB l, Pten, RANK, RANKL, Shh (TEC), Sin/Polr3e, Stat3, Tnfrsfl la/RANK, Traf6, Wnt3a (TEC), Wnt4 (TEC), Bmpr2, Fgfr2, Bmprla (TEC), Cyr61, E2F2 (TEC), IGF-1, IL-22, IL-23, KGF (Fgf7), l lb-HSD2, Fgf21, IL10, IL2 (stroma), leptin (TEC), Meisl, Cd44, hGH (GHl and GH2), Nfkb2, BMP4, Klf3, Isll, Ptma (thymosin alpha- 1), Smad4, IL6, or Ihh.

In embodiments, the positive regulator of thymic function comprises a T-cell growth factor, e.g., BMP4, Klf3, Shh (T-cell), IL6, CD70, Cldn4, D114, Egrl, Egr2, Egr3, EphB2, Flt3, Flt3L, Foxol, FoxP3, Gata3, Gfil, Ghrl (ghrelin), GnRH, Icaml, Id3, Ifnarl, IL-12b (IL12 p40 subunit), IL-15, IL18, IL2, IL21, IL-7, K , Lefl, leptin, Nfat5, Nfatcl, Notch 1, prolactin, Ragl, Rag2, Runxl, Satbl, SCF (K ), Tcfl, Tshb (thyrotropin beta chain), Tslp, Vcaml, Wnt3a, Wnt4, Zfp3611, or Zfp3612. In embodiments, the positive regulator of thymic function comprises a T-cell activity factor, e.g., E2F2. In embodiments, the positive regulator of thymic function comprises a T-cell growth factor receptor, e.g., Ihh, CD 127 (non-soluble IL-7R), CD27, Crlf2 (TSLP-R), Fzdl, FzdlO, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Ptchl, Ptch2, or IL-7Ralpha. In embodiments, the positive regulator of thymic function comprises a T-cell migration factor, e.g., Ccll9, Ccl21, CCL25, Ccr7, Ccr9, Cxcll2, Cxcr4, S 1PR1 (S 1P1), Sele (E- selectin, CD62E, ELAM-1, or LECAM2), Sell (CD62L), or Selp (P-selectin). In embodiments, the positive regulator of thymic function comprises a TEC function factor, e.g., AIRE, beta(5t)/Psmbl 1, Fezf2, or Prssl6. In embodiments, the positive regulator of thymic function comprises a TEC growth factor, e.g., Isll, BMP4 (TEC), Cbx4, Cdh5 (VE cadherin), E2F3, Foxnl, Fspl, Kl (Klotho), Ltbr, NFkB l, Pten, RANK, RANKL, Shh (TEC), Sin/Polr3e, Stat3, Tnfrsfl la/RANK, Traf6, Wnt3a (TEC), or Wnt4 (TEC). In embodiments, the positive regulator of thymic function comprises a TEC growth factor receptor, e.g., Bmpr2, Fgfr2, or Bmprla (TEC). In embodiments, the positive regulator of thymic function comprises a TEC proliferation factor, e.g., Cyr61, E2F2 (TEC), IGF-1, IL-22, IL-23, or KGF (Fgf7). In embodiments, the positive regulator of thymic function comprises a thymus homeostasis factor, e.g., l lb-HSD2, Fgf21, IL10, IL2 (stroma), Leptin (TEC), or Meisl.

In other embodiments, the thymic function factor is a negative regulator of a thymic function, e.g., the thymic function factor is selected from Shh, Bmprla, Gata2, Lif, Atf3, E2F4, PPARgamma, Tbata, Tbxl, Tgfbr2, AR, ASC, Axin, NLRP3, gpl30, Tnfrsfl lb, E2F2, BMP4, Ihh, or IL6. In embodiments, the negative regulator of thymic function comprises a T-cell growth factor, e.g., BMP4, Shh (T-cell), Bmprla, Gata2, IL6, or Lif. In embodiments, the negative regulator of thymic function comprises a TEC growth factor, e.g., Atf3, E2F4, PPARgamma, Tbata, Tbxl, Tgfbr2, or Tnfrsf 1 lb. In embodiments, the negative regulator of thymic function comprises a thymus homeostasis factor, e.g., AR, ASC, Axin, or NLRP3. In embodiments, the negative regulator of thymic function comprises a T-cell activity factor, e.g., E2F2.

In embodiments, the thymic function factor is a positive and/or negative regulator of a thymic function, e.g., the thymic function factor is selected from BMP4, IL6, Ihh, E2F2, or Shh (T-cell).

In embodiments, the thymic function factor is selected from TCR (e.g., a TCR comprising an alpha and a beta chain, a TCR comprising a gamma chain and a delta chain, a TCR comprising a CD3 chain, a TCR comprising a zeta chain, a TCR comprising a

complementarity determining region, or a TCR comprising a T cell co-receptor (e.g., CD4 or CD8)), BMP4, BMP4 (TEC), Bmprla (TEC), Bmpr2, Ccl21, CCL25, CD127 (non-soluble IL- 7R), Cxcll2, D114, E2F2 (TEC), E2F3, Fgf21, Flt3L, Foxnl, Ghrl (ghrelin), GnRH, hGH (GH1 and GH2), IL10, IL21, IL-22, IL-23, IL-7, IL-7Ralpha, KGF (Fgf7), Kitl, leptin, leptin (TEC), prolactin, Ptma (thymosin alpha- 1), or SCF (Kitl). In embodiments, a TCR includes a TCR that binds to an epitope presented on an MHCI molecule, an epitope presented on an MHCII molecule, or an epitope presented on an MHCIII molecule

Thymic Function Modulators

In some embodiments of any of the methods described herein, an agent that modulates, e.g., increases or decreases, thymic function is also referred to as a thymic function modulator. In some embodiments, the thymic function modulator increases or decreases the expression or activity of one or more thymic function factors described herein.

In some embodiments of any of the methods described herein, the thymic function modulator comprises a nucleic acid molecule (e.g., DNA, mRNA or an inhibitory RNA), a peptide, an antibody molecule (e.g., an antibody or antigen binding fragment thereof), or a small molecule.

In some embodiments of any of the methods described herein, the thymic function modulator increases the expression or activity of one or more thymic function factors described herein, e.g., listed in Table 1. In one embodiment, the increase in expression or activity of the one or more thymic function factors is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, increased as compared to a reference expression or activity level of the one or more thymic function factors. In one embodiment, the reference expression or activity level is the expression or activity level of the one or more thymic function factors prior to administration of the thymic function modulator.

In some embodiments of any of the methods described herein, the thymic function modulator decreases the expression or activity of one or more thymic function factors described herein, e.g., listed Table 1. In one embodiment, the decrease in expression or activity of one or more thymic function factors is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold , 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, decreased as compared to a reference expression or activity level of the one or more thymic function factors. In one embodiment, the reference expression or activity level is the expression or activity level of the one or more thymic function factors prior to administration of the thymic function modulator.

In some embodiments of any of the methods described herein, the thymic function modulator is itself a thymic function factor (or fragment thereof), e.g., a thymic function factor described herein, e.g., in Table 1.

In one embodiment of any of the methods described herein, the thymic function modulator comprises a nucleic acid molecule. In one embodiment, the thymic function modulator comprises a DNA molecule or a RNA molecule. In one embodiment, the thymic function modulator comprises a RNA molecule, e.g., an inhibitory RNA or an RNA therapeutic that encodes a protein. In one embodiment, the thymic function modulator comprises an inhibitory or agonistic antibody.

In one embodiment of any of the methods described herein, the thymic function modulator comprises an mRNA molecule (e.g., an RNA therapeutic) that encodes one or more of the thymic function factors described herein, e.g., listed in Table 1, or a functional fragment thereof. In one embodiment, the mRNA molecule encodes an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence listed in Table 1, or a functional fragment thereof. In one embodiment, the mRNA molecule encodes an amino acid sequence that differs by no more than 20, 10, 5, 4, 3, 2, or 1 amino acids to an amino acid sequence listed in Table 1. In one embodiment, the mRNA molecule encodes an amino acid sequence comprising an amino acid sequence listed in Table 1, or a functional fragment thereof.

In one embodiment of any of the methods described herein, the thymic function modulator comprises an inhibitory RNA, e.g., an interfering RNA (RNAi) molecule. In one embodiment, the RNAi molecule is selected from a siRNA, a short hairpin RNA, or a microRNA. In one embodiment, the interfering RNA molecule decreases or inhibits expression of one or more of the thymic function factors listed in Table 1.

In one embodiment of any of the methods described herein, the thymic function modulator comprises an mRNA molecule that encodes an antibody molecule (e.g., an antibody or an antigen binding fragment thereof) that targets (e.g., specifically binds to) one or more of the thymic function factors described herein, e.g., listed in Table 1.

In one embodiment of any of the methods described herein, the thymic function modulator comprises a guide RNA sequence that enables CRISPR-mediated gene editing of one or more of the thymic function factors described herein, e.g., listed in Table 1.

In one embodiment of any of the methods described herein, the thymic function modulator comprises an mRNA molecule that encodes a zinc finger nuclease (ZFN) that targets (e.g., cleaves) the sequence encoding one or more thymic function factors described herein, e.g., listed in Table 1.

In one embodiments of any of the methods described herein, the thymic function modulator comprises an epigenetic modifying agent, e.g., an epigenetic modifying agent described herein.

In some embodiments of any of the methods described herein, one or more additional thymic function modulators, e.g., one, two, three, four, or five additional thymic function modulators (e.g., a second, third, fourth, fifth or sixth thymic function modulator) is administered to the subject. In one embodiment, the one or more of the thymic function modulators increase or decrease the expression or activity level of the same type of thymic function factor, e.g., a T cell growth factor, a T cell growth factor receptor, a T cell proliferation factor, a T cell migration factor, a T cell activity factor, a TEC proliferation factor, a TEC growth factor, a TEC growth factor receptor, a TEC function factor, or a thymus homeostasis factor. By way of example, in an embodiment, two thymic function modulators are administered, and both thymic function modulators increase or decrease the expression or activity level of a T cell growth factor. In other embodiments where two or more thymic functional modulators are administered, two or more of the thymic function modulators increase or decrease the expression or activity level of different types of thymic function factors, e.g., a T cell growth factor, a T cell growth factor receptor, a T cell proliferation factor, a T cell migration factor, a T cell activity factor, a TEC proliferation factor, a TEC growth factor, a TEC growth factor receptor, a TEC function factor, or a thymus homeostasis factor. By way of another example, in another embodiment, two thymic function modulators are administered, and one thymic function modulator increases or decreases the expression or activity level of a T cell growth factor while the other thymic function modulator increases or decreases the expression or activity level of a TEC proliferation factor. In any of the aforementioned embodiments, the one or more thymic function modulators increase or decrease the expression or activity level of a thymic function factor described herein, e.g., listed in Table 1.

In some embodiments of any of the methods described herein, administering two or more thymic function modulators results in a synergistic effect. In one embodiment, a synergistic effect is observed when the effect of the two or more thymic function modulators is greater than the additive effect observed of each of the thymic function modulators.

In some embodiments, the method includes administering a first and a second thymic function modulator. In one embodiment, the first and second thymic function modulators are formulated in separate dosage forms. In another embodiment, the first and second thymic function modulators are formulated in the same dosage form. In one embodiment, the first and second thymic function modulators are coupled by a covalent bond, a non-covalent bond, or a chemical linkage. In one embodiment, the first and second thymic modulators are separated by a linker.

In some embodiments, the first and second thymic function modulators are each mRNA molecules (e.g., encoding a thymic function factor or functional fragment thereof). In one embodiment, the first and second thymic function modulators are coupled by a phosphodiester bond. In one embodiment, the first and second thymic function modulators are translated into a single polypeptide (e.g., a chimeric polypeptide or a polypeptide that can be cleaved into two polypeptides). In one embodiment, the first and second thymic function modulators are separated by a peptide cleavage site. In one embodiment, the first thymic function modulator is translated into a first polypeptide and the second thymic function modulator is translated into a second polypeptide.

In some embodiments, the first and second thymic function modulators are each interfering RNA molecules (e.g., a siRNA, a shRNA, or a miRNA). In one embodiment, the first and second thymic function modulators are each siRNAs, and wherein the first and second thymic function modulators are coupled by a linker, e.g., a peptide linkage or a chemical linkage, e.g., as described herein.

In some embodiments, the method includes administering more than one (e.g., two, three, four, five, or more) thymic function modulator. In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are formulated in separate dosage forms. In another embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are formulated in the same dosage form. In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are coupled to each other by a covalent bond, a non-covalent bond, or a chemical linkage. In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic modulators are separated from each other by a linker.

In some embodiments, the more than one (e.g., two, three, four, five, or more) thymic function modulators are each mRNA molecules (e.g., encoding a thymic function factor or functional fragment thereof). In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are coupled to each other by a phosphodiester bond. In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are translated into a single polypeptide (e.g., a chimeric polypeptide or a polypeptide that can be cleaved into multiple polypeptides). In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are separated from each other by a peptide cleavage site. In one embodiment, each thymic function modulator is translated into a polypeptide.

In some embodiments, the more than one (e.g., two, three, four, five, or more) thymic function modulators are each interfering RNA molecules (e.g., a siRNA, a shRNA, or a miRNA). In one embodiment, the more than one (e.g., two, three, four, five, or more) thymic function modulators are each siRNAs, and each of the thymic function modulators are coupled by a linker, e.g., a peptide linkage or a chemical linkage, e.g., as described herein.

Assessing Thymic Involution

In some embodiments of any of the methods described herein, the methods further include assessing one or more markers of thymic structure or function (e.g., one or markers of thymus involution). In some embodiments, the methods include identifying the presence of a thymus tissue after treatment in subjects where no thymus or thymus tissue was detectable prior to treatment.

In some embodiments, the method includes measuring the size (e.g., length, width, and/or thickness), opacity, and/or density of the anterior mediastinal shadow, e.g., to assess a decrease or a reverse in thymic involution. In one embodiment, measuring the size of the anterior mediastinal shadow includes measuring the length, measuring the width, and/or measuring the thickness of the anterior mediastinal shadow. In one embodiment, the size, opacity, and/or density of the anterior mediastinal shadow determined from an imaging technique. Examples of imaging techniques include ultrasound, X-ray, CAT scan, MRI, or PET (e.g., FDG avidity via PET). In one embodiment, a decrease or reverse in thymic involution is characterized by an increase, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more, in size (e.g., length and/or width) of the anterior mediastinal shadow, as compared to a reference size of the anterior mediastinal shadow. In one embodiment, a decrease or reverse in thymic involution is characterized by an increase, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more increase, in the density of the anterior mediastinal shadow, as compared to a reference density of the anterior mediastinal shadow. In one embodiment, a decrease or reverse in thymic involution is characterized by an increase, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more increase, in the opacity of the anterior mediastinal shadow, as compared to a reference opacity of the anterior mediastinal shadow. In such embodiments, the reference size, density, and/or opacity of the anterior mediastinal shadow is the size, density, and/or opacity of the anterior mediastinal shadow in the subject prior to administration of the thymic function modulator. In some embodiments, the method includes assessing one or more markers of thymic involution. In one embodiment, the marker of thymic involution is selected from: the level of stromal cells; the level of non-stromal cells; the level of adipocytes; the level of non-adipocytes; T cell diversity; TCR repertoire diversity; T cell clonality; or T cell diversity and T cell clonality. In one embodiment, a decrease or a reverse in thymic involution is characterized by a decrease in the level or number of adipocytes, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more decrease, e.g., as compared to prior to treatment. In one embodiment, a decrease or a reverse in thymic involution is characterized by a increase in the level or number of non-adipocytes, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more decrease, e.g., as compared to prior to treatment. In one embodiment, a decrease or a reverse in thymic involution is characterized by an increase in T cell diversity, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more increase in T cell diversity, e.g., as compared to prior to treatment. In one embodiment, a decrease or a reverse in thymic involution is characterized by an increase in TCR repertoire diversity, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more increase in TCR repertoire diversity, e.g., as compared to prior to treatment. In one embodiment, a decrease or a reverse in thymic involution is characterized by a decrease in T cell clonality, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more decrease in T cell clonality, e.g., as compared to prior to treatment. In one embodiment, a decrease or a reverse in thymic involution is characterized by an increase in T cell clonality, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more increase in T cell clonality, e.g., as compared to prior to treatment. In one embodiment, a decrease or a reverse in thymic involution is characterized by an increase in T cell or TCR repertoire diversity, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more increase in T cell or TCR repertoire diversity, and a decrease or increase in T cell clonality, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more decrease or increase in T cell clonality, e.g., as compared to prior to treatment.

Methods of Treating

In another aspect, the invention features methods of modulating, e.g., treating physiological senescence, e.g., reversing, reducing the rate of, or delaying physiological senescence, in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby treating the subject. In some embodiments, physiological senescence is associated with one or more of muscle atrophy/degeneration; decrease in bone health (e.g., bone strength and/or density); immunosenescence (e.g., decrease in immune responsiveness); cardiac disease; uncontrolled cell growth (e.g., tumorigenesis).

In some embodiments, a subject experiencing physiological senescence has a disease or condition associated with muscle atrophy/degeneration; decrease in bone health (e.g., bone strength and/or density); immunosenescence (e.g., decrease in immune responsiveness); cardiac disease; uncontrolled cell growth (e.g., tumorigenesis). In one embodiment, the subject has a muscular degenerative disorder (e.g., a myopathy, muscle wasting, or sarcopenia); amyotrophic lateral sclerosis (ALS); osteoporosis and related diseases; a cardiac disease (e.g., myocardial infarction, atherosclerosis); a cancer (e.g., a solid cancer, such as uterine cancer, colon cancer, ovarian cancer, rectal cancer, skin cancer, stomach cancer, lung cancer, non-small cell carcinoma of the lung, breast cancer, cancer of the small intestine, testicular cancer, cancer of the anal region, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, rectal cancer, renal-cell carcinoma, liver cancer, cancer of the esophagus, melanoma, cutaneous or intraocular malignant melanoma, uterine cancer, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, cancer of the adrenal gland, bone cancer, pancreatic cancer, cancer of the head or neck, epidermoid cancer, carcinoma of the endometrium, carcinoma of the vagina, cervical cancer, sarcoma, uterine cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, carcinoma of the cervix squamous cell cancer, carcinoma of the fallopian tubes, sarcoma of soft tissue, cancer of the urethra, carcinoma of the vulva, cancer of the kidney or ureter, carcinoma of the renal pelvis, spinal axis tumor, cancer of the penis, cancer of the bladder, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, metastatic lesions of said cancers, and/or combinations thereof; or a hematological cancer, e.g., a leukemia or lymphoma, e.g., a hematological cancer such as acute lymphoid leukemia (ALL); one or more chronic leukemias, e.g., chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL), B cell prolymphocytic leukemia; B-cell acute Lymphoid Leukemia ("BALL"); T-cell acute Lymphoid Leukemia ("TALL"); blastic plasmacytoid dendritic cell neoplasm; Follicular lymphoma; diffuse large B cell lymphoma; non-Hodgkin lymphoma; Hodgkin lymphoma; Burkitt's lymphoma; malignant lymphoproliferative conditions; Hairy cell leukemia; small cell- or large cell-follicular lymphoma; mantle cell lymphoma; MALT lymphoma; marginal zone lymphoma; multiple myeloma; myelodysplasia; myelodysplastic syndrome; Waldenstrom macroglobulinemia;

plasmablastic lymphoma; and plasmacytoid dendritic cell neoplasm); or a metabolic disease (e.g., diabetes, e.g., type I diabetes, or obesity).

In some embodiments, the method further includes monitoring the muscle function, bone health, cardiac health, and/or immune responsiveness of the subject.

In another aspect, the invention features methods of treating a thymic injury in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby treating the subject.

In one embodiment, the injury is induced by a disease, a drug treatment, irradiation, or other environmental factor. In one embodiment, the injury is induced by drug is a cytoreductive or chemo therapeutic drug.

In embodiments, the injury is an acute injury. In other embodiments, the injury is a chronic injury.

In another aspect, the invention features methods of enhancing, e.g., increasing, an immune response, e.g., an anti-cancer immune response, a vaccine response, or an immune response to an infection (e.g., chronic infection), in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby enhancing the immune response in the subject.

In some embodiments, the cancer is selected from a hematological cancer or a solid cancer such as lung cancer, non-small cell lung cancer (NSCLC), skin cancer, melanoma, cervical cancer, uterine cancer, ovarian cancer, breast cancer, pancreatic cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, sarcoma, lymphoma, and brain cancer. In embodiments, the method comprises administering the thymic function modulator in combination with an immunotherapy, e.g., cancer immunotherapy, e.g., cancer immunotherapy described herein.

In embodiments, the infection is a chronic infection or an acute infection. In

embodiments, the infection is a bacterial or viral infection. In another aspect, the invention features methods of treating an infectious disease in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby treating the subject.

In some embodiments, the infectious disease is a bacterial infection. In some

embodiments, the infectious disease is a viral infection. In one embodiment, the infection is a chronic infection or an acute infection. In one embodiment, the infection is a parasitic infection.

In another aspect, the invention features methods of treating a subject wherein the subject has an implanted thymus, comprising administering an effective amount of a thymic function modulator described herein, thereby treating the subject. In one embodiment, the subject has, e.g., is experiencing, physiological senescence.

In another aspect, the invention features methods for replacing or increasing thymic function in a subject comprising administering an effective amount of a thymic function modulator described herein in combination with a thymus transplant, thereby replacing and/or augmenting thymic function in the subject.

In embodiments, the thymic function modulator is administered prior to the thymus transplant. In embodiments, the thymic function modulator is administered after the thymus transplant. In embodiments, the thymic function modulator is administered during the thymus transplant treatment, e.g., within 1 day, e.g., within 24 h, 12 h, 6 h, 4 h, 2 h, or less of the thymus transplant.

In accordance with any method described herein, a thymus cell, tissue, or organ can be transplanted into a subject, e.g., as described in Markert et al. Clin. Immunol. 135.2(2010):236- 46 or Sachs et al. Transpl. Immunol. 21.2(2009): 101-105. For example, a thymus cell, tissue, or organ can be transplanted into the quadriceps muscles of the subject.

In another aspect, the invention features methods for decreasing an immune response in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby decreasing the immune response in the subject. In another aspect, the invention features methods for preventing transplant rejection in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby preventing transplant rejection in the subject.

In another aspect, the invention features methods for treating an autoimmune disease in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby treating the autoimmune disease in the subject.

In embodiments, the method comprises inducing negative selection in the subject, e.g., inducing clonal deletion and/or inducing Treg generation in the subject.

In embodiments, the transplant comprises a cell or organ transplant. In embodiments, the cell comprises a stem cell, e.g., hematopoietic stem cell and/or mesenchymal stem cell. In embodiments, the organ comprises a lung, heart, eye, liver, or kidney.

In embodiments, the autoimmune disease is rheumatoid arthritis, j venile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, or pernicious anemia.

In another aspect, the invention features methods for reducing or preventing thymic involution in a subject comprising administering an effective amount of a thymic function modulator described herein, thereby reducing or preventing thymic involution in the subject.

In embodiments, the thymic involution is caused by aging, a drug or treatment, and/or a disease. In embodiments, the drug or treatment comprises a chemotherapy, radiation, orimmunotherapy. In embodiments, the disease comprises a cancer or an infection, e.g., chronic or acute infection. In embodiments, the cancer is selected from a hematological cancer or a solid cancer, e.g., a hematological or solid cancer described herein, e.g., lung cancer, non-small cell lung cancer (NSCLC), skin cancer, melanoma, cervical cancer, uterine cancer, ovarian cancer, breast cancer, pancreatic cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, sarcoma, lymphoma, or brain cancer. In embodiments, the infection is a bacterial infection or a viral infection.

Dosing and Administration

In some embodiments, the thymic function modulator is administered in a single dose.

