CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Serial No. 63/197,188, filed on June 4, 2021, and of U.S. Patent Application Serial No. 63/210,709, filed on June 15, 2021. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. AG045779, awarded by National Institute of Aging. The government has certain rights in the invention.

SEQUENCE LISTING

This document includes a Sequence Listing that has been submitted electronically as an ASCII text file named 07039-2055W01_ST25.txt. The ASCII text file, created on May 19, 2022, is 6 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.

BACKGROUND

1. Technical Field

This document relates to methods and materials for treating cancer in a subject and improving efficacy of adoptive immune cell therapy by engineering the immune cells (e.g., chimeric antigen receptor T (CAR-T) cells or tumor-infiltrating lymphocytes) to have reduced expression of a VPS39 polypeptide and administering the engineered immune cells to a subject.

2. Background Information

Adoptive transfer of immune cells is a personalized form of cancer therapy. In adoptive transfer, T cells can be harvested from a patient, grown in vitro and selected, for example, for recognition of the tumor. The T cells also can be manipulated before cell transfer to enhance antitumor immunity. See, e.g., Rosenberg and Restifo, Science , 348(6230):62-68 (2015).

SUMMARY

This document is based, at least in part, on reducing expression of Vps39 in immune cells (e.g., chimeric antigen receptor T cells or tumor-infiltrating lymphocytes). Targeting VPS39 can be useful in clinical settings where adoptive T cell therapy is applied, such as treatment with CAR-T cells or tumor-infiltrating T cells.

In some embodiments, a method for treating cancer (e.g., melanoma) in a subject is provided. The method can include administering, to the subject, engineered immune cells, wherein the immune cells have reduced expression of a VPS39 polypeptide. The engineered immune cells can be chimeric antigen receptor T cells or tumor infiltrating lymphocytes.

This document also features a method of increasing efficacy of adoptive cell transfer in a subject. The method includes administering to the subject (e.g., a subject having cancer), chimeric antigen receptor T cells having reduced expression of a VPS39 polypeptide.

In another aspect, this document features an immune cell comprising an inactivated VPS39 gene. The immune cell can be a T cell, a chimeric antigen receptor T cell, or a tumor infiltrating lymphocyte.

This document also features an isolated immune cell that includes a disrupted nucleic acid encoding a VPS39 polypeptide, wherein the cell does not express an endogenous VPS39 polypeptide. The immune cell can be a T cell, a chimeric antigen receptor T cell, or a tumor infiltrating lymphocyte.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figures 1A- IK. Lysosome-independent activation of mTORCl in naive CD4 T cell responses. (Figure 1 A) Naive CD4 T cells were activated with anti-CD3/anti-CD28 beads for 5 days followed by DQ-BS A treatment for 6 hours. Fluorescence of cleaved DQ-BSA was analyzed by flow cytometry to determine lysosomal activities (left). Phospho-S6Kl (Thr389) protein expression was determined by Western blotting (right). Results are from ten young (Y, 20-35 years) and ten old (O, 65-85 years) healthy individuals. Intensities of p-S6Kl protein expression were normalized to b-actin and are shown relative to the mean from young individuals; comparison by two-tailed unpaired t- test. (Figure IB) Naive CD4 T cells from young individuals were activated for 5 days with the last 2 days in the presence of vehicle, chloroquine (CQ) or bafilomycin A1 (BafAl). Lysosomal activities (left) and mTORCl activities (right) were determined as in (Figure 1 A). (Figure 1C) Naive CD4 T cells from young individuals were transfected with control or TFEB siRNA and activated for 5 days. Lysosomal activities (left) and mTORCl activities (right) were determined. (Figure ID) Heat map showing expression differences of genes involved in the amino acid signaling arm of the mTORCl pathway, comparing the transcriptome of day 5-activated naive CD4 T cells from old and young adults (Accession number: SRA: SRP158502). (Figure IE) SLC7A5 and SLC7A1 transcripts from day 5-activated naive CD4 T cells from twelve 20-35 year-old and twelve 65-85 year-old healthy adults. Results are expressed relative to the mean from young individuals; comparison by two-tailed unpaired t-test. (Figure IF) Chromatin accessibility at SLC7A5 gene in human Epstein-Barr (EBV), varicella-zoster (VZV) and influenza (Flu) virus-specific CD4 T cells from young and old adults. Averaged tracks (young n=3, old n=4) at SLC7A5 show increased peak in highlighted (red) region. (Figure 1G) SLC7A5 and SLC7A1 transcripts in samples from (Figure IB) and (Figure 1C). (Figure 1H) SLC7A5 and c-MYC protein expression in samples from (Figure IB) and (Figure 1C). (Figure II) c-MYC protein expression in samples from (Figure 1 A). (Figure 1 J) GSEA comparing fold transcript differences in young compared with old naive CD4 T cells on day 5 after stimulation (Accession number: SRA: SRP158502) with that of “HALLMARK MY C TARGETS” . (Figure IK) SLC7A5 and c-MYC protein expression in day 5-stimulated naive CD4 T cells from three old individuals transfected with control or MYC siRNA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS, not significant.