In some embodiments, the thymic function modulator is administered in multiple, e.g., two or more, doses. In one embodiment, the thymic function modulator is administered once a day, once every two days, once every 5 days, once a week, once every other week, or once a month. In one embodiment, multiple doses of the thymic function modulator are administered over a period of 1 month, 2 months, 3 months, 4 months, 6 months, or more. In another embodiment, multiple doses of the thymic function modulator are administered over a period of 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days or less.

In some embodiments, a first treatment regimen comprising one or more doses of a first thymic function modulator and a second treatment regimen comprising one or more doses of a second thymic function modulator are administered simultaneously or sequentially to the subject. In one embodiment, the first treatment regimen and the second treatment regimen are

administered simultaneously. In one embodiment, the first treatment regimen and the second treatment regimen are overlapping. In one embodiment, the first treatment regimen is initiated prior to, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, the initiation of the second treatment regimen. In one embodiment, the second treatment regimen is initiated prior, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, to the initiation of the first treatment regimen. In one embodiment, the first treatment regimen is completed prior to, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, initiation of the second treatment regimen. In one embodiment, the second treatment regimen is completed prior to, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, the initiation of the first treatment regimen. In some embodiments, a first treatment regimen comprising one or more doses of a first thymic function modulator and one or more additional treatment regimens, e.g., a second, third, fourth, or fifth treatment regimen, wherein each additional treatment regimen comprises one or more doses of an additional thymic function modulator, e.g., second, third, fourth, or fifth thymic function modulator, are administered simultaneously or sequentially to the subject. In one embodiment, the first treatment regimen and the one or more, e.g., second, third, fourth, or fifth, treatment regimens are administered simultaneously. In one embodiment, the first treatment regimen and the one or more, e.g., second, third, fourth, or fifth, treatment regimens are overlapping. In one embodiment, the one (e.g., the first) treatment regimen is initiated prior to, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, the initiation of another (e.g., the second, third, fourth, and/or fifth) treatment regimen. In one embodiment, the one (e.g., the first) treatment regimen is completed prior to, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, the initiation of another (e.g., the second, third, fourth, and/or fifth) treatment regimen.

In some embodiments, the method further includes monitoring the subject for one or more of the following: anterior mediastinal shadow size (e.g., length, width, and/or thickness); anterior mediastinal shadow density; anterior mediastinal shadow opacity; level of adipocytes and/or non-adipocytes; level of stromal and/or non-stromal cells; T cell clonality; T cell diversity or TCR repertoire diversity; or T cell clonality and T cell diversity or TCR repertoire diversity.

In some embodiments, doses of the thymic function modulator are administered until one or more of the following occurs: an increase in a parameter associated with anterior mediastinal shadow (e.g., size, density, and/or opacity); an increase in the level of non-adipocyte cells and/or a decrease in adipocyte cells; an increase in the level of stromal cells and/or a decrease in the level of non-stromal cells; a decrease in T cell clonality; an increase in T cell diversity or TCR repertoire diversity; or a decrease in T cell clonality and an increase in T cell diversity or TCR repertoire diversity.

In some embodiments, the thymic function modulator is administered intravenously. In embodiments, the thymic function modulator is administered orally. In some embodiments, the thymic function modulator is administered intrathymically. In embodiments where more than one thymic function modulator is administered, one thymic function modulator is administered intravenously and another thymic function modulator is administered intrathymically. In some embodiments, the thymic function modulator is administered in combination with an additional therapeutic agent. In one embodiment, the additional therapeutic agent is selected from: an immunomodulatory agent, an antimicrobial, or a chemotherapy. In

embodiments, the thymic function modulator is administered in combination with an additional treatment, e.g., a radiation or surgery.

Subjects

In some embodiments of any of the methods described herein, the subject has or is experiencing physiological senescence. In one embodiment, physiological senescence comprises muscle atrophy/degeneration, inadequate bone strength/density, immunosenescence (age-related decrease in immune responsiveness or menopause), or cardiac disease.

In some embodiments of any of the methods described herein, the subject has a disease or condition associated with muscle atrophy/degeneration, inadequate bone strength/density, immunosenescence (age-related decrease in immune responsiveness or menopause), or cardiac disease.

In some embodiments of any of the methods described herein, the subject has an acute thymic injury.

In some embodiments of any of the methods described herein, the subject has drug or radiation induced thymic damage.

In some embodiments of any of the methods described herein, the subject is an adult subject, e.g., a geriatric subject, e.g., a subject older than 60, 65, 70, 75, 80, 85, 90.

In some embodiments of any of the methods described herein, the subject is a juvenile subject, e.g., less than 18 years old.

In some embodiments of any of the methods described herein, the subject does not have a detectable thymus before treatment.

In some embodiments of any of the methods described herein, the subject has an implanted thymus tissue, e.g., has an implanted thymus.

In some embodiments of any of the methods described herein, the subject has had a thymic transplant.

In some embodiments of any of the methods described herein, the subject has undergone, is undergoing, or will undergo a cell (e.g., stem cell, e.g., hematopoietic stem cell) transplant. In embodiments, the method comprises administering a thymic function factor or thymic function modulator (e.g., a KGF mRNA (e.g., modified or synthetic mRNA)) to the subject. In embodiments, the transplant occurs subsequent to or prior to (e.g., 1, 2, 3, or more days before or after) the administration of the thymic function factor or thymic function modulator (e.g., a KGF mRNA (e.g., modified or synthetic mRNA)). In embodiments, the transplant occurs within 24 hours (e.g., within 24, 12, 10, 8, 6, 4, 3, 2, 1 hour or less) of the administration of the thymic function factor or thymic function modulator (e.g., a KGF mRNA (e.g., modified or synthetic mRNA)). In embodiments, the method further comprises administering a second agent (e.g., somatropin) to the subject. In embodiments, the second agent (e.g., somatropin) is administered after (e.g., daily for 1, 2, 3 or more months after) the transplant and/or the thymic function factor or thymic function modulator (e.g., a KGF mRNA (e.g., modified or synthetic mRNA)) is administered before (e.g., 5, 4, 3, 2, 1 day or less before) the transplant.

In another aspect, the invention features methods of treating a subject having a thymus related disease or condition, or aging-related disease or condition (e.g., a condition described herein) comprising administering an effective amount of a thymic function modulator described herein, thereby treating the subject.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure relates to compositions comprising thymic function modulators and methods of use. Without wishing to be bound by theory, it is believed that thymic function modulators improve one or more functions of the thymus and can, e.g., lead to rejuvenation of thymus activity. Accordingly, thymic function modulators are useful for treating diseases and conditions associated with a loss or reduction of thymic activity. A reduction in thymic activity can occur, e.g., due to age (e.g., thymic involution) or stresses (e.g., trauma, drug effects, and diseases). Such diseases and conditions can be related to or include: physiological senescence, autoimmunity, cardiac disease, cancer, infectious disease, muscle atrophy/degeneration, decrease in bone health, immunosenescence, thymic injury, and other diseases/conditions described herein. For example, featured herein are therapeutic methods for decreasing or reversing thymic involution. Also featured herein are methods of enhancing the function/lifetime of a thymus transplant. Further embodiments are described in greater detail herein. Definitions:

As used herein, a "thymic function modulator" is an agent that modulates, e.g., increases or decreases, thymic function. Determination of thymic function is described herein, e.g., in the "Measurement of thymus function" section. Thymic function modulators are described in greater detail herein, e.g., in the "Thymic function modulators" section.

As used herein, a "thymic function factor" is a molecule, e.g., protein, peptide, polypeptide, or nucleic acid molecule (e.g., oligonucleotide, e.g., DNA, RNA, or mRNA, or gene), that is associated with thymic function. In embodiments, expression, e.g., high

expression, of a thymic function factor is associated with increased or decreased thymic function. In embodiments, lack of or low expression of a thymic function factor is associated with increased or decreased thymic function. Thymic function factors are described in greater detail herein, e.g., in the "Thymic function factors" section.

As used herein, an "antibody molecule" is a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody molecule can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region: (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody molecule" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab') 2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). In some embodiments, antibodies are recombinant human or humanized antibodies.

As used herein, a "combination therapy" or "administered in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous

administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection while a second therapeutic agent of the combination may be

administered orally.

As used herein, the term "pharmaceutical composition" refers to a medicinal or pharmaceutical formulation that contains one or more active ingredient as well as one or more excipients and diluents to enable the active ingredient(s) suitable for the method of

administration. The pharmaceutical composition of the present invention includes

pharmaceutically acceptable components that are compatible with the agents described herein. The pharmaceutical composition is typically in aqueous form for intravenous or subcutaneous administration. In embodiments, a pharmaceutical composition or pharmaceutical preparation is a composition or preparation produced under good manufacturing practices (GMP) conditions, having pharmacological activity or other direct effect in the mitigation, treatment, or prevention of disease, and/or a finished dosage form or formulation thereof and is for human use.

As used herein, the terms "increasing" and "decreasing" refer to modulating resulting in, respectively, greater or lesser amounts, function or activity of a metric relative to a reference. For example, subsequent to administration of a thymic function modulator, the amount of a marker of thymic function may be increased or decreased in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more relative to the amount of the marker prior to administration. Generally, the metric is measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least one week, one month, 3 months, 6 months, after a treatment regimen (e.g., a therapy described herein) has begun.

"Treatment" and "treating," as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy).

The term "synergy" or "synergistic" means a more than additive effect of a combination of two or more agents (e.g., a combination therapy described herein) compared to their individual effects. In certain embodiments, synergistic activity is present when a first agent produces a detectable level of an output X, a second agent produces a detectable level of the output X, and the first and second agents together produce a more-than-additive level of the output X.

The term "heterologous thymic capability," as used herein, refers to the ability to carry out one or more thymic functions, e.g., the presence of a cell or tissue that has a thymic function, e.g., a thymic cell or tissue. In embodiments, the thymic cell or tissue comprises a thymocyte, epithelial thymic cell (e.g., of the cortex or medulla), stromal cell and/or a dendritic cell (e.g., a conventional dendritic cell or a plasmacytoid dendritic cell). In embodiments, the thymic cell or tissue can be autologous or allogeneic. In embodiments, the thymic cell or tissue can be suitable for implantation. In embodiments, the thymic cell or tissue has been implanted in a subject. In embodiments, the thymic cell or tissue is evaluated by assessing certain thymic activities, functions, markers or characteristics, e.g., using a method described herein, e.g., in the

"Measurement of thymus function" section herein.

As used herein the term "peptide linker" refers to an amino acid sequence (e.g., synthetic amino acid sequence) that connects or links two polypeptide sequences, e.g., that links two polypeptide domains. As used herein the term "synthetic" refers to amino acid sequences that are not naturally occurring. In embodiments, peptide linkers connect two amino acid sequences via peptide bonds, e.g., in a linear sequence. In one embodiment, a peptide linker connects a biologically active moiety to a second moiety in a linear sequence. In another embodiment, a peptide linker connects two biologically active moieties.

As used herein, the terms "linked," "fused", or "fusion", are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. Any method of chemical conjugation (e.g., using heterobifunctional crosslinking agents) can be used. For example, two or more molecules, e.g., nucleic acid molecules (e.g., mRNAs or inhibitory nucleic acid molecules), small molecules, and/or peptides/polypeptides can be linked, e.g., using a linker, e.g., chemical linker or peptide linker. As used herein, the term "genetically fused," "genetical ly linked" or "genetic fusion" refers to the co-linear, covalent linkage or attachment of two or more proteins, polypeptides, or fragments thereof via their individual peptide backbones, e.g., through genetic expression of a single polynucleotide molecule encoding those proteins, polypeptides, or fragments. Such genetic fusion results in the expression of a single contiguous genetic sequence. Exemplary genetic fusions are in frame, i.e., two or more open reading frames (ORFs) are fused to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single polypeptide containing two or more protein segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). In this case, in some embodiments, the single polypeptide is cleaved during processing to yield dimeric molecules comprising two polypeptide chains.

Percent identity in the context of two or more polypeptide sequences or nucleic acids, refers to two or more sequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 60% identity, e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms or by manual alignment and visual inspection. In some cases, the identity (or substantial identity) exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Biol.

48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Thymic Involution

In some aspects, featured herein are methods for decreasing or reversing thymic involution in a subject. Thymic involution is a process in which the thymus shrinks in size. Thymic involution occurs gradually with age or may be the result of a stress, e.g., trauma or disease. During the neonatal and developmental period of a mammal, the size and function of the thymus is at its greatest. Involution progresses as a mammal ages, leading to lower thymic function, e.g., less thymopoiesis (T cell maturation) and fewer naive T cells (which are self- tolerant and responsive to foreign antigens but have not been stimulated by a foreign agent). During involution, thymic tissue becomes replaced with adipose tissue. Thymic involution can also occur in a more acute fashion due to factors other than age, e.g., stresses such as therapies (e.g., chemotherapy), pregnancy, diseases or pathological conditions such as infections, and malnutrition.

Thymic function factors

Functional characteristics of thymic function factors

Thymic function factors modulate thymic function and can be characterized by one or more properties described herein. A thymic function factor can itself be a thymic function modulator.

In embodiments, a thymic function factor is a positive regulator of thymic function (e.g., increases one or more activity of the thymus). In other embodiments, a thymic function factor is a negative regulator of thymic function (e.g., decreases one or more activity of the thymus). In embodiments, a thymic function factor can be both a positive and a negative regulator of thymic function. Exemplary positive and/or negative regulators of thymic function are listed in Table 1.

In embodiments, a thymic function factor increases the proliferation or cell count of stromal cells or non-adipocyte cells. Proliferation/cell count can be determined by proliferation assays, e.g., Ki67 staining or PCNA staining.

In embodiments, a thymic function factor changes the stromal/non-stromal cellular balance in the thymus. In embodiments, the stromal/non-stromal cellular balance is determined by a method such as ultrasound, histological analysis, or imaging (e.g., PET imaging, e.g., FDG avidity via PET imaging). In embodiments, a thymic function factor increases or decreases the stromal/non-stromal cellular balance, e.g., increases or decreases the ratio of thymic epithelial cells to thymocytes; or increases or decreases the ratio of adipocytes to thymocytes.

In embodiments, a thymic function factor increases the production and/or level of a thymic hormone. In embodiments, a thymic hormone is associated with the size of the thymus. An exemplary thymic hormone is thymulin. The level of a thymic hormone can be determined by an assay, e.g., a rosette inhibition assay, e.g., as described in Consolini et al. Haematologica 77.3(1992):243-47. In embodiments, a thymic function factor increases the level of a thymic hormone by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50- fold, 100-fold, 1000-fold, or more, e.g., relative to a reference level. In embodiments, a reference level is the level of the thymic hormone in a sample (e.g., a cell or tissue sample, e.g., from a subject) lacking the thymic function factor or a sample (e.g., a cell or tissue sample, e.g., from a subject) prior to addition/administration of the thymic function factor.

In embodiments, a thymic function factor modulates a T cell phenotype, e.g., a peripheral T cell phenotype. An exemplary peripheral T cell phenotype includes a thymic emigrant profile, e.g., percent of T cells that have unique T cell receptors (TCRs) (e.g., TCR diversity), percent of T cells that are FoxP3 positive, percent of T cells that are regulatory T cells (Tregs), percent of cells that are positive for a CD45RA isoform (e.g., CD45RA), percent of cells that are CD4 positive and CD25 positive, or percent of T cells that are cytotoxic T lymphocytes (CTLs). In embodiments, thymic emigrant profile can be determined by quantifying and/or comparing the levels of different T cell populations. In embodiments, a thymic function factor increases the number of Tregs (or percent of cells that are Tregs), e.g., thereby decreasing autoimmunity. In embodiments, a thymic function factor decreases the number of Tregs (or percent of cells that are Tregs), e.g., thereby decreasing the progression or severity of a cancer (e.g., decreasing the number of cancer cells.

In embodiments, a thymic function factor modulates the polyclonality of a B cell response. In embodiments, polyclonality of a B cell responses can be determined by measuring changes in B-cell isotype distribution, polyclonal antibody titers to an antigen, and/or polyclonal antibody titers to one or more thymus -dependent antigens. B-cell isotypes can be detected and/or quantified by assays such as flow cytometry, e.g., fluorescence activated cell sorting (FACS). Antibody titer can be measured by assays such as ELISAs, cytometric bead array, and other kits/methods. ELISAs are plate based assays to quantify soluble proteins, including immunoglobulins (see, e.g., Pathak et al.; 1997; Immunology Methods Manual, vol. 2. 1.

Lefkovitz, ed. Academic Press, Inc., San Diego, p. 1056-1075). Cytometric Bead Array (CBA) is a bead-based immunoassay that allows multiplexed quantitation of soluble and intracellular proteins including immunoglobulin and can be combined with further markers that are used for phenotyping (see, e.g., Morgan et al.; Clin Immunol. 2004). CBAs and ELISAs allow evaluation of immunoglobulin isotypes in serum, plasma or cell culture supernatants. Other methods, e.g., commercially available kits, are available in the art to perform individual isotype determination of secreted immunoglobulins. Populations of B cells can be quantified using FACS, as described in Allman D, Pillai S. Peripheral B cell subsets. Curr Opin Immunol. 2008;20: 149-157. Also, high-throughput DNA sequencing allows determination of the antibody repertoire encoded by B cells in the blood or lymphoid organs and can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development, and to understand autoimmunity (see, e.g., Georgiou et al. 2014. Nature Biotechnology). The detection limit is an estimated 1 in a million (see, e.g., Wu et al. 2014. Clin Cancer Res.).

In embodiments, a thymic function factor modulates thymocyte flux. Thymocyte flux can be determined by measuring the T-Cell Receptor Excision Circles (TRECs) per number of cells, e.g., over a period of time, e.g., over about 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, or more.

Exemplary thymic function factors are listed in Table 1. In embodiments, a thymic function factor is associated with, e.g., results in, one or more effects described in Table 1.

Structural characteristics of thymic function factors

In embodiments, a thymic function factor comprises a peptide, polypeptide, protein, or nucleic acid molecule (e.g., oligonucleotide, RNA, mRNA (e.g., modified or synthetic mRNA), DNA, or gene or gene fragment).

In embodiments, a thymic function factor comprises a ligand (e.g., secreted ligand, a non- secreted ligand (i.e., membrane-bound)), a transmembrane protein (e.g., receptor, e.g., cell surface receptor or nuclear receptor), or a nucleic acid molecule (e.g., gene, DNA, RNA, or mRNA) encoding same. Examples of a ligand (e.g., secreted ligand, a non-secreted ligand (i.e., membrane-bound)), a transmembrane protein (e.g., receptor, e.g., cell surface receptor or nuclear receptor), are shown in Table 1.

In some embodiments, one or more thymic function factors, e.g., one, two, three, four, or five thymic function factors (e.g., a second, third, fourth, fifth or sixth thymic function factor) is administered to the subject. In one embodiment, the one or more of the thymic function factors are associated with increased or decreased activity level of a thymic function as described herein. In embodiments, a thymic function factor comprises a hormone, a transcription factor, an enzyme (e.g., proteinase, protease, phosphatase), a chemokine, a cell matrix protein, cytoplasmic protein, or a nucleic acid molecule (e.g., gene, DNA, RNA, or mRNA) encoding same.

Examples of a hormone, a transcription factor, an enzyme (e.g., proteinase, protease,

phosphatase), a chemokine, a cell matrix protein, cytoplasmic protein are listed in Table 1.

In embodiments, a thymic function factor comprises a growth factor (e.g., T cell growth factor, thymic epithelial cell (TEC) growth factor, thymocyte growth factor, T cell subset specific growth factor (e.g., Tregs, naive T cells, or memory T cells), a growth factor receptor (e.g., a T cell growth factor receptor, a TEC growth factor receptor, a proliferation factor (e.g., T cell proliferation factor, thymic epithelial cell (TEC) proliferation factor), cell migration factor (e.g., T cell migration factor), T cell activity factor, TEC function factor, and/or a thymus homeostasis factor, or a nucleic acid molecule (e.g., gene, DNA, RNA, or mRNA) encoding same.

A T cell growth factor is involved in T cell development and/or maturation, e.g., involved in proliferation of thymocytes, commitment to T-cell lineage, and/or positive and/or negative selection of immune cells. Exemplary T cell growth factors include but are not limited to BMP4, Bmprla, CD70, Cldn4, DM, Egrl, Egr2, Egr3, EphB2, Flt3, Flt3L, Foxol, FoxP3, Gata2, Gata3, Gfil, Ghrl (ghrelin), GnRH, Icaml, Id3, Ifnarl, IL-2, IL-6, IL-7, IL-12b (IL12 p40 subunit), IL- 15, IL-18, IL-21, K , Klf3, Lefl, leptin, Lif, Nfat5, Nfatcl, Notch 1, Prolactin, Ragl, Rag2, Runxl, Satbl, SCF (K ), Shh (T-cell), Tcfl, TCR (any TCR variant), Tshb (thyrotropin beta chain), Tslp, Vcaml, Wnt3a, Wnt4, Zfp3611, and Zfp3612. Exemplary TCR variants include a TCR comprising an alpha and a beta chain, a TCR comprising a gamma chain and a delta chain, a TCR comprising a CD3 chain, a TCR comprising a zeta chain, a TCR comprising a

complementarity determining region, and a TCR comprising a T cell co-receptor (e.g., CD4 or CD8). In embodiments, a TCR includes a TCR that binds to an epitope presented on an MHCI molecule, an epitope presented on an MHCII molecule, or an epitope presented on an MHCIII molecule.

In some embodiments, the T cell growth factor is selected from at least one of Flt3, Flt3L, Foxol, FoxP3, Gfil, Ghrl (ghrelin), GnRH, Icaml, Id3, Ifnarl, IL-2, IL-6, IL-7, IL-12b (IL12 p40 subunit), IL-15, IL-18, IL-21, KM, Klf3, Lefl, leptin, Lif, and SCF (KM). In some embodiments, the T cell growth factor is selected from at least one of IL-7, IL-21, Delta-like 4 (DLL4), Flt3L, SCF (Kitl), and miR-29a. In some embodiments, one or more the T cell growth factors, e.g., one, two, three, four, or five T cell growth factors (e.g., a second, third, fourth, fifth or sixth T cell growth factor) is administered to the subject. In some embodiments, the T cell growth factor may also include at least one selected from IL-15, IL-2, IL-12, IL-18, and IFNy. Examples of a T cell growth factor are listed in Table 1.

A TEC growth factor is involved in TEC development, differentiation, and/or

proliferation. Exemplary TEC growth factors include but are not limited to Atf3, BMP4 (TEC), Cbx4, Cdh5 (VE cadherin), E2F3, E2F4, Foxnl, Fspl, Fstll, Ml, Kl (Klotho), Ltbr, NFkB l, PPARgamma, Pten, RANK, RANKL, Shh (TEC), Sin/Polr3e, Stat3, Tbata, Tbxl, Tgfbr2, Tnfrsfl la /RANK, Tnfrsf 1 lb, Traf6, Wnt3a (TEC), and Wnt4 (TEC). Examples of a TEC growth factor are listed in Table 1. In some embodiments, the TEC growth factor enhances thymus function by modulating, e.g., increasing, e.g., enhancing, proliferation, survival and/or generation of thymic epithelial cells. In some embodiments, the TEC growth factor is selected from at least one of FGF21, FGF7 (KGF), FGF8, FGF10, IL-22, Wnt4, Bmp4, RANKL, LTa, CL40L, Foxnl, leptin, IGF-1, GH, (Ghrl) ghrelin, GnRH, and NGF. In some embodiments, one or more the TEC growth factors, e.g., one, two, three, four, or five TEC growth factors (e.g., a second, third, fourth, fifth or sixth TEC growth factor) is administered to the subject.

A T cell growth factor receptor binds to (e.g., is a receptor for) a T cell growth factor. Exemplary T cell growth factor receptors include but are not limited to CD 127 (non-soluble IL- 7R), CD27, Crlf2 (TSLP-R), Fzdl, FzdlO, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Ihh, IL-7R alpha, Ptchl, and Ptch2. Examples of a T cell growth factor receptor are listed in Table 1.

A TEC growth factor receptor binds to (e.g., is a receptor for) a TEC growth factor. Exemplary TEC growth factor receptors include but are not limited to Bmpr2, Fgfr2, and bmprla (TEC). Examples of a TEC growth factor receptor are listed in Table 1.

A T cell migration factor is involved in early thymic progenitor (ETP) homing, thymocyte migration within the thymus, and/or T-cell export. ETPs are a population of cells comprising about 0.01% of total thymocytes; they are the more immature T-cell precursors in the thymus and can expand to repopulate thymocyte populations, e.g., after intrathymic transfer. In embodiments, ETPs have the following phenotype: lin ^low CD44 ⁺ cKit ^high CD25 ^" . Without wishing to be bound by theory, it is believed that one or more factors are involved in Treg egress and migration. For example, one factor that may be required for Treg thymic egress is sphingosine-1 phosphate receptor- 1 (S 1P1). It is believed that mature thymocytes follow a S 1P1 gradient, secreted at least in part by the pericytes surrounding the blood vessels at the thymic

corticomedullary junction (CMJ). S 1P1 expression in thymocytes is believed to be regulated by KLF2. In some examples, factors believed to be involved in Treg migration (e.g., into tissues and/or tumors) include CCL20 (e.g., which binds to CCR6 receptor expressed on Tregs), CCR6, CXCLl-3 (e.g., which binds to CXCR2 receptor expressed on Tregs), CXCR2, IL8 (e.g., which binds to CXCR1 receptor expressed on Tregs), CXCR1, CCL2, CCL4, CCL5, CCL22, CXCL8, and CXCLIO. Exemplary T cell migration factors include but are not limited to CC119, Ccl21, CCL25, Ccr7, Ccr9, Cxcll2, Cxcr4, S 1PR1 (S 1P1), Sele (E-selectin, CD62E, ELAM-1, or LECAM2), Sell (CD62L), Selp (P-selectin), CCL20, CCR6, CXCLl-3, CXCR2, IL8, CXCR1, CCL2, CCL4, CCL5, CCL22, CXCL8, and CXCLIO. In some embodiments, one or more the T cell migration factors, e.g., one, two, three, four, or five T cell migration factors (e.g., a second, third, fourth, fifth or sixth T cell migration factor) is administered to the subject. Examples of a T cell migration factor are listed in Table 1.

A T cell activity factor is involved in T cell function. An exemplary T cell activity factor includes E2F2. In some embodiments, one or more the T cell activity factors, e.g., one, two, three, four, or five T cell activity factors (e.g., a second, third, fourth, fifth or sixth T cell activity factor) is administered to the subject. Examples of a T cell activity factor are listed in Table 1. Assessment of T cell function is described in greater detail in the "T cell function section" herein.

A TEC proliferation factor is involved in TEC proliferation, e.g., in an adult thymus. Exemplary TEC proliferation factors include but are not limited to Cyr61, E2F2 (TEC), IGF-1, IL-22, IL-23, and KGF (Fgf7). In some embodiments, one or more the TEC proliferation factors, e.g., one, two, three, four, or five TEC proliferation factors (e.g., a second, third, fourth, fifth or sixth TEC proliferation factor) is administered to the subject. Examples of a TEC proliferation factor are listed in Table 1.

A thymic function factor is involved in one or more thymic functions. Exemplary thymic function factors include but are not limited to AIRE, beta(5t)/Psmbl l, Fezf2, HLA (e.g., any HLA variant/polymorph, e.g., HLA-A, HLA-B, HLA-B27, HLA-B47, HLA-C, HLA-E, HLA-F, HLA-G, p2-microglobulin, HLA-DM (e.g., HLA-DMA1 and/or HLA-DMB 1), HLA-DO (e.g., HLA-DOA1 and/or HLA-DOB 1), HLA-DP (e.g, HLA-DPA1 and/or HLA-DPB 1), HLA-DQ (e.g., HLA-DQA1 and/or HLA-DQB 1), HLA-DQ2, HLA-DQ8, HLA-DR (e.g., HLA-DRA, HLA-DRB l, HLA-DRB3, HLA-DRB4, and/or HLA-DRB5), HLA-DR2, HLA-DR3, HLA-DR4, and/or an HLA encoding a component of the complement system), a Major Histocompatibility Complex (MHC) molecule (e.g., Class I MHC molecule (MHC I), e.g., comprising one or more polypeptides encoded by a HLA- A, HLA-B, HLA-C, HLA-G, and/or HLA-E gene; Class II MHC molecule (MHC II), e.g., comprising one or more polypeptides encoded by a HLA-DP, HLA-DQ, and/or HLA-DR gene; or Class III MHC molecule (MHC III), e.g., comprising a polypeptide involved in inflammation, e.g., a component of the complement system (e.g., C2, C4, or factor B), tumor necrosis factor (TNF)-a, lymphotoxin-a, lymphotoxin-β, or a heat shock protein), and Prssl6. Examples of a thymic function factor are listed in Table 1.

In embodiments, an HLA comprises a HLA Class I allele (e.g., HLA-A, -B, -C, -E, -F, or -G allele), a HLA Class I Pseudogene (e.g., HLA-H, -J, -K, -L, -P, -T, -U, -V, -W, -X, or -Y), HLA Class II allele (e.g., HLA-DRA, -DRB, -DQA1, -DQB 1, -DPA1, -DPB 1, -DPB2, -DMA, - DMB, -DOA, -DOB), or HLA Class II DRB allele (e.g., HLA-DRB l, -DRB2, -DRB 3, -DRB4, - DRB 5, -DRB6, -DRB7, -DRB 8, -DRB 9). In embodiments, an HLA Class I allele comprises an HLA Class I allele described at http://hla.alleles.org/alleles/classl.html (accessed as of June 9, 2016), incorporated herein by reference. In embodiments, an HLA comprises an HLA Class II allele described at http://hla.alleles.org/alleles/class2.html (accessed as of June 9, 2016), incorporated herein by reference.

A thymus homeostasis factor is involved in homeostatic maintenance of the thymus. Exemplary thymus homeostasis factors include but are not limited to l lb-HSD2, AR, ASC, Axin, Fgf21, ILIO, IL2 (stroma), Leptin (TEC), Meisl, and NLRP3. In some embodiments, one or more the thymus homeostasis factors, e.g., one, two, three, four, or five thymus homeostasis factors (e.g., a second, third, fourth, fifth or sixth thymus homeostasis factor) is administered to the subject. Examples of a thymus homeostasis factor are listed in Table 1.

Additional exemplary thymic function factors include Cd44, gpl30, hGH (GH1 and GH2), Nfkb2, Ptma (thymosin alpha- 1), Smad4, Smad6, and Tmpo (thymopoetin), e.g., as described in Table 1.

Exemplary thymic function factors include but are not limited to those described in Table 1. The thymic function factors listed in Table 1 can include the nucleic acid molecule (DNA or RNA, e.g., mRNA) or polypeptide (e.g., translated protein). In embodiments, a thymic function factor comprises a polypeptide/protein or nucleic acid molecule homologous to a thymic function factor described herein, e.g., in Table 1. In embodiments, a thymic function factor comprises an amino acid sequence or nucleic acid sequence that is substantially identity to, e.g., having at least 60% (e.g., at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more, e.g., 100%) identity to the amino acid sequence or nucleic acid sequence of a thymic function factor described herein, e.g., in Table 1.

Table 1 : Amino acid sequences or nucleic acid sequences of thymic function factors

form

TEC expansion and

NM_000618, HSC derived

NM_001111283, progenitor TEC ligand,

IGF-1 NM 001111284 Pos reconstitution proliferation secreted

NR_126505,

NM_001302825,

NM_001302824,

NM_001302823,

NM_001302822, hormone, NM_001302821, T-cell proliferation, secreted, NM_001134941, increased ETPs, encodes NM_016362, restoration of thymic ghrelin- NM_001134946, architecure, t-cell obestatin NM_001134945, increased thymus growth preproprotei

Ghrl Ghrelin NM 001134944 Pos size factor n

NM_172175, t-cell

NM_000585, growth ligand,

IL-15 NR 037840 Pos thymoctye survival factor secreted t-cell

component of IL7R growth

same as IL7R and TSLP receptor factor

IL-7Ralpha (CD 127) Pos complex receptor receptor

proliferation of

TECs, activaiton of TEC ligand,

Cyr61 NM_001554 Pos AKT signalling proliferation secreted involved in

premature ageing,

increased IL7 TEC growth

Kl (Klotho) NM_004795 Pos expression factor enzyme

ligand, secreted and t-cell non-

NM_000899, ETP survival and growth secreted

Kitl NM_003994 Pos proliferation factor forms t-cell

T-cell lineage growth ligand,

D114 NM_019074 Pos commitment factor secreted master regulator of TEC growth transcription

Foxnl NM_003593 Pos TECs factor factor expansion of ETPs t-cell

and immature growth ligand,

Wnt4 NM 030761 Pos thymocyte factor secreted

TEC proliferation,

Wnt4 potential Foxnl TEC growth ligand, (TEC) NM 030761 Pos regulation factor secreted

NM_001270987, mTEC development TEC growth

Ltbr NM 002342 Pos and function factor receptor NM_001277990,

NM_000609,

NM_001178134,

NM_199168, t-cell chemokine,

Cxcll2 NM_001033886 Pos chemokine migration secreted decoy receptor for

RANKL, mTEC TEC growth

Tnfrsf 1 lb NM_002546 Neg homeostasis factor receptor

NM_001278268,

NM_003839,

NM_001270950,

Tnfrsfl la/ NM_001270949, increase in mTEC TEC growth

RANK NM_001270951 Pos numbers factor receptor positive selection t-cell

(TCR costimulatory growth

Cldn4 NM_001305 Pos activity) factor receptor survival and

proliferation of early

NM_001127216, thymocyte, control t-cell

NM_001127215, of Treg growth transcription

Gfil NM_005263 Pos differentiation factor factor t-cell

deletion of growth

Ifnarl NM_000629 Pos autoreactive T cells factor receptor

E2F2 TEC transcription (TEC) NM_004091 Pos TEC proliferation proliferation factor neg regulator of

peripheral T-cell

proliferation and t-cell transcription

E2F2 NM_004091 Neg/Pos autoimmunity activity factor

TEC growth transcription

E2F4 NM_001950 Neg repressor of Foxnl factor factor

NM_001949. activator of Foxnl TEC growth transcription

E2F3 NM_001243076 Pos expression factor factor

NM_213662,

NM_003150, mTEC development TEC growth transcription

Stat3 NM_139276 Pos and homeostasis factor factor

VE TEC growth

Cdh5 cadherin NM_001795 Pos factor receptor

TEC growth

Bmprla BMP4/2 receptor, factor recept

(TEC) NM_004329 Pos thymus size or receptor thymocyte

homeostasis (neg t-cell

regulation of growth

Bmprla NM_004329 Neg proliferation) factor receptor possible role in TEC TEC growth transcription l NM_002202 Pos differentiation factor factor

TEC growth

factor

Bmpr2 NM_001204 Pos BMP receptor receptor receptor block in HSC

NM_001145662, differentiation t-cell

NM_001145661, towards lymphoid growth transcription

Gata2 NM_032638 Neg lineage factor factor

Bmp4 antagonists,

proinflamatory TEC growth

Fstll NM_007085 molecule factor secreted progression through

beta selection, t-cell

positive selection, growth transcription

Egrl NM_001964 Pos thymocyte survival factor factor

NM_001199881, t-cell

NM_001199880, overlapping growth transcription

Egr3 NM_004430 Pos functions with Egrl factor factor

NM_001321037,

NM_001136178, DP to SP and NKT

NM_000399, cell, some t-cell

NM_001136179, overlapping function growth transcription

Egr2 NM_001136177 Pos with Egrl/3 factor factor

NM_001030287,

NM_001674,

NM_001040619,

NM_001206484,

NM_001206488, null show decreased TEC growth transcription

AtG NM_001206486 Neg involution phenotype factor factor t-cell

growth transcription

KIG NM_016531 Pos positive selection factor factor

T-cell lineage t-cell

NM_001002295, commitment and growth transcription

Gata3 NM_002051 Pos development factor factor

NM_001195470,

NM_001131010,

NM_002971,

NM_001322876,

NM_001322875,

NM_001322874, positive selection,

NM_001322873, Treg numbers and t-cell

NM_001322872, function, negative growth matrix

Satbl NM_001322871 Pos selection factor protein

NM_016269,

NM_001166119, NKT development, t-cell

NM_001130714, TCRalpha growth transcription

Lefl NM_001130713 Pos expression factor factor t-cell

NM_001306179, growth transcription

Tcfl NM_000545 Pos T-cell specification factor factor same as TEC growth

RANK Tnfrsf 11 a Pos mTEC development factor receptor mTEC development ligand,

NM_033012, through RANK TEC growth membrane

RANKL NM_003701 Pos signaling factor bound NM_138711, overexpression nuclear

NM_015869, copies thymic receptor,

PPARgam NM_138712, involution, involved TEC growth transcription ma NM_005037 Neg in adipogenesis factor factor

TEC transcription

AIRE NM_000383 Pos TRA expression function factor

TRA expression

(separate from TEC transcription

Fezf2 NM_018008 Pos AIRE) function factor

HLA (e.g.,

any

variant/poly TEC

morph) function receptor

MHC I/II

and all

known TEC

alleles function receptor ligand, anderson, t-cell membrane proliferation of DN growth bound or

SCF Kitl same as Kitl Pos thymocytes factor secreted

NM_001201359, anderson, ETP t-cell

CCL25 NM_005624 Pos attractiong migration chemokine t-cell

NM_001114377, growth transcription

FoxP3 NM_014009 Pos Treg development factor factor t-cell

TCR (any growth

variant) factor receptor

NM_001278675,

NM_172390,

NM_001278673,

NM_172388,

NM_001278672,

NM_001278670,

NM_172389,

NM_172387, t-cell

NM_006162, beta selection, DN to growth transcription

Nfatcl NM_001278669 Pos SP factor factor

NM_001113178,

NM_006599,

NM_138714,

NM_138713, thymocyte survival t-cell

NM_173214, and transition from growth transcription

Nfat5 NM_173215 Pos beta selection to DP factor factor

NM_001165412, mTEC

NM_003998, differentiation and TEC growth transcription

Nfkbl NM_001319226 Pos function factor factor

NM_002502,

NM_001261403,

NM_001077494, required for normal

NM_001288724, negative selection of transcription

Nfkb2 NM_001322934 Pos CD8+ T-cell factor t-cell

beta selection, growth transcription

Id3 NM_002167 Pos positive selection factor factor

Treg function, iTreg

induction and

function,

homeostasis and life

span of naive T cells, t-cell

thymocyte growth transcription

Foxol NM_002015 Pos survival/proliferation factor factor beta(5t)/Ps TEC enzyme mbl l NM_001099780 Pos antigen presentation function (proteinase)

NM_001079821,

NM_001243133,

NM_001127461,

NM_001127462,

NM_004895, reduced thymic thymus

NLRP3 NM_183395 Neg involution in null homeostasis receptor

NM_145182, thymus

ASC NM_013258 Neg similar to Nlrp3 homeostasis cytoplasmic involved in

adipogenesis of

thymus,

overexpression

NM_181050, promotes thymocyte thymus

Axin NM_003502 Neg apoptosis homeostasis cytoplasmic thymocyte

proliferation,

protects against t-cell

NM_001083111, thymic involution in growth hormone,

GnRH NM_000825 Pos case of insult factor secreted protection against

stress induced TEC

damage (particularly

Leptin mTEC), increased thymus hormone,

(TEC) NM_000230 Pos IL7 expression homeostasis secreted

DN proliferation,

protection of DP

against endotoximia

induced apoptosis, t-cell

naive T cell growth hormone,

Leptin NM_000230 Pos proliferation factor secreted

NM_000515,

NM_022560,

NM_022559,

NM_022558, restoration of HSCs

NM_022557, in bone marrow,

hGH (GH1 NM_022556, thymus recovery hormone, and GH2) NM_002059 Pos after insult secreted thymocyte

development and

proliferation, t-cell

NM_021803, accelerated thymic growth ligand,

IL21 NM_001207006 Pos recovery post GC factor secreted thymocyte survival

and proliferation; t-cell

NM_001163558, counteracts growth hormone,

Prolactin NM_000948 pos glucocorticoid factor secreted

Thyrotrop activates calcium t-cell

in (beta NM_001277991, signaling and growth hormone,

Tshb chain) NM_000549 pos cyclicAMP signaling factor secreted downregulation of

D114 (and IL7,

Ccl25), thymocyte

survival (mediated

through TECs), TEC

NM_000044, proliferation (non thymus

AR NM_001011645 Neg cell autonomous) homeostasis receptor

NM_001244701, role in beta selection t-cell

NM_001244698, and regulation of growth transcription

Zfp3611 NM_004926 Pos Notch 1 mRNA factor factor role in beta selection t-cell

and regulation of growth transcription

Zfp3612 NM_006887 Pos Notch 1 mRNA factor factor required for T-cell t-cell

lineage commitment growth

Notch 1 NM_017617 Pos and differentiation factor receptor

NM_001318243,

NM_001318243, ligand, NM_152710, TEC proliferation TEC growth membrane

Tbata NM_001318241 Neg and function factor bound reduction in thymic ligand, glucocorticoid thymus membrane l lb-HSD2 NM_000196 Pos expression homeostasis bound

NM_003276,

NM_001307975,

thymopeot NM_001032284,

Tmpo in NM_001032283 induction of CD90 enzyme

T-cell proliferation

and funciton (in

periphery and

tissues), HLA-DR

thymosin NM_001099285, expression in human hormone,

Ptma alpha- 1 NM_002823 Pos APCs secreted thymus cellularity,

NM_000314, architecture, enzyme NM_001304717, decreased thymocyte TEC growth (phosphatas

Pten NM_001304718 pos numbers, factor e)

NM_000193,

NR_132319, TEC development, (- NR_132318, ve) modulation of TEC growth ligand,

Shh (TEC) NM_001310462 pos MHCII expression factor secreted expansion and

differentiation of

NM_000193, DN; neg regulation

NR_132319, of pre-TCR t-cell

Shh (T- NR_132318, dependent DN growth ligand, cell) NM_001310462 mixed differentiation to DP factor secreted TEC enzyme

Prssl6 NM_005865 Pos antigen presentation function (protease) mTEC

differentiation,

NM_004620, thymic DC TEC growth

Traf6 NM_145803 pos differentiation factor enzyme dendritic cell

activity, T cell

development,

NM_138551, thymocyte t-cell

NM_033035, progenitor and T-cell growth ligand,

Tslp NR_045089 Pos proliferation factor secreted thymocyte

proliferation and

differentiation,

deletion of t-cell

autoreactive ClassII growth ligand,

IL2 NM_000586 pos restricted thymocytes factor secreted possible role in

maintenance of

thymic

IL2 monocyte/macropha thymus ligand,

(storma) NM_000586 pos ges and cTEC homeostasis secreted differentiation of

CDl lb+ DCs from

DN thymocytes,

functions together

with IL2 and IL12 - t-cell

NM_001562, phenotypic change in growth ligand,

IL18 NM_001243211 pos thymocyte factor secreted anti-ageing effect on thymus ligand,

¾f21 NM_019113 pos thymus homeostasis secreted

NM_019554, proliferation and TEC growth

Fspl NM_002961 pos maturation of TECs factor cytoplasmic

NM_001258036,

NM_001258035,

NM_001258034,

NM_001258033,

NM_018119, mTEC development TEC growth

Sin/Polr3e NR_047581 pos and maintenance factor enzyme

NM_080647, negative regulation

NM_080646, of Foxnl TEC growth transcription

Tbxl NM_005992 neg transcription factor factor deletion leads to transcription

Smad4 NM_005359 Pos thymus hypoplasia factor mTEC proliferation

and compartment

NM_003242, size, Treg TEC growth

Tgfbr2 NM_001024847 neg development factor receptor required for

thymocyte

proliferation (at

physiological levels), t-cell

NM_000600, causes loss of DP at growth ligand,

IL6 NM_001318095 Pos/Neg higher levels factor secreted required for

thymocyte

proliferation (at

physiological levels), t-cell

NM_002309, causes loss of DP at growth ligand,

Lif NM_001257135 neg higher levels factor secreted

NM_001190981,

NM_175767,

NM_002184, mediator of IL6,

gpl30 NR_120480 neg LPS, Lif activities receptor protection against

stress induced TEC thymus ligand,

IL10 NM_000572 Pos damage homeostasis secreted

NM_001001890, t-cell

NM_001754, IL7R expression, growth transcription

Runxl NM_001122607 Pos thymocyte survival factor factor

T-cell progenitor and

NM_001309193, thymocyte migration,

NM_001309192, differentiation and t-cell

NM_017449, interaction with growth

EphB2 NM_004442 Pos TECs factor receptor

TEC proliferation TEC growth

Cbx4 NM_003655 Pos and function factor enzyme t-cell

Ccl21 NM_002989 Pos chemokine migration chemokine positive selection

(costimulation and

thymocyte survival), t-cell ligand, thymocyte numbers growth membrane

Icaml NM_000201 Pos and proportion factor bound

NM_001199834, t-cell ligand,

NM_001078, growth membrane

Vcaml NM_080682 Pos positive selection factor bound neg regulator of

NM_005585, BMP and TGFbeta transcription

Smad6 NR_027654, signaling factor expressed in

progenitor-like TECs

in adult thymus,

important for thymus transcription

Meisl NM_002398 Pos homeostasis homeostasis factor temporarily required

during thymocyte

development for t-cell

TCR gene growth

Ragl NM_000448 Pos rearrangement factor enzyme temporarily required

during thymocyte

NM_001243786, development for t-cell

NM_001243785, TCR gene growth

Rag2 NM_000536 Pos rearrangement factor enzyme recruitment and entry

of hematopoietic t-cell

Selp P-selectin NM_003005 Pos progenitors migration receptor E-selectin,

CD62E, recruitment and entry

ELAM-1, of hematopoietic t-cell

Sele LECAM2 NM_000450 Pos progenitors migration receptor export of naive T- cells, naive T-cells

trafficking to

NM_000655, secondary lymphoid t-cell

Sell CD62L NR_029467 Pos organ migration receptor recruitment and entry

of hematopoietic

NM_031200, progenitors; DN-DP

NM_006641, transition; CD4 t-cell

Ccr9 NM_001256369 Pos differentiation migration receptor

NM_001001389,

NM_000610,

NM_001202556,

NM_001202555,

NM_001001392,

NM_001001391,

NM_001001390,

Cd44 NM_001202557 Pos receptor t-cell

NM_004119, growth

Flt3 NR_130706 Pos receptor for Flt3L factor receptor receptor for Cxcll2,

seeding of

NM_003467, progenitors to t-cell

Cxcr4 NM_001008540\ Pos thymus migration receptor migration to outer

cortex and from t-cell ligand,

Ccll9 NM_006274 Pos cortex to medulla migration secreted

NM_001301718,

NM_001301717,

NM_001301716,

NM_001301714, receptor for Cell 9 t-cell

Ccr7 NM_001838 Pos and Ccl21 migration receptor

NM_001320730, t-cell

S1PR1 S 1P1 NM_001400 Pos ETP export migration receptor

NM_000141,

NM_023029,

NM_001144918,

NM_001144916,

NM_022970,

NM_001144917,

NM_001144914,

NM_001144915,

NM_001144913,

NM_001144919, TEC growth

NM_001320658, Fgf7 and FgflO factor

Fgfr2 NM_001320654 Pos receptor receptor receptor early stages of T-cell t-cell ligand,

Wnt3a NM_033131 Pos development growth secreted factor

Wnt3a TEC growth ligand,

(TEC) NM_033131 Pos TEC development factor secreted t-cell

growth

factor

Fzdl NM_003505 Pos Wnt receptor receptor receptor t-cell

growth

factor

Fzd2 NM_001466 Pos Wnt receptor receptor receptor t-cell

growth

NM_017412, factor

Fzd3 NM_145866 Pos Wnt receptor receptor receptor t-cell

growth

factor

Fzd4 NM_012193 Pos Wnt receptor receptor receptor t-cell

growth

factor

Fzd5 NM_003468 Pos Wnt receptor receptor receptor

NM_001317796,

NR_133921, t-cell

NM_001164616, growth

NM_001164661 factor

Fzd6 5, NM_003506 Pos Wnt receptor receptor receptor t-cell

growth

factor

Fzd7 NM_003507 Pos Wnt receptor receptor receptor t-cell

growth

factor

Fzd8 NM_031866 Pos Wnt receptor receptor receptor t-cell

growth

factor

Fzd9 NM_003508 Pos Wnt receptor receptor receptor t-cell

growth

factor

FzdlO NM_007197 Pos Wnt receptor receptor receptor promoting

thymopoiesis before t-cell

pre-TCR signalling growth

but negatively factor ligand,

Ihh NM_002181 Pos/Neg regulating later steps receptor secreted NM_000264,

NM_001083602,

NM_001083604,

NM_001083605, t-cell

NM_001083607, receptor for growth

NM_001083606, hedgehog signalling factor

Ptchl NM_001083603 Pos ligands receptor receptor t-cell

receptor for growth

NM_001166292, hedgehog signalling factor

Ptch2 NM_003738 Pos ligands receptor receptor thymocyte t-cell

development and growth

differentiation, Treg factor

CD27 NM_001242 Pos survival receptor receptor thymocyte

development and t-cell ligand, differentiation, Treg growth membrane

CD70 NM_001252 Pos survival factor bound

dendritic cell

activity, T cell

development, t-cell

NM_001012288, thymocyte growth

NM_022148, progenitor and T-cell factor

Crlf2 TSLP-R NR_110830 Pos proliferation receptor receptor

Assays to measure expression (level) of thymic function factors

The expression or level of a thymic function factor can refer to the level of mRNA or protein.