Figures 2A - 2H. Naive CD4 T cells from old adults lose lysosomal function but maintain mTORCl activity after activation on day 5. (Figure 2A and Figure 2B) Naive CD4 T cells from young and old individuals were activated with anti-CD3/anti-CD28 beads for 5 days followed by DQBSA treatments for 6 hours. Fluorescence of cleaved DQ-BSA was analyzed by flow cytometry to determine lysosome activities (Figure 2A). Phospho-S6Kl (Thr389) protein expression (right) was determined by Western blotting (Figure 2B). (Figure 2C) Naive CD4 T cells were activated for 5 days in the presence of increasing doses of chloroquine (CQ), bafilomycin A1 (BafAl) or JPH203. The percentage of live cells was determined as the frequency of Annexin V negative population. Mean ± SD of four experiments. (Figures 2D-2G) Naive CD4 T cells were activated for 5 days with the last 2-day culture with vehicle, chloroquine (CQ) or bafilomycin A1 (BafAl). Alternatively, cells were transfected with control or TFEB siRNA and activated for 5 days. (Figure 2D) Lysosomal activities were determined as in (Figure 2 A). (Figure 2E) mTORCl activity was determined as in (Figure 2B). (Figure 2F) TFEB protein silencing were confirmed by Western blotting on day 5 after activation. (Figure 2G) AKT phosphorylation was determined by Western blotting on day 5 after activation. **p < 0.01; NS, not significant. (Figure 2H) Naive CD4 T cells from three old individuals were activated and treated with CQ as in (Figure 2D). SLC7A5 and c-MYC protein expression were determined. Comparison by two-tailed paired t test. *p < 0.05, **p < 0.01; NS, not significant.

Figures 3 A - 3H. SLC7A5-dependent mTORCl activation in naive CD4 T cell responses from old adults. (Figures 3 A and 3B) Longitudinal analysis of SLC7A5 gene (Figure 3 A) and protein (Figure 3B) expression in naive CD4 T cells from four young and four old individuals after stimulation. (Figure 3C) SLC7A5 protein expression in ex v/vo-sorted naive CD4 T cells and central memory (CM) CD4 T cells from four young and four old individuals. Statistical significance by two-tailed unpaired t test. (Figure 3D) Dose dependent analysis of mTORCl activities after JPH203 inhibition as shown by the phosphorylation of S6RP. Mean ± SD of four experiments. (Figures 3E and 3F) Naive CD4 T cells from young and old individuals were transfected with control or SLC7A5 siRNA and activated with anti-CD3/anti-CD28 beads for 3 or 5 days. Reduced SLC7A5 protein expression after silencing was confirmed on day 3 -stimulated cells from young and old individuals (Figure 3E). mTORCl activities were determined by flow cytometry- based analysis of intracellular p-S6RP (S235/S236) (Figure 3F). Comparison by two- tailed paired t test. (Figures 3G and 3H) Naive CD4 T cells from young individuals were transfected with control vector (pCMV6) or SLC7A5 vector (pSLC7A5, both Origene) and activated with anti-CD3/anti-CD28 beads for 5 days. SLC7A5 protein overexpression was confirmed on day 5 (Figure 3G). mTORCl activities were determined by flow cytometry-based analysis of intracellular p-S6RP (S235/S236) (Figure 3H). Comparison by two-tailed paired t test. *p < 0.05, **p < 0.01; NS, not significant.