The level, e.g., mRNA level, of a thymic function factor can be determined by a number of assays, e.g., Northern blots, PCR, RT-PCR, or fluorescence in situ hybridization. The level, e.g., protein level, of a thymic function factor can be determined by a number of assays, e.g., Western blot, mass spectrometry (MS), liquid chromatography (LC) (e.g., LC-MS), ELISA, or histological methods (e.g., immunohistochemistry).

In embodiments, the level of a thymic function factor is determined in a sample, e.g., a sample from a subject. The sample can comprise a cell or tissue sample. In embodiments, the sample comprises a blood, serum, urine, or tissue (e.g., biopsy) sample.

Assays to measure activity of thymic function factors

The activity of a thymic function factor can include one or more of the effects listed in Table 1, corresponding to each thymic function factor. In embodiments, the activity of an enzyme thymic function factor is determined using an assay for catalytic activity of the enzyme. In embodiments, the activity of a non-enzyme thymic function factor can be determined by assessing a downstream effect of the factor. For example, if the thymic function factor is in a signaling pathway that leads to the increase or decrease of a particular molecule, e.g., polypeptide, nucleic acid, or metabolite, the activity of the factor can be measured by measuring the level of the particular molecule.

Thymic function modulators

Functions of thymic function modulators

Thymic function modulators are used in the methods (e.g., therapeutic methods or ex- vivo culture methods) described herein. In embodiments, a thymic function modulator modulates, e.g., increases or decreases, a thymic function (e.g., in vivo). For example, a thymic function modulator improves a thymus function, e.g., a thymus function described herein, e.g., one or more of: increases the number of T cells in a subject; increases the number and/or relative ratio of a T cell subset (e.g., Tregs, CD4 positive T cells, and/or CD8 positive T cells) in a subject, improves a T cell function in a subject; and/or increases T cell migration in a subject. In embodiments, a thymic function modulator modulates the function of dendritic cells associated with thymus tissue (e.g., modulates antigen presentation of a dendritic cell, modulates the inflammatory properties of a dendritic cell, and/or modulates the cytokines secreted by a dendritic cell). In embodiments, a thymic function modulator modulates, e.g., increases or decreases, the level and/or activity of a thymic function factor, e.g., a thymic function factor described herein, e.g., in Table 1. In embodiments, a thymic function modulator results in one or more effects listed in Table 1.

In embodiments, a thymic function modulator modulates one or more thymic function factors, e.g., one or more thymic function factors described herein, e.g., in Table 1. In embodiments, a thymic function modulator modulates the level and/or activity of a thymic function factor selected from a T cell growth factor, T cell growth factor receptor, T cell migration factor, T cell activity factor, TEC proliferation factor, TEC growth factor, TEC growth factor receptor, TEC function factor, or thymus homeostasis factor, e.g., as described in Table 1. In embodiments, a thymic function modulator modulates the level and/or activity of a thymic function factor selected from a chemokine, cytoplasmic protein, enzyme (e.g., phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), matrix protein, receptor (e.g., cell surface receptor or nuclear receptor), or transcription factor, e.g., as described in Table 1.

In some embodiments, one or more thymic function modulators, e.g., one, two, three, four, or five thymic function modulators (e.g., a second, third, fourth, fifth or sixth thymic function modulator) is administered to a subject. In one embodiment, administration of one or more of the thymic function modulators increases or decreases expression of or activity level of a thymic function factor, e.g., a T cell growth factor, a T cell growth factor receptor, a T cell proliferation factor, a T cell migration factor, a T cell activity factor, a TEC proliferation factor, a TEC growth factor, a TEC growth factor receptor, a TEC function factor, or a thymus homeostasis factor.

In embodiments, a thymic function modulator increases the level and/or activity of a thymic function factor (e.g., a positive regulator of thymic function, e.g., described in Table 1) by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, increased as compared to a reference expression or activity level of the one or more thymic function factors. In

embodiments, a thymic function modulator decreases the level and/or activity of a thymic function factor (e.g., a negative regulator of thymic function, e.g., described in Table 1) by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, decreased as compared to a reference expression or activity level of the one or more thymic function factors. In one embodiment, the reference expression or activity level is the expression or activity level of the one or more thymic function factors prior to administration of the thymic function modulator.

In embodiments, a thymic function modulator can be itself a thymic function factor described herein, e.g., in Table 1.

In embodiments, a thymic function modulator comprises a polypeptide/protein or nucleic acid molecule homologous to a thymic function modulator described herein, e.g., in Table 1. In embodiments, a thymic function modulator comprises an amino acid sequence or nucleic acid sequence that is substantially identity to, e.g., having at least 60% (e.g., at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more, e.g., 100%) identity to the amino acid sequence or nucleic acid sequence of a thymic function modulator described herein, e.g., in Table 1. Structural characteristics of thymic function modulators

A thymic function modulator can comprise a number of different modalities. In embodiments, a thymic function modulator comprises a nucleic acid molecule (e.g., DNA molecule or RNA molecule, e.g., mRNA, guide RNA (gRNA), or inhibitory RNA molecule (e.g., siRNA, shRNA, or miRNA), or a hybrid DNA-RNA molecule), a small molecule, a peptide, or a polypeptide (e.g., an antibody molecule, e.g., an antibody or antigen binding fragment thereof). In embodiments, the nucleic acid molecule, peptide, polypeptide, or antibody molecule can be modified. For example, the modification can be a chemical modification, e.g., conjugation to a marker, e.g., fluorescent marker or a radioactive marker. In other examples, the modification can include conjugation to a molecule that enhances the stability of the thymic function modulator (e.g., stability in vivo and/or in cell/tissue culture).

Polypeptides

In embodiments, a thymic function modulator described herein comprises a thymic function factor polypeptide or functional fragment or derivative thereof. In embodiments, a thymic function modulator is a polypeptide listed in Table 1, wherein the primary sequence of the thymic function modulator is provided by reference to accession number. Methods of making a therapeutic polypeptide are routine in the art. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology:

Fundamentals and Applications, Springer (2013).

Preferred methods for producing a thymic function modulator polypeptide involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of appropriate promoters. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

Various mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include CHO cells, COS cells, HeLA and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologies

Manufacturing (Advances in Biochemical Engineering/Biotechnology), Springer (2014).

Purification of protein therapeutics is described in Franks, Protein Biotechnology:

Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).

Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

Antibodies

In embodiments, the thymic function modulator is an antibody or antigen binding fragment thereof. The making and use of therapeutic antibodies against a target antigen (e.g., a thymic function factor described herein) is known in the art. See, for example, the refernces cited hereinabove, as well as Zhiqiang An (Editor), Therapeutic Monoclonal Antibodies: From Bench to Clinic. 1st Edition. Wiley (2009) and Greenfield (Ed.), Antibodies: A Laboratory Manual. 2d ed. Spring Harbor Laboratory Press (2013) for methods of making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques.

Modified mRNA

In embodiments, the thymic function modulator comprises an mRNA molecule, e.g., a modified mRNA molecule encoding a thymic function factor. In embodiments, the mRNA molecule increases the level (e.g., protein and/or mRNA level) and/or activity of a thymic function factor, e.g., a positive regulator of thymus function. In embodiments, the mRNA molecule encodes a thymic function factor or a fragment thereof. For example, the mRNA molecule encodes a polypeptide having at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to the amino acid sequence of a thymic function factor listed in Table 1. In other examples, the mRNA molecule has at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to the nucleic acid sequence of a thymic function factor listed in Table 1. In embodiments, the mRNA molecule encodes an amino acid sequence differing by no more than 30 (e.g., no more than 30, 20, 10, 5, 4, 3, 2, or 1) amino acids to the amino acid sequence of a thymic function factor listed in Table 1. In some embodiments, the mRNA molecule comprises a fragment of a thymic function factor listed in Table 1. For example, the fragment comprises 10-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, 140- 160, 160-180, 180-200, 200-250, 250-300, 300-400, 400-500, 500-600, or more amino acids in length. In embodiments, the fragment is a functional fragment, e.g., having at least 20%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater, of an activity of a full length thymic function factor listed in Table 1. In embodiments, the mRNA molecule increases the level and/or activity of or encodes a thymic function factor (or fragment thereof) that is a positive regulator of thymus function.

The mRNA molecule can be modified, e.g., chemically. The mRNA molecule can be chemically synthesized or transcribed in vitro. The mRNA molecule can be disposed on a plasmid, e.g., a viral vector, bacterial vector, or eukaryotic expression vector. In some examples, the mRNA molecule can be delivered to cells by transfection, electroporation, or transduction (e.g., adenoviral or lentiviral transduction).

In some embodiments, the modified RNA encoding a thymic function factor of interest described herein has modified nucleosides or nucleotides. Such modifications are known and are described, e.g., in WO 2012/019168. Additional modifications are described, e.g., in

WO2015038892; WO2015038892; WO2015089511; WO2015196130; WO2015196118 and WO2015196128A2.

In some embodiments, the modified RNA encoding a polypeptide of interest described herein has one or more terminal modification, e.g., a 5'Cap structure and/or a poly-A tail (e.g., of between 100-200 nucleotides in length). The 5' cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine. In some cases, the modified RNAs also contain a 5 ' UTR comprising at least one Kozak sequence, and a 3 ' UTR. Such modifications are known and are described, e.g., in WO2012135805 and WO2013052523. Additional terminal modifications are described, e.g., in WO2014164253 and WO2016011306. WO2012045075 and WO2014093924

Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNA) which may include at least one chemical modification are described in WO2014028429.

In some embodiments, a modified mRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 '-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5'-/3'- linkage may be intramolecular or intermolecular. Such modifications are described, e.g., in WO2013151736.

Methods of making and purifying modified RNAs are known and disclosed in the art. For example modified RNAs are made using only in vitro transcription (IVT) enzymatic synthesis. Methods of making IVT polynucleotides are known in the art and are described in WO2013151666, WO2013151668, WO2013151663, WO2013151669, WO2013151670, WO2013151664, WO2013151665, WO2013151671, WO2013151672, WO2013151667 and WO2013151736.S Methods of purification include purifying an RNA transcript comprising a polyA tail by contacting the sample with a surface linked to a plurality of thymidines or derivatives thereof and/or a plurality of uracils or derivatives thereof (polyT/U) under conditions such that the RNA transcript binds to the surface and eluting the purified RNA transcript from the surface (WO2014152031); using ion (e.g., anion) exchange chromatography that allows for separation of longer RNAs up to 10,000 nucleotides in length via a scalable method

(WO2014144767); and subjecting a modified RMNA sample to DNAse treatment

(WO2014152030).

Formulations of modified RNAs are known and are described, e.g., in WO2013090648. For example, the formulation may be, but is not limited to, nanoparticles, poly(lactic-co-glycolic acid)(PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gel, fibrin hydrogel, fibrin glue, fibrin sealant, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNPs) and combinations thereof.

Modified RNAs encoding polypeptides in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are disclosed in for example, Table 6 of International Publication Nos. WO2013151666, WO2013151668, WO2013151663, WO2013151669, WO2013151670, WO2013151664, WO2013151665, WO2013151736; Tables 6 and 7 International Publication No. WO2013151672; Tables 6, 178 and 179 of International Publication No. WO2013151671; Tables 6, 185 and 186 of International Publication No

WO2013151667. Any of the foregoing may be synthesized as an IVT polynucleotide, chimeric polynucleotide or a circular polynucleotide, and each may comprise one or more modified nucleotides or terminal modifications.

Inhibitory RNA

In embodiments, the thymic function modulator comprises an inhibitory RNA molecule, e.g., that acts via the RNA interference (RNAi) pathway. For example, an inhibitory RNA molecule includes a short interfering RNA, short hairpin RNA, and/or a microRNA. A siRNA is a double- stranded RNA molecule that typically has a length of about 19-25 base pairs. A shRNA is a RNA molecule comprising a hairpin turn that decreases expression of target genes via RNAi. shRNAs can be delivered to cells in the form of plasmids, e.g., viral or bacterial vectors, e.g., by transfection, electroporation, or transduction). A microRNA is a non-coding RNA molecule that typically has a length of about 22 nucleotides. MiRNAs bind to target sites on mRNA molecules and silence the mRNA, e.g., by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA. In embodiments, the inhibitory RNA molecule decreases the level and/or activity of a negative regulator of thymic function. In other embodiments, the inhibitor RNA molecule decreases the level and/or activity of an inhibitor of a positive regulator of thymic function.

An inhibitory RNA molecule can be modified, e.g., to contain modified nucleotides, e.g., 2'-fluoro, 2'-o-methyl, 2'-deoxy, unlocked nucleic acid, 2' -hydroxy, phosphorothioate, 2'- thiouridine, 4'-thiouridine, 2'-deoxyuridine. Without being bound by theory, it is believed that certain modification can increase nuclease resistance and/or serum stability, or decrease immunogenicity .

In embodiments, the inhibitory RNA molecule decreases the level and/or activity of a thymic function factor. In embodiments, the inhibitory RNA molecule inhibits expression of a thymic function factor (e.g., inhibits translation to protein). In other embodiments, the inhibitor RNA molecule increases degradation of a thymic function factor and/or decreases the stability (i.e., half-life) of a thymic function factor. The inhibitory RNA molecule can be chemically synthesized or transcribed in vitro.

The making and use of inhibitory therapeutic agents based on non-coding RNA such as ribozymes, RNAse P, siRNAs, and miRNAs are also known in the art, for example, as described in Sioud, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). Humana Press (2010).

Gene Editing

In embodiments, the thymic function modulator comprises a component of a gene editing system. For example, the thymic function modulator introduces an alteration (e.g., insertion, deletion (e.g., knockout), translocation, inversion, single point mutation, or other mutation) in a thymic function factor, e.g., described in Table 1. Exemplary gene editing systems include the clustered regulatory interspaced short palindromic repeat (CRISPR) system, zinc finger nucleases (ZFNs), Transcription Activator-Like Effector-based Nucleases (TALEN). ZFNs, TALENs, and CRISPR-based methods are described, e.g., in Gaj et al. Trends Biotechnol.

31.7(2013):397-405.

In embodiments, the thymic function modulator comprises a guide RNA (gRNA) for use in a clustered regulatory interspaced short palindromic repeat (CRISPR) system for gene editing. In embodiments, the thymic function modulator comprises a zinc finger nuclease (ZFN), or a mRNA encoding a ZFN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) encoding a thymic function factor, e.g., a thymic function factor described in Table 1. In embodiments, the thymic function modulator comprises a TALEN, or an mRNA encoding a TALEN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) encoding a thymic function factor, e.g., a thymic function factor described in Table 1.

For example, the gRNA can be used in a CRISPR system to engineer an alteration in a gene (e.g., a thymic function factor, e.g., described in Table 1). In other examples, the ZFN and/or TALEN can be used to engineer an alteration in a gene (e.g., a thymic function factor, e.g., described in Table 1). Exemplary alterations include insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations, or other mutations. The alteration can be introduced in the gene in a cell, e.g., in vitro, ex vivo, or in vivo. In some examples, the alteration increases the level and/or activity of a thymic function factor, e.g., a positive regulator of thymic function, e.g., described in Table 1. In other examples, the alteration decreases the level and/or activity of (e.g., knocks down or knocks out) a thymic function factor, e.g., a negative regulator of thymic function, e.g., described in Table 1. In yet another example, the alteration corrects a defect (e.g., a mutation causing a defect), in a thymic function factor, e.g., described in Table 1.

CRISPR refers to a set of (or system comprising a set of) clustered regularly interspaced short palindromic repeats. A CRISPR system refers to a system derived from CRISPR and Cas (a CRISPR-associated protein) or other nuclease that can be used to silence or mutate a gene described herein (e.g., a thymic function factor or thymic function modulator, e.g., described herein).

The CRISPR system is a naturally occurring system found in bacterial and archeal genomes. The CRISPR locus is made up of alternating repeat and spacer sequences. In naturally-occurring CRISPR systems, the spacers are typically sequences that are foreign to the bacterium (e.g., plasmid or phage sequences).

The CRISPR system has been modified for use in gene editing (e.g., changing, silencing, and/or enhancing certain genes) in eukaryotes. See, e.g., Wiedenheft et al. (2012) Nature 482: 331-8. For example, such modification of the system includes introducing into a eukaryotic cell a plasmid containing a specifically-designed CRISPR and one or more appropriate Cas proteins. In embodiments, in a CRISPR system for use described herein, e.g., in accordance with one or more methods described herein, the spacers of the CRISPR are derived from a target gene sequence, e.g., a sequence from a thymic function factor or thymic function modulator, e.g., described herein.

The CRISPR locus is transcribed into RNA and processed by Cas proteins into small RNAs that comprise a repeat sequence flanked by a spacer. The RNAs serve as guides to direct Cas proteins to silence specific DNA/RNA sequences, depending on the spacer sequence. See, e.g., Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7; Pennisi (2013) Science 341: 833-836. In some examples, the CRISPR system includes the Cas9 protein, a nuclease that cuts on both strands of the DNA. See, e.g., i.d.

In certain embodiments, the CRISPR system is used to edit (e.g., to add or delete a base pair) a target gene, e.g., a thymic function factor or thymic function modulator, e.g., described herein. In other embodiments, the CRISPR system is used to introduce a premature stop codon, e.g., thereby decreasing the expression of a target gene, e.g., a thymic function factor or thymic function modulator, e.g., described herein. In yet other embodiments, the CRISPR system is used to turn off a target gene (e.g., a thymic function factor or thymic function modulator, e.g., described herein) in a reversible manner, e.g., similarly to RNA interference. In embodiments, the CRISPR system is used to direct Cas to a promoter of a thymic function factor or thymic function modulator, e.g., described herein, for example, thereby blocking an RNA polymerase sterically.

In embodiments, a CRISPR system can be generated to edit a thymic function factor or thymic function modulator (e.g., described herein), using technology described in, e.g., U.S. Publication No.20140068797, Cong (2013) Science 339: 819-823, Tsai (2014) Nature

Biotechnol., 32:6 569-576, U.S. Patent No.: 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359.

In some embodiments, the CRISPR interference (CRISPRi) technique can be used for transcriptional repression of specific genes, e.g., a gene encoding a thymic function modulator or thymic function factor described herein. In CRISPRi, an engineered Cas9 protein (e.g., nuclease-null dCas9, or dCas9 fusion protein, e.g., dCas9-KRAB or dCas9-SID4X fusion) can pair with a sequence specific guide RNA (sgRNA). The Cas9-gRNA complex can block RNA polymerase, thereby interfering with transcription elongation. The complex can also block transcription initiation by interfering with transcription factor binding. The CRISPRi method is specific with minimal off-target effects and is multiplexable, e.g., can simultaneously repress more than one gene (e.g., using multiple gRNAs). Also, the CRISPRi method permits reversible gene repression.

In embodiments, CRISPR-mediated gene activation (CRISPRa) can be used for transcriptional activation, e.g., of one or more genes described herein, e.g., genes encoding a thymic function modulator or thymic function factor described herein. In the CRISPRa technique, dCas9 fusion proteins recruit transcriptional activators. For example, dCas9 can be used to polypeptides (e.g., activation domains) such as VP64 or the p65 activation domain (p65D) and used with sgRNA (e.g., a single sgRNA or multiple sgRNAs), to activate a gene or genes, e.g., endogenous gene(s). Multiple activators can be recruited by using multiple sgRNAs - this can increase activation efficiency. A variety of activation domains and single or multiple activation domains can be used. In addition to engineering dCas9 to recruit activators, sgRNAs can also be engineered to recruit activators. For example, RNA aptamers can be incorporated into a sgRNA to recruit proteins (e.g., activation domains) such as VP64. In some examples, the synergistic activation mediator (SAM) system can be used for transcriptional activation. In SAM, MS2 aptamers are added to the sgRNA. MS2 recruits the MS2 coat protein (MCP) fused to p65AD and heat shock factor 1 (HSF1).

The CRISPRi and CRISPRa techniques are described in greater detail, e.g., in

Dominguez et al. Nat. Rev. Mol. Cell Biol. 17(2016):5-15, incorporated herein by reference. In addition, dCas9-mediated epigenetic modifications and simultaneous activation and repression using CRISPR systems, as described in Dominguez et al., can be used to modulate a thymic function modulator or thymic function factor described herein.

Epigenetic modifying agents

In embodiments, the thymic function modulator comprises an epigenetic modifying agent. An epigenetic modifying agent is an agent that modifies chromatin structure, e.g., directly and/or indirectly, and in some cases affects gene expression and function. For example, an epigenetic modifying agent modifies an epigenetic status of a cell (e.g., phenotype or gene expression in the cell that is caused by mechanisms other than alterations in DNA sequence). For example, an epigenetic status of a cell includes DNA methylation, histone modification(s) and RNA-associated silencing. Without wishing to be bound by theory, it is believed that epigenetic alterations in the genome can contribute to certain diseases, e.g., cancer initiation and progression. For example, abnormal DNA methylation and histone hypoacetylation in promoter regions of important genes can lead to gene silencing.

In embodiments, an epigenetic modifying agent involves sequence-specific targeting of an epigenetic enzyme (e.g., an enzyme that generates or removes epigenetic marks, e.g., acetylation and/or methylation, e.g., histone modifying enzyme). In embodiments, an epigenetic modifying agent comprises a DNA-sequence-specific targeting system in which an epigenetic enzyme is linked (e.g., fused) to a DNA binding domain. In embodiments, such an epigenetic modifying agent modulates (e.g., downregulates or upregulates) gene expression, e.g., expression of a thymus function factor or thymus function modulator described herein. Exemplary epigenetic enzymes include DNA methyltransferases, DNA methylases, histone methyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine- specific histone demethylase 1 (LSDl), histone-lysine-N-methyltransferase (Setdbl), euchromatic histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), viral lysine methyltransferase (vSET), histone methyltransferase (SET2), and protein-lysine N-methyltransferase (SMYD2). Examples of such epigenetic modifying agents are described, e.g., in de Groote et al. Nuc. Acids Res.

(2012): 1-18.

In embodiments, an epigenetic modifying agent is a small molecule. Exemplary small molecule epigenetic modifying agents are described, e.g., in Lu et al. J. Biomolecular Screening 17.5(2012):555-71, e.g., at Table 1 or 2, incorporated herein by reference. In embodiments, an epigenetic modifying agent comprises vorinostat, romidepsin. In embodiments, an epigenetic modifying agent comprises an inhibitor of class I, II, III, and/or IV histone deacetylase (HDAC). In embodiments, an epigenetic modifying agent comprises an activator of SirTI. In

embodiments, an epigenetic modifying agent comprises Garcinol, Lys-CoA, C646, (+)-JQI, I- BET, BICI, MS 120, DZNep, UNC0321, EPZ004777, AZ505, AMI-I, pyrazole amide 7b, benzo[d] imidazole 17b, acylated dapsone derivative (e.e.g, PRMTI), methylstat, 4,4'-dicarboxy- 2,2'-bipyridine, SID 85736331, hydroxamate analog 8, tanylcypromie, bisguanidine and biguanide polyamine analogs, UNC669, Vidaza, or decitabine.

In embodiments, an epigenetic modifying agent comprises a construct described in Koferle et al. Genome Medicine 7.59(2015): 1-3 (e.g., at Table 1), incorporated herein by reference.

In embodiments, an epigenetic modifying agent inhibits DNA methylation, e.g., is an inhibitor of DNA methyltransferase (e.g., is 5-azacitidine and/or decitabine). In embodiments, an epigenetic modifying agent modifies histone modification, e.g., histone acetylation, histone methylation, histone sumoylation, and/or histone phosphorylation. In embodiments, the epigenetic modifying agent is an inhibitor of a histone deacetylase (e.g., is vorinostat and/or trichostatin A). In embodiments, the epigenetic modifier comprises sodium phenyl butyrate (SDB), lipoic acid (LA), quercetin, valproic acid, hydralazine, bactrim, green tea extract (e.g., epigallocatechin gallate (EGCG)), curcumin, sulforphane and/or allicin/diallyl disulfide. In embodiments, an epigenetic modifying agent modifies, e.g., increases or decreases, the expression level of a thymic function factor (e.g., a thymic function factor described herein), or a thymic function modulator (e.g., a thymic function modulator described herein).

In embodiments, the thymic function modulator comprises an aptamer. An aptamer can be made up of nucleic acids (e.g., DNA, RNA, modified DNA, modified RNA, or combinations thereof) and/or peptides. Aptamers form three-dimensional structures that bind specifically to a target molecule, inhibiting or activating the target molecule. For example, an aptamer can be generated using systematic evolution of ligands using exponential enrichment (SELEX). In embodiments, the aptamer binds to a thymic function factor, e.g., described in Table 1. For example, the aptamer decreases the activity of a thymic function factor, e.g., a negative regulator of thymic function, e.g., described in Table 1. In other examples, the aptamer increases the activity and/or stability of a thymic function factor, e.g., a positive regulator of thymic function, e.g., described in Table 1.

In embodiments, the thymic function modulator comprises an antibody molecule or an mRNA encoding an antibody molecule. In embodiments, the antibody molecule binds to a thymic function factor, e.g., described in Table 1. For example, the antibody molecule decreases the activity of a thymic function factor, e.g., a negative regulator of thymic function, e.g., described in Table 1. In other examples, the antibody molecule increases the activity and/or stability of a thymic function factor, e.g., a positive regulator of thymic function, e.g., described in Table 1.

In embodiments, the thymic function modulator comprises a peptide or polypeptide, e.g., a thymic function factor or fragment thereof (e.g., functional fragment thereof) described in Table 1. In embodiments, the peptide or polypeptide comprises an amino acid sequence having at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or more) identity to an amino acid sequence of a thymic function factor described in Table 1, e.g., a positive regulator of thymic function described in Table 1. In embodiments, a peptide has a molecular weight between about 700 to about 5000 Daltons. In embodiments, a polypeptide has a molecular weight between about 5000 and about 500,000 Daltons, e.g., between about 5000 and about 150,000 Daltons. Peptides and polypeptides can be naturally occurring, recombinant or chemically synthesized.

In embodiments, the thymic function modulator comprises a small molecule. For example, the small molecule binds to a thymic function factor, e.g., described in Table 1. For example, the small molecule inhibits the activity of a thymic function factor, e.g., a negative regulator of thymic function, e.g., described in Table 1. For example, the small molecule increases the activity of a thymic function factor, e.g., a positive regulator of thymic function, e.g., described in Table 1. A small molecule has a molecular weight of between about 50 and about 1000 Daltons, e.g., between about 100 Daltons and about 700 Daltons.

Measurement of thymus function

Methods described herein may include the assessment of certain thymic activities, functions or characteristics. For example, methods described herein can modulate (e.g., increase or decrease, as desired) T cell exhaustion, TILs, T-cell diversity, T cell clonality, thymocytes, thymic epithelial cells, thymic stromal cells, and thymus size. Accordingly, subjects being treated may be assessed for one or more such thymic activities, functions or parameter before, during and/or after treatment.

In some embodiments, patients are assessed before treatment, e.g., to establish a baseline level of a particular thymic parameter. In some such instances, a patient may be selected for treatment based on a pre-treatment assessment described herein. In some embodiments, a patient is assessed after a first administration of the therapy, e.g., one or more times (e.g., 2, 3, 4, 5, 7, 10, 15 or more times) during the period encompassing the treatment regimen. In some such embodiments, a patient may be monitored for disease treatment or progression based on assessments during the period encompassing the treatment regimen, e.g., where an increase in a thymic function parameter is intended by treatment, such assessments may be used to confirm the effect of treatment, or to determine whether to stop or continue treatment. In other embodiments, a patient is assessed after the period encompassing a treatment regimen is complete. The results of such assessments may be used, e.g., to determine if the patient should undergo a different treatment, continue with another round of the same treatment regimen, or another action determined by the patient' s health care provider.

Thymus function (e.g., extent of thymic involution, thymic damage, thymic regeneration, or decrease/reverse in thymic involution or damage) can be determined a number of methods.

In some embodiments, an increase in thymus tissue size/mass after treatment in a subject is indicative of a decrease/reverse in thymic involution. Thymus size

Generally speaking, the size and cellular composition of a human thymus is proportional to its output and level of activity. Thymus size can be assessed by direct

visualization in vivo, ex vivo, or intraoperatively, as well as through a wide variety of histological processing techniques known to the art, including but not limited to hematoxylin and eosin staining. Thymus size can also be assessed using biomedical imaging techniques such as ultrasound, magnetic resonance imaging (MRI or MR imaging), computed tomography (CT), and position emission tomography (PET) (PMID: 21700977). Ultrasound can be used to measure the size and/or shape of the thymus. For example, ultrasound can be used to determine the size and/or shape of each of the two lobes of the thymus. Ultrasound permits the imaging of individual parts of the thymus, e.g., medulla, cortex, interlobular septum, and blood vessels. See, e.g., Han et al. Pediatr. Radiol. 31(2001):474-79; and Nasseri et al. RadioGraphics

30(2010):413-28. The size of the thymus can be expressed, e.g., in terms of volume and/or a Thymus index, e.g., as described in Hasselbalch et al. Eur. Radiol. 6.5(1996):700-03; and Varga et al. Surg. Radiol. Anat. 33(2011):689-95. Recently, 18F-FDG PET imaging is an important tool for the visualization and staging of human cancer. In infants and young adults, the thymus's extensive cellularity and metabolic activity results in physiologic uptake of FDG, although this disappears during adolescence as the thymus involutes. (PMID 8896924) In contrast, transient thymic hyperplasia has been sporadically observed following chemotherapy, particularly for testicular carcinoma or malignant lymphoma; additionally, a retrospective study showed that in a subset of adult patients who experience chemotherapy-induced thymic hyperplasia, the hyperplasia is detectible via increased FDG uptake, rendering thymus FDG uptake an imaging biomarker for enhanced thymic cellularity (PMID 11337547).

In certain embodiments, thymus size is increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to prior to treatment.

In some embodiments, anterior mediastinal mass is a marker of thymic function (e.g., extent of thymic involution or extent of decrease/reverse in thymic involution). A larger, longer, thicker, denser, and/or more opaque anterior mediastinal mass is indicative of a decrease/reverse in thymic involution. Accordingly, methods described herein include in some embodiments measuring the size, (e.g., length, width, and/or thickness), opacity, and/or density of the anterior mediastinal shadow in a subject, e.g., before, during, and/or after treatment with a therapy, e.g., a thymic function modulator, e.g., described herein. Anterior mediastinal shadow can be measured by imaging techniques such as ultrasound, X-ray, CT scan, MRI, or PET (e.g., FDG avidity via PET). In some embodiments, a thymic function modulator, e.g., described herein, increases the size (e.g., length, width, and/or thickness), opacity, and/or density of the anterior mediastinal shadow in a subject, compared to a reference value. In embodiments, the thymic function modulator increases the size, opacity, and/or density of the anterior mediastinal shadow by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value. In embodiments, an increase (e.g., of at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more) in size, opacity, and/or density of the anterior mediastinal shadow in a subject (compared to a reference value) is indicative of an improvement in thymic function, e.g., a decrease or reverse in thymic involution.

In embodiments, the reference value is the size (e.g., length, width, and/or thickness), opacity, and/or density of the anterior mediastinal shadow in the subject prior to administration with a thymic function modulator, e.g., described herein.

Thymic stromal cells

Another marker of thymic function, e.g., thymic involution, is the level of stromal cells in the thymus. Stromal cells in the thymus include dendritic cells and epithelial cells of the thymic medulla and cortex. Stromal cells are involved in the selection of a self-tolerant and functional T cell repertoire.

Thymic stromal cells are the non-thymocyte cells within the thymus that mediate the thymus's system for 'training' T lineage progenitor T cells as developing thymocytes for proper binding of a T cell receptor recognizing 'self in the context of a peptide; this

compartment includes fibroblasts, epithelium, endothelium, and dendritic cells (Gray et al., J Immun., 2007). The main stromal compartments responsible for interacting with developing thymocytes are lined with TECs. Among other cell types, thymic stromal cells also comprise fibroblasts; fibroblasts are also known to play a role in guiding thymocyte development. Thymic stromal cells are heterogeneous and can be assessed by determining the relative or absolute quantity of the subsets comprising thymic stromal cells (e.g., fibroblasts, TECs, endothelium). For example, the stromal cell subset comprising thymic fibroblasts can be assessed through the use of the monoclonal antibody MTS-15 as known to the art (Gray et al., J. Immun., 2007).

In certain embodiments, thymic stromal cells (e.g., one or more or all of:

fibroblasts, TECs, endothelium) are increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to prior to treatment.

In embodiments, a thymus function modulator, e.g., described herein, e.g., in Table 1, increases the level of stromal cells (e.g., dendritic cells, medulla epithelial cells, and/or cortex epithelial cells) in the thymus of a subject, e.g., by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value. In embodiments, a thymus function modulator, e.g., described herein, e.g., in Table 1, increases the percentage of stromal cells (e.g., dendritic cells, medulla epithelial cells, and/or cortex epithelial cells) in a population of cells in a thymic sample, e.g., by at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100- fold, or more, compared to a reference value.

In embodiments, an increase (by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40- fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value) in the level of stromal cells in the thymus in a subject after administration of a thymus function modulator, e.g., described herein, e.g., in Table 1, is indicative of an improvement in thymic function, e.g., a decrease or reverse in thymic involution. In embodiments, an increase (by at least 1.25-fold, 1.5- fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value) in the percentage of stromal cells (e.g., dendritic cells, medulla epithelial cells, and/or cortex epithelial cells) in a population of cells in a thymic sample is indicative of an improvement in thymic function, e.g., a decrease or reverse in thymic involution.

In embodiments, the reference value is the level of the stromal cells (e.g., dendritic cells, medulla epithelial cells, and/or cortex epithelial cells) in the subject prior to administration with a thymic function modulator, e.g., described herein. In embodiments, the reference value is the percentage of stromal cells (e.g., dendritic cells, medulla epithelial cells, and/or cortex epithelial cells) in a population of cells in a thymic sample from the subject prior to administration with a thymic function modulator, e.g., described herein.

The level of stromal cells can be determined by identifying/quantifying one or more markers of stromal cells (e.g, dendritic cells, medulla epithelial cells, and/or cortex epithelial cells). For example, the level of stromal cells can be determined by using flow cytometry, e.g., FACS. For example, the level of stromal cells can be determined (e.g., using FACS) by using one or more of the cell surface markers shown in Table 2. In embodiments, the level of stromal cells is determined in a sample from the subject, e.g., a blood or tissue sample from the subject.

Table 2: Exemplary FACS/surface markers for thymic stromal cells

Another marker of thymic function, e.g., thymic involution, is the level of adipocytes in the thymus. As a thymus ages or involutes, thymic tissue degenerates into adipose tissue.

Without being bound by theory, it is believed that a decrease or reverse in thymic involution is characterized, in part, by a decrease in the level of and/or size of adipocytes in the thymus. In embodiments, a thymus function modulator, e.g., described herein, e.g., in Table 1, decreases the level of and/or size of adipocytes in the thymus of a subject, e.g., by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value. In embodiments, a decrease (by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value) in the level of and/or size of adipocytes in the thymus in a subject after administration of a thymus function modulator, e.g., described herein, e.g., in Table 1, is indicative of an improvement in thymic function, e.g., a decrease or reverse in thymic involution. In embodiments, the reference value is the level of and/or size of adipocytes in a thymic samples from the subject prior to administration with a thymic function modulator, e.g., described herein. The size of adipocytes can be the average size of adipocytes, e.g., in a sample containing a population of adipocytes, or the size of an individual adipocyte.

The level of adipocytes can be determined by identifying/quantifying one or more markers of adipocytes. For example, the level of and/or size of adipocytes can be determined by using flow cytometry, e.g., FACS. In other examples, the level of and/or size of adipocytes can be determined using histological methods, e.g., in situ hybridization or immunohistochemistry. In yet other embodiments, the level of adipocytes can be determined by using PPARy staining (e.g., to identify adipogenic fibroblasts), LipidTOX Green (Invitrogen), Oil Red O staining, and/or magnetic resonance imaging (MRI). In embodiments, the level of adipocytes is determined in a sample from the subject, e.g., a blood or tissue sample (e.g., biopsy, e.g., thymus biopsy) from the subject.

Thymocytes

Thymocytes are T-cells and T-cell progenitors found in the thymus. In the thymus, under the influence of the thymic stromal microenvironment, immature thymocytes acquire various cell surface molecules useful in their future role as mature T cells. Thymocytes can be assessed by multiple methods as described in the art. For example, thymocytes can be assessed by assaying for cell surface expression of developmentally regulated thymocyte markers, using labeled antibodies that specifically bind to these markers. For example, the most immature CD4-CD8- double-negative (DN) thymocytes give rise to CD4+CD8+ double- positive (DP) thymocytes, which give rise to mature CD4+CD8- single-positive (SP) and CD4-CD8+ SP T cells. The DN population can be further subdivided by the expression of CD44 and CD25: CD44+CD25- (DN1) cells differentiate into CD44+CD25+ (DN2) cells, which give rise to CD44-CD25+ (DN3) cells, which finally become the most mature CD44-CD25- (DN4) DN population. The DN4 cells may pass through an intermediate population expressing either coreceptor alone before becoming DP cells. This intermediate population, most commonly expressing CD8, is known as immature single positive (ISP) cells. Progression beyond the DN3 stage is dependent on successful rearrangement of a TCRP-chain gene and pre-TCR signaling, whereas differentiation from DP to mature SP cell is dependent on the expression and positive selection of an aPTCR (Von Boehmer et al., Immunol. Rev. 191: 62, 2003; Ceredig and Rolink, Nat. Rev. Immunol. 2: 888, 2002). Additionally, the cellularity of the thymus (including thymocytes) can be assessed using clinical imaging modalities such as MRI, CT, or PET, as described in the art (Brink et al., J. Nuc. Med., 2001; Ackman and Wu, Am. J. Roent, 2011).

In certain embodiments, thymocytes (e.g., one or more of: CD4+CD8+ DP thymocytes; CD4+CD8- SP thymocytes; CD4-CD8+ SP T cells; CD44+CD25- DN1 cells; CD44+CD25+ DN2 cells; to CD44-CD25+ DN3 cells; CD44-CD25- DN4) are increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to prior to treatment.

Thymic epithelial cells

Thymic epithelial cells or TECs are epithelial cells located in the thymus, including cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs). They comprise the stromal compartment of the thymus responsible for guiding developing thymocytes through various developmental stages and to maturity. Thymocytes can be assessed by multiple methods as described in the art. For example, TECs can be assessed by assaying for cell surface expression of developmentally regulated TEC markers using labeled antibodies that specifically bind these markers; such cell surface markers include Keratin 8 (K8), K5, EpCAM, MHC class I, MHC class II, CD45, CD80, CD86, CD90, CDl lc, CCL25, RANK, RANKL and CXCL12 (Gray et al., Immun. Meth., 2008). Other markers of TECs are intracellular or secreted and can be assessed using intracellular staining or other methods known to the art. Such markers include AIRE, FezF2, FoxNl, Hoxa3, proteasome subunit β5ί, proteasome subunit β5ί, BMP4, retinoic acid, Wnt, Shh, FGF, and SCF. In some case the absence of a particular marker can be useful in assessing the identity of a cell as a TEC, for example, CD45. Additionally, the cellularity of the thymus (including thymic epithelium) can be assessed using clinical imaging modalities such as MRI, CT, or PET, as described in the art (Brink et al., J. Nuc. Med., 2001; Ackman and Wu, Am. J. Roent., 2011).

In certain embodiments, thymic epithelial cells are increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to prior to treatment.

T cell function/output

A measure of thymic activity is T cell output. T cell maturation takes place in the thymus. Hematopoietic progenitor cells (e.g., a CD34 positive hematopoietic stem cell) enter the thymus, where they proliferate to generate a large population of immature thymocytes.

Immature thymocytes make distinct T cell receptor via gene rearrangement. Some T cell receptors are functional (e.g., recognize specific cell surface molecules, e.g., foreign antigens) and some T cell receptors are autoreactive (e.g., recognize self-peptides). During T cell maturation in the medulla of the thymus, immature thymocytes undergo positive selection and negative selection based on their T cell receptor specificity. These newly generated T cells are exported into the periphery and circulate through the lymphoid tissues and blood. In

embodiments, the recently exported T cells are CD4+ and CD8+. In embodiments, recently exported T cells are referred to as recent thymic emigrants and typically have undergone no more than a few cellular divisions after leaving the thymus. It is believed that the number of T cells exported into the periphery, i.e., T cell output, e.g., recent thymic emigrants, decreases during the process of thymic involution.

T cell output, e.g., over a period of time (e.g., T cell output per day, per hour, or per 12 hours) can be determined, e.g., by measuring one or more markers of recent thymic emigrants. Exemplary markers of recent thymic emigrants (RTEs) include chTl+, CD45RA, and CD31 (PECAM-1), In embodiments, a RTE has the following expression pattern: CD3 ⁺ CD62L ⁺ CD45RA ⁺ CD31 ⁺ CD44 ^_/1 °. In embodiments, a RTE can be distinguished from an older non- RTE naive CD4+ T cell, e.g., using TPK7 as a marker. See, e.g., Haines et al. J. Exp. Med. 206.2(2009):275-85. In other embodiments, a RTE can be distinguished from a mature naive T cell, e.g., by using Rag2 as a marker. See, e.g., Houston Jr. et al. J. Immunol. 181(2008):5213- 17. Another measure of T cell output, e.g., recent thymic emigrants, is the concentration of T cell receptor excision circles (TRECs). TRECs are extrachromosomal nonreplicative DNA byproducts generated during T-cell receptor (TCR) rearrangement. TRECs are expressed only in thymus -originated T cells, each of which is believed to contain a single TREC. TRECs are exported into the periphery from the thymus, and it is believed that TREC levels in the periphery are an indicator of the number of recent thymic emigrants, which is a measure of T cell output and/or T-cell recovery (e.g., after hematopoietic cell transplantation). TREC concentration can be measured using PCR, real-time PCR, quantitative-competitive PCR, or PCR-ELISA. T cell output and measurement of recent thymic emigrant and/or TREC concentration is described, e.g., in Ye et al. J. Immunol. 168(2002):4968-79 or in http://www.mayomedicallaboratories.com/test- catalog/Clinical+and+Interpretive/87959 (accessed as of June 9, 2016).

In embodiments, a thymus function modulator, e.g., described herein, e.g., in Table 1, increases the T cell output (e.g., number of recent thymic emigrants, e.g., concentration of TRECs) in a subject, e.g., by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60- fold, 80-fold, 100-fold, or more, compared to a reference value. In embodiments, an increase (by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value) in the T cell output (e.g., number of recent thymic emigrants, e.g., concentration of TRECs) in a subject after administration of a thymus function modulator, e.g., described herein, e.g., in Table 1, is indicative of an improvement in thymic function, e.g., a decrease or reverse in thymic involution. In embodiments, the reference value is the T cell output (e.g., number of recent thymic emigrants, e.g., concentration of TRECs) in a sample from the subject prior to administration with a thymic function modulator, e.g., described herein.

T cell output, e.g., markers of recent thymic emigrants, e.g., concentration of TRECs, can be determined in a sample from a subject, e.g., a blood (peripheral blood) or tissue sample. Additional indicators of thymic function (e.g., extent of thymic involution, thymic damage, thymic regeneration, or decrease/reverse in thymic involution or damage) include T cell diversity, TCR repertoire diversity, T cell clonality, and/or T cell diversity.

In embodiments, a thymus function modulator, e.g., described herein, e.g., in Table 1, increases the T cell diversity, TCR repertoire diversity, and/or T cell clonality in a subject, e.g., by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value. In embodiments, an increase (by at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, or more, compared to a reference value) in the T cell diversity, TCR repertoire diversity, and/or T cell clonality in a subject after administration of a thymus function modulator, e.g., described herein, e.g., in Table 1, is indicative of an improvement in thymic function, e.g., a decrease or reverse in thymic involution. In embodiments, the reference value is the T cell diversity, TCR repertoire diversity, and/or T cell clonality in a sample from the subject prior to administration with a thymic function modulator, e.g., described herein.

T cell exhaustion

The invention provides, inter alia, methods of decreasing T cell exhaustion. T cell exhaustion, also referred to as T cell dysfunction, refers to dysfunctional state of T cells indicated by reduced or absent production of effector cytokines IFNg, TNFa, IL-2, effector molecules perforin, granzyme A, B, or K, reduced ability of cells to proliferate, and increased expression of co-inhibitory receptors PD-1, LAG-3, TEVI-3, CTLA-4, CD160, 2B4 (CD244), BTLA, or TIGIT as described in the art (PMID: 26205583, PMID:21739672).

T cell exhaustion can be assessed by the following methods: T cells are stained for the expression of above mentioned surface receptors, or reactivated ex vivo with cognate peptide, PMA + Ionomycin, or anti-CD3, to assess their ability to produce cytokines and other effector molecules, as well as their ability to proliferate as measured by the increase of Ki-67 expression, incorporation of BrdU, or dilution of a cell dye such as CSFE or CellTrace Violet.

In certain embodiments, T cell exhaustion is increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to a reference level, e.g., the extent of T cell exhaustion prior to treatment..

Memory T cell response

In embodiments, a composition or method described herein stimulates a robust memory T cell response. This memory T cell response will aid in the clearance of pathogens or tumor cells and will prevent the occurrence of relapse.