Figures 4A- 4G. SLC7A5-dependent late endosomal mTORCl activation in naive CD4 T cell responses. (Figures 4Aand 4B) Naive CD4 T cells from young and old healthy individual were activated with anti-CD3/anti-CD28 beads for 3 days in the presence of vehicle or SLC7A5 inhibitor JPH203 (upper panel). Alternatively, cells were activated with anti-CD3/anti-CD28 beads for 5 days with the last 2 days in the presence of vehicle or JPH203 (lower panel). mTORCl activities were determined either by Western blotting of p-S6Kl (Figure 4A) or by flow cytometry of intracellular p-S6RP (S235/S236) (Figure 4B). Data are shown as one representative experiment (left) and cumulative data of three or four experiments (right). (Figure 4C) Naive CD4 T cells were activated for 3 days followed by treatment or not with an AKT inhibitor for 2 hours prior to harvesting. Endosomes were isolated and analyzed for in vitro mTORCl kinase activity toward S6K1. Total cell lysates (T) and endosome isolates (E) were analyzed by immunoblotting for indicated proteins. Data are shown as one representative of three experiments. (Figure 4D) In vitro mTORCl kinase activity of endosome isolates in day 5- stimulated naive CD4 T cells from one young and one old individual. Data are shown as one representative of three experiments. (Figure 4E) Cells were stained with anti-EEAl, anti-LAMPl and anti-mTOR. Confocal images representative of two independent experiments are shown. Scale bar, 5 pm. (Figures 4F and 4G) mTORCl activities in day 3- and day 5-stimulated naive CD4 T cells from young and old individuals after control or VPS39 silencing. Two-tailed paired t-test. The gray histogram represents isotype control. *p < 0.05, **p < 0.01; NS, not significant.

Figures 5 A - 5C. VPS39 silencing does not affect TGF-p/SMAD signaling in naive CD4 T cell responses. Naive CD4 T cells were transfected with control or VPS39 siRNAand activated for 3 days in the presence or absence of 10 ng/ml of TGF- bΐ. (Figure 5A) VPS39 protein expression after silencing. (Figure 5B) SMAD2 (pS465/pS467)/SMAD3 (sS423/pS425) were determined by flow-cytometry. Date are shown as frequencies of p-SMAD2/3 ⁺ cells and MFI of p-SMAD2/3 ⁺ . (Figure 5C) TGF- b/SMAD signaling activites were determined by an SMAD binding element (SBE) reporter assay. The normalized luciferase activity was obtained by subtracting background luminescence from the negative control followed by calculating the ratio of firefly luminescence from the SBE reporter to Renilla luminescence from the control Renilla luciferase vector. Mean ± SEM of three experiments. Comparison by two-tailed paired t test. NS, not significant.