During a T cell response to a viral infection or a tumor, antigen- specific CD 8 T cells will be activated by antigen-presenting cells and will differentiate into effector T cells. Effector CD8 T cells proliferate rapidly, produce pro-inflammatory cytokines, and kill infected target cells or tumor cells using cytotoxic molecules such as granzymes and perforin. The number of antigen- specific T cells responding to a pathogen peaks at day 8 after the initial infection and then the contraction phase of the T cell response begins. During contraction, the majority of T cells die and 5-10% persist as memory cells (PMID: 23080391). Before the contraction phase begins, T cells have already entered a molecular program of cellular death during the contraction phase or persist as memory cells. The antigen- specific cells that will eventually die during contraction are termed "short-lived effector cells" (SLECs) and the cells that will persist are termed "memory- precursor effector cells" (MPECs). During an infection, these cells can be differentiated using the cell surface markers KLRG1 and CD127 (IL-7Ra). SLECs will be KLRG1+ CD127- and MPECs will be KLRG1- CD127+ (PMID: 23080391). An increase in the percentage of cells in the MPEC population indicates a stronger memory T cell response and greater protective immunity.

In certain embodiments, memory T cells are increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to a reference level, e.g., the level of memory T cells prior to treatment. In some embodiments, MPECs comprise between 1-20%, between 2-20%, between 5-20%, between 2-15%, between 5-15%, between 7- 20%, between 7-15%, between 10-20% of T cells in a treated subject. Tumor infiltrating lymphocytes (TILs)

Tumor infiltrating lymphocytes (TIL) are populations of immune cells that are associated with tumor tissue. TIL can be made up of lymphocytes such as CD4 T cells, CD8 T cells and NK cells, and also myeloid cells such as macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSCs). These populations of cells can be enriched from single-cell suspensions of tumor tissue using density gradient centrifugation. The presence of TIL in tumors is associated with better prognosis, particularly for CD8 T cells. TILS can be assessed, identified, and/or phenotyped by direct antibody labeling and flow cytometric analysis. CD45 is often used to identify hematopoietic cells from the tumor, which can then be phenotyped for more parameters such as CD3, CD4, CD8, CDl lb, CDl lc, NK1.1. Better pheno type and function of CD8 T cells in tumors is associated with good responses to immunomodulatory antibody therapy (PD-1 and CTLA-4 pathways) and can be assessed using flow cytometry markers that have been described previously (PMID: 25754329; PMID: 23197535).

In certain embodiments, TILs are increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to prior to treatment.

T cell diversity

T cell diversity refers to the range of phenotypically, genetically, epigenetically, biochemically, and/or functionally distinct T cell populations present within a given sample or subject. T cell diversity can be assessed through a wide variety of methods known to the art. For example, T cell diversity can be assessed through qualitative and/or quantitative comparison of T cell receptor repertoire, as described herein. Importantly, T cell diversity can also be assessed by methods known to the art that do not involve and that are independent of TCR analysis. For example, high-resolution mass spectrometry can be used to map the proteome from cytotoxic T lymphocytes and use this information to identify different functional subgroups that vary in population size and cytotoxic efficacy (PMID: 26551880) Additionally, it is known to the art that engagement of different metabolic pathways and properties across subsets of T-cell populations can lead to functionally distinct T-cell populations; thus, T cell diversity can also be assessed through quantitative or qualitative assessment of such metabolic pathways and properties through a variety of methods known to the art (PMID 23746840, PMID 26261266). The diversity of cells can also be assessed from blood samples or other tissues such as biopsied tumor, using flow cytometry or mass cytometry (CyTOF), by staining cell surface markers. For example, CD4 T cells can be divided into naive (CD4+CCR7+CD45RA+CD45RO-), TH1 (CD4+CXCR3+CCR6-CD 161 -) , TH17 (CD4+CCR6+CD 161 +CXCR3 -) , TH2

(CD4+CRTH2+CXCR3 -) , Treg (CD4+CD1271oCD25+), memory (CD4+CD45RA-CD45RO+), TCM (CD4+CCR7+CD45RA-CD45RO+), TEM (CD4+CCR7-CD45RA-CD45RO+), TEMRA (CD4+CCR7-CD45RA+CD45RO-), Trl: (CD49b and Lag3 co-expression), Tfh (CXCR5, PD-1, BCL6, FoxP3-), and Tfr (CXCR5, PD-1, BCL6, FoxP3+), and CD8 T cells can be similarly divided into naive (CD8+CCR7+CD45RA+CD45RO-), TCM (CD8+CCR7+CD45RA- CD45RO+), TEM (CD8+CCR7-CD45RA-CD45RO+), TEMRA (CD8+CD45RA+CCR7- CD45RO-), Tel (IFNg expression following restimulation) Tc2 (CRHT2+) and Tel 7 (CCR5, CCR6, CD161) as known to art (PMID: 23624599, 26146074, 25177353, 7525836, 11069080, 21706005). Following treatment with the inventions described herein, the distribution of the different subsets of these cells can be measurably altered, thereby quantitatively impacting the diversity of the T cells in the patient.

In certain embodiments, T cell diversity is increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to a reference level, e.g., the T cell diversity prior to treatment.

TCR repertoire diversity

TCR repertoire diversity refers to the number of different T cell receptors (TCR) in a population of T cells; sources of diversity within TCR repertoire include unique alpha and beta TCR subunits, genetic differences stemming from V-J (TCR alpha) and V-D-J (TCR beta) recombination, as well as different terminal deoxynucleotidyl transferase (TdT) - introduced nucleotides at DNA junctions, resulting in sequence variation. (Murphy et al., Janeway's

Immunobiology, 8th Edition, 2012). Because T-cells expand clonally, in some cases a given TCR can be shared by multiple T-cells that have expanded from a common parent cell; in this situation and in similar situations, TCR repertoire diversity can additionally refer to the number of different clonal populations of T-cells that harbor distinct TCRs within a sample. An increase in TCR repertoire diversity is an indicator of either the presence of or an increased potential for a robust antigen- specific T cell response to a pathogen or tumor, as increased diversity suggests that more T cells are capable of responding to the pathogen or tumor, which is beneficial for ensuring effective control of the pathogen or tumor. Increases in repertoire diversity also suggest an environment more favorable for effective immunity, as subdominant clones with weaker TCR affinity for their antigen are also able to become activated and proliferate.

TCR repertoire diversity can be assessed by methods known to art including RNA- Sequencing, DNA- sequencing, TCR-targeted sequencing, and TCR probe-based PCR. For example, TCR repertoire profiling can measure the distribution of individual T cell clones in a population. This is done by amplifying the CDR3 region of the TCR alpha and TCR beta chains from T cell genomic DNA and next-generation sequencing the region. Since each clonal population of T cells has a unique T cell receptor, analysis of deep sequencing data from a TCR repertoire profile can yield information about the total number of different T cell clones and T- cell receptors found in the population. Both of these metrics can be measured from a single sample, given that the sample is sequenced deeply enough and the amount of input T cell genomic DNA is standardized. (US 20100035764, PMID: 26404496, PMID: 2343517, PMID: 24329790).

In certain embodiments, T cell repertoire diversity is increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to a reference level, e.g., the T cell repertoire diversity prior to treatment..

T cell clonality

T cell clonality refers to the absolute or relative size of a clonal T cell population or of several clonal T cell populations, including peripheral blood mononuclear cells, TILs, or other tissue-derived immune cells, or the magnitude of a T cell expansion giving rise to said clonal population. For example, during the course of a response to a particular antigen, a T cell that is specific for said antigen will undergo a series of receptor-mediated reactions, resulting in the division of the cell into daughter cells and establishing a clonal population of T cells expressing the same TCR. In embodiments, an increase TCR clonality is a good indicator of a robust antigen- specific T cell response to a pathogen or tumor. The term "T cell clonality" can be used to refer to the absolute or relative size of a specific T cell clonal population, or it can be used to refer to multiple clonal T cell populations simultaneously. Increases in T cell clonality can also suggest that T cells are expanding vigorously after antigen recognition, indicating that they are not functionally suppressed (e.g., exhausted). Accordingly, in some embodiments, an increase in T cell clonality (e.g., of disease-specific T cells, e.g., cancer-specific T cells) can be desirable. In some cases, a subject can achieve a state of T cell clonality in which clonal populations of T cells specific to a particular target or set of targets (e.g., cytomegalovirus or Epstein-Barr Virus) represent a disproportionately large portion of the total T-cell pool. In such cases, for example, a decrease in T cell clonality (e.g., for certain T cell populations) may be desirable.

T cell clonality can be assessed by performing T cell spectratyping on a population of T cells, performing tetramer staining on a population of T cells, sequencing at least one TCR subunit from a population of cells, staining a population of T cells with an anti-TCR antibody, performing flow cytometry, or a combination thereof. TCR sequence can be assessed by methods known to art including RNA-Sequencing, DNA-sequencing, TCR-targeted sequencing, and TCR probe-based PCR (US 20100035764 Al, PMID: 26404496, PMID: 2343517, PMID: 24329790).

TCR-Immunosequencing is a platform technology that allows the enumeration, specification and quantification of each and every T-cell in any biologic sample of interest. It is based on bias-controlled multiplex PCR and high throughput sequencing and is highly accurate, standardized, and sensitive. See, e.g., Kirsch. J. Immunother Cancer. 3.29(2015); or Carlson et al. Nat. Commun. 4(2013):2680, both incorporated herein by reference. Without wishing to be bound by theory, it is believed that the average clonal size of a naive cell is about 100-200 cells. Recent thymic emigrants in the newborn undergo three to four divisions in the periphery after TCRA gene rearrangement, thereby establishing a minimal clonal size of 10 cells. Qi et al. have described that there is an increase in T-cell clonality in the naive repertoire of the elderly; the contribution of clonally expanded T cells to the observed repertoire increased by a factor of >100 for naive CD8 and of >10 for naive CD4 T cells compared with young adults. See Qi et al. Proc. Natl. Acad. Sci. USA. 111.36(20140: 13139-44.

In certain embodiments, T cell clonality is increased or decreased in a subject, as determined by these methods, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared to a reference level, e.g., the clonality prior to treatment. In embodiments, the T cell clonality is increased or decreased, e.g., as determined by TCR-immunosequencing, by 1.5-fold to 1000-fold, e.g., 1.5- fold to 10-fold, 10-fold to 20-fold, 20-fold to 50-fold, 50-fold to 100-fold, 100-fold to 250-fold, 250-fold to 500-fold, or 500-fold to 1000-fold, compared to a reference level, e.g., the clonality prior to treatment.

Other markers of thymic function include the formation of a plasma cell, development of an antigen- specific T cell, and/or formation of a thymic organoid. In other embodiments, the marker of thymic structure/function comprises the formation of, or modulation (e.g., increase or decrease) in quantity, size, function, and/or distribution of, Hassall's corpuscles in the cell(s)/tissue(s)/organoid(s), compared to before the culture step(s). In embodiments, a change in Hassall's corpuscles can be detected histologically and/or through a thymus exosome profile, e.g., as described in Skogberg et al. J. Immunol. 193(2014):2187-95, incorporated herein by reference.

The formation of a plasma cell (e.g., polyclonal plasma cell) can be determined by detection (e.g., using flow cytometry, e.g., FACS, and/or immunohistochemistry) of one or more surface markers, e.g., CD138, CD38, CD45, CD19, and/or the presence of kappa and/or lambda cytoplasmic immunoglobulin light chains in the cells. In embodiments, high expression of CD38 and CD138 (in conjunction with scatter properties of cells), e.g., in flow cytometric methods or immunohistochemical methods, can indicate presence of polyclonal plasma cells. Without wishing to be bound by theory, it is believed that CD45 and CD 19 are expressed on normal plasma cells; abnormal plasma cells lack CD19 and variably express CD45. Clonality can be confirmed based on the presence of either kappa or lambda cytoplasmic immunoglobulin light chains (or both) in the cells. In other embodiments, plasma cells (e.g., polyclonal plasma cells) can be detected using morphological analysis, e.g., in smear slides (e.g., stained with Giemsa). For example, mature plasma cells can be detected via their eccentric nuclei and basophilic cytoplasm with perinuclear halos. In some examples, morphological analysis is combined with cellular marker staining (e.g., CD138 or other cellular marker(s)).

Antigen- specific T cells can be identified and/or quantified. In embodiments, cells, e.g., T cells, are analyzed by in vitro stimulation of T cells, followed by identification of cells that are activated using one of the methods described herein. Exemplary methods include flow cytometry, intracellular cytokine staining (ICS), enzyme-linked immunospot (ELISpot), carboxyfluorescein succinimidyl ester (CFSE)-based methods, TCR sequencing, mass spectrometry based methods (e.g., CyTOF), and tetramer staining (e.g., detection of peptide- MHC multimers). In embodiments, pre-enrichment (e.g., magnetic pre-enrichment) can be used to pre-enrich rare antigen- specific T cells, e.g., in order to achieve a higher signal-to-noise ratio, increased sensitivity, and/or improved detection of antigen-specific T cells, e.g., within the naive and/or Treg compartment. ICS detects antigen-induced secretion of cytokines by blocking cytokine secretion by cells (e.g., trapping in Golgi/endoplasmic reticulum). ICS may provide more flexibility than ELISpot and can be performed on cryopreserved cells as well as fresh samples. ICS can be combined with MHC multimer staining and flow cytometry. ICS works well with Thl cells. ICS can detect a limited number of cytokines and does not permit analysis of live cells. ICS typically provides supporting evidence of antigen specificity (e.g., in combination with another method, e.g., described herein). ELISpot detects antigen-induced secretion of cytokines using an enzyme linked immunosorbent assay (ELISA) based method. ELISpot is highly reproducible and sensitive and is suited for high-throughput analysis. ELISpot detects secreted cytokines at a single cell level, measures one or two parameters at a time, and requires cell purification to identify the source of the cytokine. ELISpot works well with Thl cells and does not permit analysis of live cells. CFSE based methods utilize the dye, 5,6- carboxyfluorescein diacetate succinimidyl ester, which is distributed evenly between daughter cells (of proliferating activated T-cells). Flow cytometry can identify antigen- specific T cells through antibodies specific for surface and/or cytoplasmic markers. Flow cytometry can be used to detect rare T cell populations and provides a quantitative analysis of lymphocytes expressing a specific TCR. For example, CD 154 (CD40L) is expressed after 5-7 hours of TCR stimulation by all naive and memory CD4 T-cells. Similarly, CD137 is expressed by Treg cells after 5-7 hours of antigen stimulation. Flow cytometry can detect these markers. In TCR-seq methods, TCR clones are sequenced; this provides comprehensive information about antigen-activated T cells. CyTOF is a mass spectrometry based approach that identifies antigen specificity; it allows analysis of over 40 parameters at a single cell level with moderate throughput. Detection of peptide-MHC multimers permits direct identification of T cells according to TCR binding, independently of functional status of T cells or antigen presenting cells (APCs). In this method, up to 100 different multimers can be combined in cytometric analysis using barcoding. For example, the peptide-MHC multimer method requires knowledge of antigens and epitopes, and works for pathogens with low antigen complexity and peptides. In some examples, differences in TCR specificities among individuals or within an individual, e.g., at different stages of disease, can be detected using the peptide-MHC multimer method.

Thymic organoids can be detected by the detection of one or more thymus cell markers, e.g., as described in Table 2 or Table 4, or the detection of a thymus structure, e.g., cortex and/or medulla.

Hassall's corpuscles are histologically distinct groups of epithelial cells present in the thymic medulla that are known to engage dendritic cells and facilitate production of CD4+ CD25+ FoxP3+ regulatory T cells (i.e., natural or thymic Tregs, nTregs.) See, e.g., Watanabe et al. Nature. 436(2005): 1181-85; or Skogberg et al. J. Immunol. 193(2014):2187-95, both incorporated herein by reference. Therefore, changing the quantity, size, function, or distribution of Hassall's corpuscles can change the rate of generation of nTregs, their molecular properties, and their population in the bloodstream. In embodiments, a change in Hassall's corpuscles (e.g., in a subject treated with a therapy described herein) can be detected histologically and/or through a thymus exosome profile, e.g., as described in Skogberg et al. J. Immunol. 193(2014):2187-95, incorporated herein by reference. In embodiments, a subject treated with a therapy described herein, e.g., a thymic function modulator described herein, a change in Hassall's corpuscles can be detected, e.g., by detecting a change in the number of sjTREC+, FoxP3+, and/or nTreg cells in the bloodstream of the subject. In other examples, a change in Hassall's corpuscles can be detected, e.g., by detecting a serum biomarker such as a secreted thymus exosome with distinct molecular properties. In embodiments, a change in Hassall's corpuscles can be indicated by detecting a secreted thymus exosome as described herein and/or sjTRECs.

Combination therapies

Compositions described herein can include any combination of thymic function modulators, e.g., thymic function modulators, e.g., which can include any thymic function factor described in Table 1 or modulator thereof. Combinations can include two or more, e.g., two, three, four, five, six, seven, eight, nine, ten or more thymic function modulators, e.g., described herein.

In embodiments, combinations can include one or more of the following or any modulators thereof: (i) a chemokine listed in Table 1 with a cytoplasmic protein, enzyme (phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), matrix protein, transcription factor, receptor (e.g., cell surface receptor or nuclear receptor) or additional chemokine listed in Table 1 ;

(ii) a cytoplasmic protein listed in Table 1 with a chemokine, enzyme (phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), matrix protein, transcription factor, receptor (e.g., cell surface receptor or nuclear receptor) or additional cytoplasmic protein listed in Table l ;

(iii) an enzyme (phosphatase, protease, or proteinase) listed in Table 1 with a chemokine, cytoplasmic protein, hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), matrix protein, transcription factor, receptor (e.g., cell surface receptor or nuclear receptor) or additional enzyme listed in Table 1 ;

(iv) a hormone (e.g., secreted hormone) listed in Table 1 with a cytoplasmic protein, enzyme (phosphatase, protease, or proteinase), ligand (e.g., membrane bound or secreted ligand), matrix protein, transcription factor, receptor (e.g., cell surface receptor or nuclear receptor) or additional hormone (e.g., secreted hormone) listed in Table 1;

(v) a ligand (e.g., membrane bound or secreted ligand) listed in Table 1 with a

cytoplasmic protein, enzyme (phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), matrix protein, transcription factor, receptor (e.g., cell surface receptor or nuclear receptor) or additional ligand (e.g., membrane bound or secreted ligand) listed in Table 1 ;

(vi) a matrix protein listed in Table 1 with a cytoplasmic protein, enzyme (phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), transcription factor, receptor (e.g., cell surface receptor or nuclear receptor) or additional matrix protein listed in Table 1 ;

(vii) a transcription factor listed in Table 1 with a cytoplasmic protein, enzyme

(phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), matrix protein, receptor (e.g., cell surface receptor or nuclear receptor) or additional transcription factor listed in Table 1 ;

and/or

(viii) a receptor (e.g., cell surface receptor or nuclear receptor) listed in Table 1 with a cytoplasmic protein, enzyme (phosphatase, protease, or proteinase), hormone (e.g., secreted hormone), ligand (e.g., membrane bound or secreted ligand), matrix protein, transcription factor, or additional receptor (e.g., cell surface receptor or nuclear receptor) listed in Table 1.

In other embodiments, combinations can include one or more of the following or any modulators thereof:

(i) a T-cell activity factor listed in Table 1 with a T-cell growth factor, T-cell growth factor receptor, T-cell migration factor, TEC function factor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, thymus homeostasis factor, or additional T-cell activity factor listed in Table 1 ;

(ii) a T-cell growth factor listed in Table 1 with a T-cell activity factor, T-cell growth factor receptor, T-cell migration factor, TEC function factor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, thymus homeostasis factor, or additional T-cell growth factor listed in Table 1 ;

(iii) a T-cell growth factor receptor listed in Table 1 with a T-cell growth factor, T-cell migration factor, TEC function factor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, thymus homeostasis factor, T-cell activity factor, or additional T-cell growth factor receptor listed in Table 1 ;

(iv) a T-cell migration factor listed in Table 1 with a T-cell growth factor, T-cell growth factor receptor, TEC function factor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, thymus homeostasis factor, T-cell activity factor, or additional T-cell migration factor listed in Table 1 ;

(v) a TEC function factor listed in Table 1 with a T-cell growth factor, T-cell growth factor receptor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, thymus homeostasis factor, T-cell activity factor, T-cell migration factor, or additional TEC function factor listed in Table 1 ;

(vi) a TEC growth factor listed in Table 1 with a T-cell growth factor, T-cell growth

factor receptor, T-cell migration factor, TEC function factor, TEC growth factor receptor, TEC proliferation factor, thymus homeostasis factor, T-cell activity factor, or additional TEC growth factor listed in Table 1 ;

(vii) a TEC growth factor receptor listed in Table 1 with a T-cell growth factor, T-cell growth factor receptor, T-cell migration factor, TEC function factor, TEC growth factor, TEC proliferation factor, thymus homeostasis factor, T-cell activity factor, or additional TEC growth factor receptor listed in Table 1 ;

(viii) a TEC proliferation factor listed in Table 1 with a T-cell growth factor, T-cell growth factor receptor, T-cell migration factor, TEC function factor, TEC growth factor, TEC growth factor receptor, thymus homeostasis factor, T-cell activity factor, or additional TEC proliferation factor listed in Table 1 ; or

(ix) a thymus homeostasis factor listed in Table 1 with a T-cell growth factor, T-cell

growth factor receptor, T-cell migration factor, TEC function factor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, T-cell activity factor, or additional thymus homeostasis factor listed in Table 1.

In embodiments, the two or more thymic function modulators can be coupled together by a covalent bond, non-covalent bond, or chemical linkage, e.g., separated by a linker. In embodiments, the thymic function modulators are nucleic acid molecules (e.g., mRNAs, siRNAs, dsRNAs, shRNAs, miRNAs, or modified versions thereof) and disposed on a single nucleic acid molecule or on separate nucleic acid molecules. In the case where the thymic function modulators are disposed on the same nucleic acid molecule, e.g., mRNA, they can be separated by an IRES, e.g., they are translated into separate polypeptides. In embodiments, the thymic function modulators are polypeptides and are disposed on a single polypeptide or on separate polypeptides. In the case where the thymic function modulators are disposed on a single polypeptide, they can be separated by a linker, e.g., peptide or chemical linker. In embodiments, the linker is a cleavable linker.

For example, a peptide linker can be a sequence of amino acids that physically connects polypeptide domains. For example, a peptide linker comprises one or more of the amino acids glycine and serine (e.g., comprises repetitive modules of glycine and serine). An exemplary peptide linker is (GGGGS) _3. In other embodiments, a peptide linker comprises

(GGGXX) _n GGGGS and GGGGS(XGGGS) _n wherein n is greater than or equal to 1, and wherein X is an amino acid which reduces or eliminates the addition of a post-translational modification to the polypeptide upon expression in a host cell. In one embodiment, the peptide linker comprises the amino acid sequence (GGGGA)„GGGGS. In another embodiment, the peptide linker comprises the amino acid sequence (GGGGQ) ₂ GGGGS. In yet another embodiment, the peptide linker comprises the amino acid sequence (GGGPS) ₂ GGGGS . In still another

embodiment, the peptide linker comprises the amino acid sequence GGGGS(PGGGS) ₂ . In a further embodiment, the peptide linker consists of the amino acid sequence (GGGGA) ₂ GGGGS. In another embodiment, the peptide linker consists of the amino acid sequence

(GGGGQ) ₂ GGGGS. In another embodiment, the peptide linker consists of the amino acid sequence (GGGPS) ₂ GGGGS. In another embodiment, the peptide linker consists of the amino acid sequence GGGGS(PGGGS) ₂ .

In embodiments, a peptide linker comprises a cleavable amino acid sequence, e.g., a protease or peptidase cleavage recognition sequence. For example, the protease or peptidase cleavage recognition sequence is cleavable by Factor XIa/FVIIa, matrix metalloprotease-1, HIV- 1 protease, NS3 protease, Factor Xa, furin, or cathepsin B. See, e.g., Chen et al. Adv. Drag Deliv. Rev. 65.10(2013): 1357-69 (e.g., at Table 3), incorporated herein by reference.

In embodiments, a peptide linker comprises a sequence described in Chen et al. Adv. Drag Deliv. Rev. 65.10(2013): 1357-69 (e.g., at Table 3), incorporated herein by reference.

Peptide linkers described herein can be of varying lengths. In one embodiment, a peptide linker is from about 5 to about 75 amino acids in length. In another embodiment, a peptide linker is from about 5 to about 50 amino acids in length. In another embodiment, a peptide linker is from about 10 to about 40 amino acids in length. In another embodiment, a 15 peptide linker is from about 15 to about 35 amino acids in length. In another embodiment, a peptide linker is from about 15 to about 20-aniino acids in length. In another embodiment, a peptide linker is from about 15 amino acids in length.

Peptide linkers may be attached to the N or to the C terminus (or both) of polypeptides to which they are attached.

In some embodiments, a chemical linker suitable for use in the compositions/methods described herein is described in WO 08/109105, incorporated herein by reference. For example, a chemical linker, e.g., described in WO 08/109105, can link one or more peptides, polypeptides, antibody molecules, nucleic acid molecules, and/or small molecules. For example, a chemical linker, e.g., described in WO 08/109105, can link one or more RNA interfering molecule (e.g., siRNA, miRNA, or shRNA) and/or one or more mRNA molecule(s). Exemplary chemical linkers include but are not limited to a polymeric linking agent, e.g., a PEG, e.g.,

homobifunctional PEG linker, heterobifunctional PEG linker, homomultifunctional PEG linker, poly-(beta- amino ester) polymeric linker, PEG-PLGA-PEG triblock polymeric linker, e.g., as described in WO 08/109105. In some embodiments, a chemical linker comprises a polymer, e.g., a branched or unbranched hydrophilic polymer, e.g., polyamino acids, amino sugars, fatty acyl, glycerolipid, glycerophospholipid, sphinglipid, sterol lipid, prenol lipid, saccarolipid, polyketide, glucosamines, lipopolysaccarides, aminopolysaccarides, polyglutamic acids, poly(allylamines), polyethylene glycol (PEG), PEG derivatives, methoxy polyethylene glycol (mPEG),

polypropylene glycol (PPG), poly(lactic acid), poly(glycolic acid), poly(ethylene-co-vinyl acetate) (EVAc), N-(2- hydroxypropyl)methacrylamides (HPMA), HPMA derivatives, poly(hydroxyalkanoates), poly(2-dimethylamino)ethyl methacrylate (DMAEMA), poly(D,L lactic-co-glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), PLGA derivatives, poly(polypropylacrylic acid) (PPAA), poly(D,L-lactide)-block- methoxypolyethylene glycol (diblock), poly(ethyleneimine), poly(beta-aminoester), polyvinyl alcohol, poly(hydroxyethyl methacrylate), polyacrylamide, polyacrylic acid, polyethyloxazole, polyvinyl pyrrolidinone, and polysaccharides such as dextran, chitosan, alginates, hyaluronic acid, and any ratio of copolymers, grafted polymers, and grafted copolymers thereof, wherein said linking moiety further comprises at least two terminal reactive groups corresponding and reactive with two or more functional groups selected from the group consisting of: OH, -COOH, N-hydroxy succidimidyl ester (NHS), Imidazole amide, triazole amide, tetrazole amide, hydroxy

benzotriazole ester (HOBt), l-hydroxy-7-azabenzotriazole ester (HOAt), 2,4-dinitrophenyl ester, pentafluorophenyl ester, 2,2,2-trifluoroethyl ester, 2,2,2-trifluoroethyl thioester, acid chloride, acid bromide, 4-nitrophenyl carbonate (NPC), isocyanate, optionally substituted aldehyde, optionally substituted ketone, optionally substituted acrylate, maleimide, vinyl sulfone, and/or orthopyridyl disulfide.

Compositions provided herein can include one or more thymic function modulators, e.g., described herein, with one or more other therapy/agent. For example, compositions can include one or more of: a thymus homeostasis factor, a T-cell growth factor, T-cell growth factor receptor, T-cell migration factor, TEC function factor, TEC growth factor, TEC growth factor receptor, TEC proliferation factor, T-cell activity factor listed in Table 1, optionally in combination with one or more other therapy/agent.

In embodiments, the combination of thymic function modulators and/or other

therapies/agents can be formulated together or separately.

In addition, methods described herein can include administering more than one thymic function modulator, e.g., thymic function modulator described herein. Methods described herein can also include administering a thymic function modulator (e.g., described herein) in

combination with a different therapy or agent.

Other therapies or agents suitable for use in combination with a thymic function modulator described herein include but are not limited to a surgery, radiation, antimicrobial, or chemotherapy.

Exemplary antimicrobials include antibacterial, antiviral, antifungal, antimycobacterial, antihelmintic and/or antiprotozoan pharmaceutical compositions. In embodiments, antimicrobial compositions include Penicillins, Macrolides, Ketolides, Cephalosporins, and/or Streptomycin. In embodiments, an antimicrobial drug is selected from the group consisting of penicillin, amoxicillin, oxacillin, dicloxoacilline, clavulinic acid with a penicillin, bicillin, ticarcillin, piperacillin, taxobactam, cephalexin, cefazolin, cephaclor, ceftibuten, cefuroxime, cefprozil, cefotaxime, ceftazidime, cefepime, cifdinir, ceftriaxone, cefditoren, cefpodoxime, aztreonam, ertapenem, cefoxitin, meropenem, imipenem, erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, daptomycin, cycloserine, quinupristin, dalfopristin, streptomycin, vancomycin, linezolid and combinations thereof. In embodiments, an antimicrobial composition comprises penicillin, amoxicillin, oxacillin, dicloxoacilline, clavulinic acid with a penicillin, bicillin, ticarcillin, piperacillin, taxobactam, cephalexin, cefazolin, cephaclor, ceftibuten, cefuroxime, cefprozil, cefotaxime, ceftazidime, cefepime, cifdinir, ceftriaxone, cefditoren, cefpodoxime, aztreonam, ertapenem, cefoxitin, meropenem, imipenem, erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, daptomycin, cycloserine, quinupristin, dalfopristin, streptomycin, vancomycin, linezolid, albendazole and mebendazole. In

embodiments, exemplary antimicrobial compositions are described in Table 3.

Table 3: Antimicrobial compositions

A. Antibacterial Drugs [Penicillins, e.g., benzylpenicillin, amoxicillin, ticarcillin,

Ipiperacillin; Cephalosporins, e.g., cefpodoxime, cefuroxime, cefazolin,

|cefalor, ceftibuten, cefprozil, cefotaxime, ceftazidime, dephaloexin, cefepime, cefdinir, ceftriaxone, cefditoren; Macrolides, e.g.,

erythromycin, clarithromycin, spiramycin, roxithromycin, azithromycin; Carbapenems, e.g., imipenen, meropenem; β-lactams, e.g., meropenem, Monobactams (e.g., aztreonam), ertapenem, cefoxitin, imipenem;

Ketolides, e.g., telithromycin; Glycopeptides, e.g., vancomycin;

Lincosamides, e.g., clindamycin, lincomycin; Cyclic lipopeptide antibacterial agents, e.g., daptomycin; Streptogamins, e.g.,

quinupristin, dalfopristin; Tetracyclines, e.g., doxycycline,

minocycline, tigecycline; Diarylquinoline; Oxazolidinones, e.g., linezolid.

B. Antimycobacterial Drugs

Rifampin, Rifabutin, Cycloserine, Isoniazid, Ethambutol,

Pyrazinamide.

C. Antiviral Drugs

Cidefovir, Foscarnet, Ganciclovir, Valganciclovir, Formivirisen,

Zidovudine, Zalcitabine, Didanosine, Stavudine, Lanivudine, Tenovir, Emtricitabine, Nevirapine.

D. Antifungal Drugs

Fluconazole, Voriconazole, Itraconazole, Caspofungin,

Clotrimazole, Amphotericin B, Micafungin, Terbinafine, Naftifine, Natamycin, Butenafine, Amorolfine, Ravuconazole, Posaconazole, Flucytosine, Econazole, Enilaconazole, Miconazole, Oxiconazole, Saperconazole, Sulconazole, Terconazole, Tioconazole, Nikkomycin Z, Anidulafungin (LY303366), Nystatin, Pimaricin, Griseofulvin, Ciclopirox, Haloprogin, Tolnaftate, Undecylenate.

E. Anthelmintic Drugs

Mebendazole, Niclosamide, Praziquantel, Pyrantel, Thiabendazole, Albendazole, diethyl carbamazine, Ivermectin, Benzimidazole,

Praziquantal/B enzimidazole combination .

F. Antiprotozoan Drugs

[Pyrimethamine, Sulfadiazine, Clindamycin, Mebendazole,

[Thiabendazole, Chloroquine Exemplary chemotherapies include but are not limited to cisplatin, vinorelbine, etoposide, vinblastine, gemcitabine, docetaxel, pemetrexed, carboplatin, paclitaxel, albumin- bound paclitaxel, irinotecan, mitomycin, ifosfamide, erlotinib, bevacizumab, cetuximab, crizotinib, afatinib, ceritinib, ramucirumab, nivolumab, ceritinib, ramucirumab, topotecan, temozolomide, cyclophosphamide, doxorubicin, vincristine, or any combination thereof, e.g., for use in treating or in a subject having a cancer, e.g., lung cancer, e.g,. non-small cell lung cancer (NSCLC). In embodiments, a chemotherapy includes but is not limited to doxorubicin, cyclophosphamide, paclitaxel, doxetaxel, 5-fluorouracil, epirubicin, methotrexate, trastuzumab, pertuzumab, carboplatin, fluorouracil, or any combination thereof, e.g., for use in treating or in a subject having a cancer, e.g., a breast cancer. In embodiments, a chemotherapy includes but is not limited to cyclophosphamide, etoposide, busulfan, fludarabine, carmustine, etoposide, cytosine arabinoside, melphalan, thiotepa, anti-thymocyte globulin, or any combination thereof, e.g., for use in treating or in a subject having a hematological cancer, e.g., a lymphoma or leukemia, e.g., non-Hodgkin or Hodgkin lymphoma or in a subject who has had, is undergoing, or will undergo a cell transplant, e.g., hematopoietic cell transplant. In embodiments, one or more of chemotherapy, e.g., described herein, is combined with irradiation (e.g., total body irradiation or total lymphoid irradiation). Additional chemotherapeutic agents are described below.

In embodiments, the two or more treatments in the combination therapy are administered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has otherwise ceased. In some embodiments, the administration of the combination of treatments is simultaneous or concurrent, e.g., delivery of the second treatment begins while the delivery of one treatment is still ongoing, such that there is an overlap in administration. In other embodiments, the administration of the combination of treatments is sequential, e.g., delivery of a second treatment begins after delivery of one treatment ceases.

The two or more treatments (e.g., thymic function modulators and/or other

agents/therapies) can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, one thymic function modulator described herein can be administered first, and a different thymic function modulator can be administered second, or the order of administration can be reversed. In other embodiments, for sequential administration, one thymic function modulator described herein can be administered first, and a different therapy/agent can be administered second, or the order of administration can be reversed.

In embodiments, treatment can be more effective due to the combination of treatments. In embodiments, a second treatment is more effective when administered in combination with the first treatment than in the absence of the first treatment. For example, an equivalent effect is seen with a lower dose of the second treatment, or the second treatment reduces a symptom to a greater degree, than if the second treatment were administered in the absence of the first treatment. In other examples, the combination of treatment reduces a symptom to a greater degree than either treatment delivered in the absence of the other. The effect of the multiple (e.g., two or more, e.g., three, four, five or more) treatments can be partially additive, wholly additive, or greater than additive. In some cases, the effect of the combination is synergistic.

Methods of treatment/use

Provided herein are compositions and methods for treating a thymus injury, e.g., acute or chronic thymus injury, e.g., using a thymus function modulator, e.g., described herein, e.g., described in Table 1. In embodiments, a thymus injury comprises a drug-induced thymus injury or a radiotherapy induced thymus injury. In embodiments, a thymus injury includes a wounded thymus. For example, the drug can be a cytoreductive drug or a chemotherapeutic drug, e.g., alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methyl ame famines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); deIta-9-tetrahydrocannabinoI (dronabinol,

MARINQL®); beta-lapachone; iapachol: colchicines; betulinic acid; a camptothecin (including the synthetic analogue topolecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; caliystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin;

podophyllinic acid: teniposide; cryptoph eins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1);

eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as

chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfanide, mechlorethamine, mechlorethamine oxide hydrochloride, melphaian, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitros ureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aciacinomysins, actinomycin, authramycin, azaserine, bleomycins,

cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including

ADRIAMYCIN®, morpholino-doxorubicin, cyanomoipholino-doxorubicin, 2-pyrroIino- doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorabicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drornostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucii; bisantrene; edatraxate; defofamine; deraecolcine; diaziquone; elfomiithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarabicin; losoxantrone; 2-ethyihydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;

raannoraustine; mitobronitol; mitolactoi; pipobroman; gacytosine; arabinoside ("Ara-C");

thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine;

mercaptopurine; methotrexate; platinum analogs such as cisplatin and carbopladn; vinblastine (VELBAN®); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine

(ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; cyclosporine, sirolimus, rapamycin, ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin

(ELOXATIN™) combined with 5-FU, leucovovin; anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (FARESTON®); anti-progesterones; estrogen receptor down- regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX®); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and triptereiin; other anti-androgens such as flutamide, nilutaniide and bicaiutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles,

aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate

(FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate

(ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g.,

LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-lH-pyrazol-l- yl)benzenesulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Exemplary chemo therapeutics are also described at https://www.navigatingcancer.com/library/all/chemotherapy_dr ugs (accessed as of June 9, 2016), incorporated herein by reference.

In embodiments, a thymus injury can be acute, e.g., transient during treatment or infection or a physiological state (e.g., with a drug,radiation, Trypanosoma cruzi infection, pregnancy infection, pregnancy). For example, an acute thymus injury subsides (e.g., symptoms of thymus injury decrease in number and/or degree) within 1 year or less (e.g., 1 year, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day or less) after cessation of administration of a treatment (e.g., drug or radiation) or after resolution of an infection (e.g., Trypanosoma cruzi infection) or a physiological state (e.g., pregnancy). In other embodiments, a thymus injury can be chronic. For example, in a chronic thymus injury, symptoms and/or degree of symptoms persist for a period of time, e.g., at least 2 weeks, e.g., at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or more.

Also provided herein are compositions and methods for replacing thymic function. For example, a thymic function modulator, e.g., described herein, is administered to a subject in combination with a thymic transplant. In embodiments, the combination of the thymic function modulator(s) with the thymic transplant increases the function/activity/lifetime of the

transplanted thymus compared to the level of function/activity/lifetime that would be seen if the thymic transplant occurred in the absence of a thymic function modulator. Thymic

function/activity can be assessed using methods described herein, e.g., in the "Measurement of thymus function" section. Exemplary dosing of the thymic function modulator in combination with a thymic transplant is described in greater detail in the Pharmaceutical compositions, administration, dosing section herein.

Also provided herein are compositions and methods for treating physiological senescence, e.g., reversing or delaying physiological senescence. The physiological senescence can be associated with muscle atrophy or degeneration, decrease in bone health (e.g., bone strength and/or density), immunosenescence (e.g., decrease in immune responsiveness), cardiac disease, or uncontrolled cell growth (e.g., tumorigenesis). In embodiments, treating physiological senescence includes rejuvenating bone, muscle, and/or heart tissue. In embodiments, senescence is characterized by a deterioration of one or more structures and/or functions of a cell, tissue, organ, or system of an organism. In embodiments, senescence can be due to aging (e.g., aging-associated senescence). In other embodiments, senescence can be non- aging associated, e.g., can be due to factors or processes other than aging/passage of time, e.g., can be therapy-induced (e.g., drug-induced, surgery-induced, and/or radiation-induced), disease- induced, and/or injury-induced. Immunosenescence is a type of senescence characterized by deterioration of the immune system, e.g., decreased immune responsiveness. Immunosenescence can be aging-associated or non-aging associated. Disease of aging can include those associated with immune aging (e.g., immunosenescence) or those not associated (directly associated) with immune aging. Accordingly, provided herein are compositions and methods for treating diseases of aging (e.g., osteoporosis, sarcopenia, amyotrophic lateral sclerosis (ALS) (e.g., sporadic or familial ALS), obesity, or diabetes). Also provided herein are compositions and methods for treating diseases of aging, e.g., immune aging (e.g., immunosenescence). For example, provided herein is a method of reducing physiological senescence, e.g., reducing aging, e.g., rejuvenating bone and/or muscle, by administering a thymic function modulator, e.g., described herein. The reduction in physiological senescence/aging is at least 10%, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, or more, compared to the extent of senescence/aging in a subject that has not been administered the thymic function modulator or in the subject prior to administration with the thymic function modulator. Methods of quantifying senescence/aging include methods of assessing presence/progression of a condition/disease such as sarcopenia, osteoporosis, T-cell contraction, poor health/cardiac output, and/or reduced insulin sensitivity.

For example, the presence of or progression of osteoporosis can be detected by measuring bone density, e.g., by radiological assessments, and/or bone turnover markers. For example, bone density can be measured using dual-energy X-ray absorptiometry (DXA). For example, bone density can be measured using peripheral DXA (peripheral DXA) (which can measure bone mass at the forearm, finger, and heel), single-energy X-ray absorptiometry (SXA) (which can measure the heel or wrist), dual photon absorptiometry (DPA) (which can measure the spine, hip, or total body, single photon absorptiometry (SPA) (which can measure the wrist), quantitative computed tomography (QCT) (which can measure the spine or hip), peripheral QCT (PQCT) (which can measure the forearm), and/or quantitative ultrasound (QUS) (which can measure the heel or finger). The World Health Organization has defined threshold values for osteoporosis, where the reference measurement is derived from bone density measurements in a population of healthy young adults (T- score). Normal hip bone density corresponds to a T- score of -1 or above; osteopenia corresponds to a T-score lower than -1 and greater than -2.5; osteoporosis corresponds to a T-score of -2.5 or lower; and severe osteoporosis corresponds to a T-score of - 2.5 or lower and presence of at least one fragility fracture. See, e.g., World Health Organisation. Assessment of fracture risk and its implication to screening for postmenopausal osteoporosis: Technical report series 843. Geneva: WHO, 1994; and Kanis J. Diagnosis of osteoporosis and assessment of fracture risk. Lancet 2002;359: 1929-36.

In some examples, sarcopenia can be diagnosed using DXA and/or measurement of gait speed. For example, sarcopenia is characterized by low muscle mass and low muscle strength and/or low physical performance. Muscle mass can be measured using computed tomography (CT), magnetic resonance imaging (MRI), DXA, bio-electrical impedance analysis (BIA), determination of total or partial body potassium per fat-free soft tissue, and/or anthropmetry. Muscle strength can be determined by handgrip strength, knee flexion/extension, and/or peak expiratory flow. Physical performance can be determined by assessment of short physical performance battery (SPPB), usual gait speed, timed get-up-and-go test, cardiac stress test, and/or stair climb power test. See, e.g., Muscaritoli M, Anker S Argiles J, et al (2010) Consensus definition of sarcopenia, cachexia and pre-cachexia: Joint document elaborated by Special Interest Groups (SIG). "Cachexia-anorexia in chronic wasting diseases" and "nutrition in geriatrics". Clinical Nutrition 29(2): 154-9; Sarcopenia: European consensus on definition and diagnosis, Report of the European Working Group on Sarcopenia in Older People," Age and Ageing Advance Access originally published online on April 13, 2010, Age and Ageing 2010 39(4):412-423; and/or Mithal A, Bonjour J-P, Boonen S, Burckhardt P, Degens H, El Hajj Fuleihan G, Josse R, Lips P, Morales Torres J, Rizzoli R, Yoshimura N, Wahl D.A., Cooper C, Dawson-Hughes B(2011) Impact of nutrition on muscle strength and performance in older adults. Osteoporosis International.

Insulin response/sensitivity and/or diabetes (e.g., Type I or Type II diabetes) can be determined by measuring fasting and 2-hour plasma glucose (both after 75 g oral tolerance test), AIC levels, islet autoantibody levels, insulin autoantibody levels, autoantibodies to GAD65, autoantibodies to tyrosine phosphatases IA-2/IA-2beta, and/or autoantibodies to zinc transporter 8 (ZnT8). See, e.g., Diabetes Care 2015;38(Suppl. 1):S8-S 16.

Obesity can be detected by basal/resting metabolic rate tests.

T cell contraction can be detected by TCR diversity analysis, e.g., spectratyping, TCR- Sequencing, and/or TREC analysis, e.g., described in greater detail herein.

ALS can be diagnosed by using electromyography (EMG) (which detects electrical activity in muscles), measuring nerve conduction velocity (NCV) (e.g., which can distinguish peripheral neuropathy or myopathy from ALS), and/or MRI (e.g., of the brain and spinal cord) (e.g., to rule out problems other than ALS, such as a spinal cord tumor, herniated disk in the neck, syringomyelia, or cervical spondylosis). In some examples, ALS is characterized by degeneration of lower motor neurons (in the spinal cord and brainstem), degeneration of upper motor neurons (in the brain), progressive spread of signs within a region to other regions, and the absence of evidence of other diseases that may explain the observed clinical/electrophysiological signs.

In other examples, provided herein is a method of treating diseases associated with aging or physiological senescence (e.g., osteoporosis, sarcopenia, ALS (e.g., sporadic or familial ALS), obesity, or diabetes), by administering a thymic function modulator, e.g., described herein. In embodiments, treatment results in the amelioration or disappearance of one or more symptoms of the disease.

Provided herein are compositions and methods for reducing or preventing thymic involution (e.g., thymic involution from aging, from therapies, or from diseases). In

embodiments, methods described herein include administering a thymic function modulator, e.g., described herein, to reduce or prevent thymic involution. The extent of thymic involution, e.g., before, during, and/or after treatment, can be assessed by determining thymic function, e.g., using methods described herein.

Also provided herein are compositions and methods for increasing an immune response. In embodiments, methods described herein include administering a thymic function modulator, e.g., described herein, to increase an immune response in a subject. For example, the immune response is an immune response to a cancer, a vaccine response, or an immune response to an infection, e.g., chronic infection. Exemplary cancers include hematological cancers or solid cancers. Exemplary solid cancers include uterine cancer, colon cancer, ovarian cancer, rectal cancer, skin cancer, stomach cancer, lung cancer, non-small cell carcinoma of the lung, breast cancer, cancer of the small intestine, testicular cancer, cancer of the anal region, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, rectal cancer, renal-cell carcinoma, liver cancer, cancer of the esophagus, melanoma, cutaneous or intraocular malignant melanoma, uterine cancer, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, cancer of the adrenal gland, bone cancer, pancreatic cancer, cancer of the head or neck, epidermoid cancer, carcinoma of the endometrium, carcinoma of the vagina, cervical cancer, sarcoma, uterine cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, carcinoma of the cervix squamous cell cancer, carcinoma of the fallopian tubes, sarcoma of soft tissue, cancer of the urethra, carcinoma of the vulva, cancer of the kidney or ureter, carcinoma of the renal pelvis, spinal axis tumor, cancer of the penis, cancer of the bladder, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, metastatic lesions of said cancers, and/or combinations thereof. In embodiments, the cancer is a hematological cancer, e.g., a leukemia or lymphoma. Exemplary hematological cancers include acute lymphoid leukemia (ALL); one or more chronic leukemias including, e.g., chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL), B cell

prolymphocytic leukemia; B-cell acute Lymphoid Leukemia ("BALL"); T-cell acute Lymphoid Leukemia ("TALL"); blastic plasmacytoid dendritic cell neoplasm; Follicular lymphoma; diffuse large B cell lymphoma; non-Hodgkin lymphoma; Hodgkin lymphoma; Burkitt's lymphoma; malignant lymphoproliferative conditions; Hairy cell leukemia; small cell- or large cell-follicular lymphoma; mantle cell lymphoma; MALT lymphoma; marginal zone lymphoma; multiple myeloma; myelodysplasia; myelodysplastic syndrome; Waldenstrom macroglobulinemia;

plasmablastic lymphoma; and plasmacytoid dendritic cell neoplasm.

Vaccines include vaccines against an infection or cancer vaccines. For example, a vaccine comprises an antigen of interest and an adjuvant and/or matrix, e.g., carrier protein matrix, e.g., as described in US 8642042, or US 8,980,288 incorporated herein by reference. In embodiments, the antigen is derived from a pathogen, e.g., a pathogen described herein. In embodiments, the antigen is derived from a cancer, e.g., a cancer described herein. In embodiments, the vaccine comprises an attenuated microorganism, e.g., expressing an immunogenic peptide. In some examples, cancer vaccines can include antigen presenting cells (APCs) loaded with one or more peptide epitopes from a cancer antigen, e.g., mesothelin, NY- ESO-1, Folate Binding Protein, HER2/neu, IL-13Ra2, MAGE-A1, and/or EphA2. Exemplary cancer vaccines are described in WO 2014127296 Al, US 20160114018, US 20160114017 , US 20160101169, US 20160058854 , US 20160030536 , US 20160022792 , US 20160015796, US 20150359867, US 20150306197, US 20150297698, US 20150297696, US 20150273034, US 20150258185, US 20150250864, US 20150202273, incorporated herein by reference. Infections can be acute or chronic. Infections can include bacterial infections or viral infections.

In embodiments, persistent infection is caused by a pathogen from one of the 3 major categories:

i) viruses, including the members of the Retroviridae family such as the lentiviruses (e.g. Human immunodeficiency virus (HIV) and deltaretroviruses (e.g., human T cell leukemia virus I (HTLV-I), human T cell leukemia virus II (HTLV-II)); Hepadnaviridae family (e.g. hepatitis B virus (HBV)), Flaviviridae family (e.g. hepatitis C virus (HCV)), Adenoviridae family (e.g. Human Adenovirus), Herpesviridae family (e.g. Human cytomegalovirus (HCMV), , Epstein- Barr virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), varicella- zoster virus), PapiUomaviridae family (e.g. Human Papillomavirus (HPV)), Parvoviridae family (e.g. Parvovirus B 19), Polyomaviridae family (e.g. JC virus and BK virus), Paramyxoviridae family (e.g. Measles virus), Togaviridae family (e.g. Rubella virus) as well as other viruses such as hepatitis D virus;

ii) bacteria, such as those from the following families: Salmonella (e.g. S. enterica Typhi), Mycobacterium (e.g. M. tuberculosis and M. leprae), Yersinia (Y. pestis), Neisseria (e.g. N. meningitides, N. gonorrhea), Burkholderia (e.g. B. pseudomallei), Brucella, Chlamydia, Helicobacter, Treponema, Borrelia and Pseudomonas; and

iii) parasites, such as Leishmania, Toxoplasma, Trypanosoma, Plasmodium, Schistosoma, or Encephalitozoon.

In embodiments, a thymic function modulator (e.g., an RNA based thymic function modulator) is administered in combination with a cancer immunotherapy. Without wishing to be bound by theory, it is believed that such a combination increases adaptive immunity in the subject, as RNAs induce an innate immunity response. In embodiments, such a combination is synergistic, e.g., in terms of the anti-cancer immune response elicited, compared to either the thymic function modulator or the cancer immunotherapy alone. Exemplary cancer

immunotherapies include cell-based therapies (e.g., chimeric antigen receptor (CAR)-expressing immune cells), antibody therapies (e.g., cytotoxic or radioactive antibodies, cytokine therapies (e.g., interferons or interleukins), and immune checkpoint inhibitors.

Also provided herein are compositions and methods for decreasing an immune response, e.g., to prevent rejection of a transplant, or to induce negative selection, e.g., to treat an autoimmune disease. In embodiments, methods described herein comprise administering a thymic function modulator, e.g., described herein. Transplants can include organ transplants, e.g., lung, heart, eye, liver, or kidney transplant. Exemplary autoimmune diseases include but are not limited to rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, primary biliary cirrhosis, celiac disease, psoriatic arthritis, ankylosing spondylitis, Guillain-Barre syndrome, ALS, and pernicious anemia.

Subject characteristics

In accordance with any method described herein, a subject comprises a mammal, e.g., a human (e.g., a male or female of any age group, e.g., an adult subject (e.g., young adult, middle- aged adult, or senior adult) or pediatric subject (e.g., infant, child, or adolescent); primate (e.g., cynomolgus monkey or rhesus monkey); cattle; pig; horse; sheep; goat; cat; or dogs.

In embodiments, the subject is an adult subject, e.g., a subject at least 18 years or older, e.g., 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 years or older. In embodiments, the subject is a geriatric subject, e.g., older than 60, 65, 70, 75, 80, 85, or 90 years of age. In embodiments, the subject is a pediatric subject, e.g., younger than 18 years, e.g., younger than 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, or younger.

In embodiments, the subject has an implanted thymus.

In embodiments, the subject has a cancer, e.g., a hematological cancers or solid cancer, such as lung cancer, non-small cell lung cancer (NSCLC), skin cancer, melanoma, cervical cancer, uterine cancer, ovarian cancer, breast cancer, pancreatic cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, sarcoma, lymphoma, and brain cancer. In embodiments, the cancer is a solid cancer. Exemplary solid cancers include breast cancer, uterine cancer, colon cancer, ovarian cancer, rectal cancer, skin cancer, stomach cancer, non-small cell carcinoma of the lung (NSCLC), cancer of the small intestine, testicular cancer, cancer of the anal region, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, rectal cancer, renal-cell carcinoma, liver cancer, cancer of the esophagus, melanoma, cutaneous or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, cancer of the adrenal gland, bone cancer, pancreatic cancer, cancer of the head or neck, epidermoid cancer, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the cervix squamous cell cancer, carcinoma of the fallopian tubes, sarcoma of soft tissue, cancer of the urethra, carcinoma of the vulva, cancer of the kidney or ureter, carcinoma of the renal pelvis, spinal axis tumor, cancer of the penis, cancer of the bladder, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, metastatic lesions of said cancers, and/or combinations thereof. In embodiments, the cancer is a hematological cancer, e.g., a leukemia or lymphoma. Exemplary hematological cancers include acute lymphoid leukemia (ALL); one or more chronic leukemias including, e.g., chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL), B cell prolymphocytic leukemia; B-cell acute Lymphoid Leukemia ("BALL"); T-cell acute Lymphoid Leukemia ("TALL"); blastic plasmacytoid dendritic cell neoplasm; Follicular lymphoma; diffuse large B cell lymphoma; non- Hodgkin lymphoma; Hodgkin lymphoma; Burkitt's lymphoma; malignant lymphoproliferative conditions; Hairy cell leukemia; small cell- or large cell-follicular lymphoma; mantle cell lymphoma; MALT lymphoma; marginal zone lymphoma; multiple myeloma; myelodysplasia; myelodysplastic syndrome; Waldenstrom macroglobulinemia; plasmablastic lymphoma; and plasmacytoid dendritic cell neoplasm.

In embodiments, the subject has an infection, e.g., a chronic infection or acute infection. In embodiments, the infection is bacterial or viral.

In embodiments, the subject has an autoimmune disease, e.g., rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, primary biliary cirrhosis, celiac disease, psoriatic arthritis, ankylosing spondylitis, Guillain-Barre syndrome, ALS, and/or pernicious anemia.

Pharmaceutical compositions, administration, dosing

Provided herein are pharmaceutical compositions comprising one or more thymic function modulators, e.g., described herein. Pharmaceutical compositions can comprise a thymic function modulator combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Pharmaceutical compositions can comprise a buffer, e.g., phosphate buffered saline or neutral buffered saline; proteins; polypeptides/amino acids, such as glycine; pantioxidants; chelating agents, e.g., EDTA or glutathione; carbohydrates, e.g., glucose, sucrose, mannose, dextrans, or mannitol; adjuvants such as aluminum hydroxide; and

preservatives.

Compositions described herein can be formulated for intravenous, oral, or subcutaneous administration. The administration of the compositions described herein can be carried out in manners such as by ingestion, transfusion, aerosol inhalation, injection, implantation, or transplantation. In embodiments, the compositions described herein can be administered to a subject orally or parenterally (e.g., intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracisternally, intratumorally, intravasally, intradermally). In embodiments, the compositions described herein can be infused or injected into the subject. In embodiments, the compositions can be administered to the site of a target tissue, for example, intravenously or intra-arterially into a blood vessel supplying a tumor. The compositions described herein can be administered directly into the thymus, lymph node, tumor, or site of infection.

The dose and dosing frequency can be determined by factors such as the condition of the patient and the type and severity of the patient's disease. In embodiments, a thymic function modulator is dosed chronically, e.g., for an extended or indefinite period of time, e.g., at least 2 weeks, e.g., at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or more. In embodiments, a thymic function modulator is dosed transiently, e.g., for a period of time no longer than 1 year, e.g., 1 year, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or less. For example, a thymic function modulator is dosed until a thymic function improves and/or a symptom of a disease described herein subsides.

Without wishing to be bound by theory, it is believed that a thymic function modulator (e.g., described herein) can boost or maintain function of a transplanted thymus in a subject. Accordingly, methods described herein include dosing (e.g., long-term dosing) of a thymic function modulator (e.g., described herein) in a subject that has undergone, will be undergoing, or is undergoing a thymic (e.g., thymic cell and/or tissue) transplant). In embodiments, the thymic function modulator (e.g., described herein) is administered for an extended or indefinite period of time, e.g., at least 2 weeks, e.g., at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or more. In embodiments, administration of the thymic function modulator (e.g., described herein) commences before or after, or simultaneously with transplantation of the thymus cell/tissue. In embodiments, administration of the thymic function modulator (e.g., described herein) commences before, e.g., at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or more) before, the transplantation of the thymus cell/tissue. In

embodiments, administration of the thymic function modulator (e.g., described herein) commences after e.g., at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or more) after, the transplantation of the thymus cell/tissue. In embodiments,

administration of the thymic function modulator (e.g., described herein) commences within 5 months, e.g., within 5 months, 4 months, 3 months, 2 months, 1 month, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 24 h, 12 h, 6 h 3 h 1 h or less, of the transplantation of the thymus cell/tissue.

In embodiments, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant. Exemplary contaminants include an endotoxin, replication competent lentivirus (RCL), VSV-G nucleic acid, HIV gag, p24, mycoplasma, mouse antibodies, bovine serum albumin, bovine serum, pooled human serum, culture media components, a bacterium, vector packaging cell or plasmid components, and a fungus.

An effective amount of a thymic function modulator can be determined by a physician with consideration of individual differences in disease, weight, age, extent of

degeneration/infection/tumor growth/metastasis/senescence/autoimmunity/thymic injury, and condition of the subject.

In some embodiments, the subject is assessed for the level of the thymic function modulator or other agent at periodic times during the treatment regimen, e.g., to ensure that the thymic function modulator or other agent is present at a threshold level over the course of the therapy. In one example, the agent is administered in a controlled release formulation. The overall period of time over which a particular treatment regimen is followed by a subject may vary depending on the response and health of the subject but typically is at least one month, 6 weeks, 2 months, 3 months, 6 months, 9 months, a year, 18 months, 2 years or more.

In certain embodiments, the agents (e.g., thymic function modulator or other agent) described herein are administered in doses of 0.01-10 mg/kg, 0.05-5 mg/kg, 0.1-5 mg/kg, 0.2-5 mg/kg, 0.5-5 mg/kg, 0.5-1 mg/kg, 0.5-5 mg/kg, 0.5-10 mg/kg, 1-10 mg/kg, 1-5 mg/kg, or any combination thereof.

All references and publications cited herein are hereby incorporated by reference.

The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

Example 1; Treatment of complications associated with stem cell transplant using mRNA for KGF

In a clinical trial, patients undergoing hematopoietic stem cell transplantation are randomized into two groups, an experimental treatment group and a control group. The experimental treatment group receives an agent described herein, e.g., a synthetic KGF mRNA, e.g., the therapeutic regimen described below, whereas the control group receives standard-of- care. Synthetic mRNA for KGF is administered to experimental treatment group patients undergoing stem cell transplantation, where such cells are obtained from any source of hematopoietic cell lineage, including bone marrow, peripheral blood, or umbilical cord blood. Administration of KGF mRNA is systemic through intravenous injection. The schedule for dosing KGF mRNA is similar to the FDA approved schedule for administration of KGF protein.

Briefly, KGF mRNA is administered on days -3, -2, -1 prior to, and on days 0, +1, +2 after stem cell transplant. The recovery of patients' thymus size and function and lymphocyte subsets are analyzed monthly for the first three months after transplant followed by analyses every 3 months until one-year post transplant. In the experimental treatment group,

administration of KGF mRNA is expected to lead to increased intra-thymic levels of the protein, leading to increased signalling in TECs through KGF receptor FGFR2B. Such increased signalling is expected to lead to a smaller reduction in thymus size and density in KGF treated patients as compared to untreated controls, as well as a faster recovery of thymus size and function. Thymus size and density are measured through standard CT (computed tomography) or MRI (magnetic resonance imaging) scans. The thymus function and output in both experimental and control patients are determined by withdrawing peripheral blood from patients for quantification of clinical biomarkers, including TREC count, peripheral CD4 ⁺ and CD8 ⁺ T-cell numbers, and peripheral CD4 ⁺ and CD8 ⁺ naive T-cell numbers, using standard techniques. Naive T-cells are identified based on cell surface markers, including CD3, CD45RA, CD45RO, CCR7, CD31, CD62L, PTK7 and others.

As compared to the control group, patients treated with KGF are expected to show higher TREC counts, higher numbers of CD4 and CD8 naive T-cells, and higher total CD4 and CD8 T- cell numbers in peripheral blood. The T-cell receptor repertoire diversity, another measure of thymus output, in treated and untreated patient groups, is determined using standard sequencing- or PCR-based methods, e.g., as described in Woodworth et al. Genome Med. 5.10(2013):98. Patients treated with KGF are expected to show higher TCR repertoire diversity compared to the untreated group. It may additionally be expected that the patient group treated with KGF shows a reduction in one or more of: the number of post-transplant infections, duration of infections, and infection related morbidity and mortality, relative to the control group.

Example 2: Multiple drugs/spatially separated dosing

In this example, sequential dosing of different mRNA therapeutics during the stem cell transplant procedure is used for promoting protection of the thymus against the toxic effects of chemotherapy and radiation therapy prior to transplant, and to accelerate thymus function recovery post-transplant and reduce post-transplantation co-morbidities including infection. In a clinical trial, patients undergoing hematopoietic stem cell transplantation are randomized into two groups, an experimental treatment group and a control group. The treatment group receives the therapeutic regimen described below, whereas the control group receives standard-of-care.

The mRNA therapeutic administered to the experimental treatment group prior to transplant is mRNA for KGF. KGF mRNA is administered through intravenous injections on days -3, -2, and -1 prior to transplant. The mRNA therapeutic administered to the experimental treatment group post-transplant is mRNA for human growth hormone (somatropin). Growth hormone (GH) mRNA is subcutaneously administered daily for 3 months after transplant.

Thymus size and density are measured through standard CT (computed tomography) or MRI (magnetic resonance imaging) scans. The thymus function and output in patients treated with KGF & GH and the control group are determined by withdrawing peripheral blood from patients for quantification of clinical biomarkers, including TREC count, peripheral CD4 ⁺ and CD8 ⁺ T- cell numbers, and peripheral CD4 ⁺ and CD8 ⁺ naive T-cell numbers, using standard techniques. Naive T-cells are identified based on cell surface markers, including CD3, CD45RA, CD45RO, CCR7, CD31, CD62L, PTK7 and others.

Patients treated with KGF and GH are expected to show higher TREC counts, higher numbers of CD4 and CD8 naive T-cells, and higher total CD4 and CD8 T-cell numbers in peripheral blood. The T-cell receptor repertoire diversity, another measure of thymus output, in treated and untreated patient groups, is determined using standard sequencing- or PCR-based methods, e.g., as described in Woodworth et al. Genome Med. 5.10(2013):98. Patients treated with KGF and GH are expected to show higher TCR repertoire diversity compared to the untreated group. Additionally, the patient group treated with KGF and GH may show a reduction in one or more of: post-transplant infections, duration of infections, and infection related morbidity and mortality relative to the control group.

Example 3: Treatment of irradiation-induced lymphopenia in mice by increasing thymic T cell proliferation and survival

Cytoreductive conditioning regimens such as chemotherapy or irradiation that are used in the context of allogeneic bone marrow transplantation (BMT) elicit deficits in innate and adaptive immunity, which predispose patients to infections. As such, transplantation outcomes depend on the successful reconstruction of immune competence. Restoration of a normal peripheral T-cell pool after hematopoietic cell transplantation (HCT) is a slow process that requires de novo production of naive T cells in a functionally competent thymus.

To study reconstitution of the peripheral immune system after BMT, the well-described clinically relevant MHC-matched BMT model is used (Schroeder et al. Disease Models & Mechanisms 2011 4: 318-333). Lethally irradiated (9.5 Gy) C57BL/6 CD45.1 mice are injected intravenously with 1x10 T cell depleted bone marrow cells from C57BL/6 CD45.2 mice (Frasca et al. Bone Marrow Transplantation (2000) 25, 427-433).

Irradiated mice are injected subcutaneously, every other day for 10 days with IL-7, IL-21, and a combination thereof, at previously titrated concentration.

The kinetics of peripheral T cell reconstitution is measured by weekly flow cytometric analysis of blood cells. Tail blood samples are collected once a week starting on day 5 after BMT and are stained for CD3, CD4, CD8, CD31, CD45.1, CD45.2, TCRy5, FoxP3, CD56, CD 19, and a cell viability dye before acquisition on a flow cytometer.

Mice are euthanized at week 2, 4 or 6 post BMT for analyzing the number and

composition of thymic T cells, as well as the composition and function of peripheral immune cells. Thymi are removed, dissociated and stained for CD3, CD45.1, CD45.2, CD44, CD28, CD127, CD25, FoxP3, TCRb, CD8 and a viability dye. These markers allow the detection of newly produced donor versus host derived thymocytes at the different stages of differentiation. Cells from peripheral blood, lymph nodes and spleens are stained for CD3, CD4, CD8, CD31, CD45.1, CD45.2, TCRy5, FoxP3, CD56, CD19 and a viability dye, for assessing the amount of donor versus host derived T cells that have been newly exported from the thymus. Function of peripheral T cells is assessed by analyzing in vitro proliferation and cytokine production in response to polyclonal stimulation. For intracellular cytokine staining, splenocytes are isolated and stimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. Cells are stained for CD3, CD4, CD8, CD45.1 and CD45.2 and fixed using a fixation/permeabilization buffer (eBioscience) and processed according to manufacturer's suggestion (see eBioscience protocol B2 for intracellular staining) to determine the T cell composition in each spleen. After intracellular staining for the effector cytokines IL-17, IFNy and IL-2 that are a surrogate for T cell function, cells are acquired and analyzed by flow cytometer. Proliferation is assessed by staining T cells with a proliferation dye (CellTrace Violet) according to the manufacturer' s instructions. Labeled cells are cultured (100,000 cells/ 200 μΐ) in 96-well microtiter plates with coated CD3 antibody (1 μg/ml) and soluble CD28 antibody (1 μg/ml). Proliferation is measured by flow cytometry on day 3 after staining with antibodies for CD3, CD4, CD8, CD45.1, and CD45.2 and a viability dye.

A treatment with IL-7 and/or IL-21 accelerates the thymic production of T cells, as reflected by an increase of thymocytes at different stages of differentiation and an accelerated reconstitution of the peripheral T cell compartment that is reflected by a faster increase of functional T cells in the periphery (blood and lymphatic organs such as spleen and lymph nodes).

Example 4: Treatment of immunodeficient mice by enhancing thymic T cell development and function

Immunodeficient mice engrafted with human immune systems provide an exciting model to study human immunobiology in an in vivo setting without placing patients at risk. Co- implantation of human hematopoietic stem cells (HSCs) with autologous fetal liver and thymic tissues into immunodeficient mice create a humanized model with optimal human T cell development. Humanized mice are generated using human HSC derived from a number of sources, including umbilical cord blood, G-CSF mobilized peripheral blood, bone marrow and fetal liver. Co-implantation of human fetal thymic tissues with autologous fetal liver derived HSC provide human thymic epithelium that is essential for T cell education. See for example, Methods Mol Biol. 2014; 1185: 267-278.

To study reconstitution of the peripheral immune system after BMT, the model described herein is used. NOO-Prkdc ^scid IL2rg ^TmlWjl (NSG) mice between 8 to 12 weeks of age are irradiated with 200 cGy, 1mm pieces of fetal thymic and liver are inserted into the kidney capsule at the posterior lateral side with a scalpel, and 2xl0 ⁵ CD34+ fetal liver hematopoietic cells are injected intravenously, see, Methods Mol Biol. 2014; 1185: 267-278.

Mice are injected subcutaneously, every other day for 10 days with SCF alone, IL-7 alone, KGF alone, and a combination of SCF/IL-7/KGF, at previously titrated concentrations.

The kinetics of peripheral T cell reconstitution is measured by weekly flow cytometric analysis of blood cells. Tail blood samples are collected once a week starting on day 5 after BMT and are stained for CD3, CD4, CD8, CD31, CD45.1, CD45.2, TCRy5, FoxP3, CD56, CD 19, and a cell viability dye before acquisition on a flow cytometer.

Mice are euthanized at week 2, 4 or 6 post surgery and injection for analyzing the number and composition of transplanted T cells, as well as the composition and function of peripheral immune cells. Tissue transplants are removed, dissociated and stained for CD3, CD29 (HLA- A2), CD44, CD28, CD127, CD25, FoxP3, TCRb, CD8 and a viability dye. These markers allow the detection of newly produced human thymocytes at the different stages of differentiation. Cells from peripheral blood, lymph nodes and spleens are stained for CD3, CD4, CD8, CD31, CD29 (HLA-A2), TCRy5, FoxP3, CD56, CD19 and a viability dye, for assessing the amount of T cells. Function of peripheral T cells is assessed by analyzing in vitro proliferation and cytokine production in response to polyclonal stimulation. For intracellular cytokine staining, splenocytes are isolated and stimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. Cells are stained for CD3, CD4, CD8 and CD29 and fixed using a

fixation/permeabilization buffer (eBioscience) and processed according to manufacturer's suggestion (see eBioscience protocol B2 for intracellular staining) to determine the T cell composition in each tissue transplant. After intracellular staining for the effector cytokines IL- 17, IFNy and IL-2 that are a surrogate for T cell function, cells are acquired and analyzed by flow cytometer. Proliferation is assessed by staining T cells with a proliferation dye (CellTrace Violet) according to the manufacturer's instructions. Labeled cells are cultured (100,000 cells/ 200 μΐ) in 96-well microtiter plates with coated CD3 antibody (1 μg/ml) and soluble CD28 antibody (1 μg/ml). Proliferation is measured by flow cytometry on day 3 after staining with antibodies for CD3, CD4, CD8 and CD29 and a viability dye.

SCF augments the number of early stage CD4-CD8- thymocytes, while IL-7 increases the number of CD4-CD8-, CD4+CD8+, CD4+CD8- and CD8+CD4- thymocytes and accelerates the reconstitution of the peripheral T cell compartment that is reflected by a faster increase of functional T cells in the periphery (blood and lymphatic organs such as spleen and lymph nodes). KGF has a positive impact on the number of thymic epithelial cells (TECs) as well as the number of thymocytes and peripheral T cells.