Figures 6A- 6F. Sustained activation of late endosomal mTORCl suppresses lysosomal activities in naive CD4 T cell responses. (Figures 6A-6F) Naive CD4 T cells from young (Y) and old (O) individuals were activated for 5 days. Indicated inhibitor or vehicle control was added for the last 2 days of culture (Figures 6A-6B and Figures 6E- 6F). Alternatively, cells were transfected with control or VPS39 siRNAand then activated with anti-CD3/anti-CD28 beads for 5 days (Figures 6C-6D). (Figure 6A, Figure 6C, Figure 6E, and Figure 6F) Lysosomal cathepsins expressions were determined by qRT- PCR (left); results are normalized to control samples using 18S rRNA as internal control; mean ± SD of four experiments; comparison by two-tailed paired t-test. Lysosomal activities were determined by flow cytometry-based analysis of cells treated with 5 pg/mL of DQ-BSA for 6 hours. Results are shown as representative histograms (middle) and cumulative data from four experiments (right, two-tailed paired t-test). The gray histogram represents DQ-BSA free samples. (Figures 6B and 6D) Cells were treated with DQ-BSA (green) and stained with anti-pS6RP (red). Confocal images representative of two independent experiments show an inverse relationship between mTORCl and lysosomal activity. Scale bar, 20 pm. *p < 0.05, **p < 0.01; NS, not significant.

Figures 7A- 7C. Effects of mTORCl inhibition on TFEB expression and phosphorylation. (Figures 7Aand 7B) Naive CD4 T cells from three old individuals were activated for 3 days in the presence of vehicle or indicated inhibitor. (Figure 7C) Alternatively, cells were transfected with control or VPS39 siRNA and activatated for 3 days. pTFEB (S211), pAKT (S473), total FOX01 protein and total TFEB protein were determined byimmune blotting. TFEB transcripts were determined by quantitative RT- PCR. Comparison by two-tailed paired t test. *p < 0.05, **p < 0.01.

Figures 8A - 8J. Sustained activation of late endosomal mTORCl prevents PD-1 from lysosomal degradation. (Figures 8A-8D) Naive CD4 T cells from young and old individuals were activated with anti-CD3/anti-CD28 beads for 5 days with the last 2 days in the presence of vehicle or indicated inhibitor (Figures 8B and 8C). Alternatively, cells were transfected with control or silencing RNA and activated for 5 days (Figures 8 A and 8D). Representative histograms showing cell surface protein expression of PD-1 (left) and cumulative data of cell surface protein expression (middle) and gene expression (right) of PD-1; comparison by two-tailed paired t-test. (Figures 8E-8G) Cell surface expression (Figure 8E), intracellular expression (Figure 8F) and cell surface/intracellular PD-1 expression ratio (Figure 8G) after control or VPS 39 silencing in day 3 -stimulated naive CD4 T cells from old individuals. (Figure 8H) Control or 17fV3 -silenced, day 5- stimulated naive CD4 T cells from old individuals were treated with 5 pg/ml cycloheximide (CHX) to inhibit de novo PD-1 synthesis. Total PD-1 protein normalized to b-actin expression are shown as relative to non-treatment. Mean ± SEM of three experiments. (Figure 81) Longitudinal analysis of cell surface protein expression of PD-1 in naive CD4 T cells from ten young and ten old individuals. Mean ± SEM were compared by two-tailed unpaired t-test. (Figure 8J) PD-1 gene expression comparison between day 5-stimulated young and old naive CD4 T cells. Two-tailed unpaired t-test. The gray histogram represents isotype control. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Figures 9A- 9E. Sustained activation of late endosomal mTORCl impairs expansion of naive CD4 T cells from old adults. (Figure 9A) Naive CD4 T cells from older individuals labeled with CellTrace Violet (CTV) were transfected with control or VPS39 siRNAand stimulated with anti-CD3/anti-CD28 beads for 5 days in the presence or absence of PD-Ll-Fc and anti-human IgG Fc antibody to crosslink PD-1. Representative histograms (left), summary data of proliferation indices (middle) and cell numbers per culture (right); comparison by two-tailed paired t-test. (Figure 9B) CTV- labeled naive CD4 T cells from eight young and eight old individuals were activated by anti-CD3/anti-CD28 beads for 6 days with the last 3 day in the presence or absence of PD-1 crosslinking. Representative histograms (left), summary data of proliferation indices (middle) and cell numbers per culture (right); comparison by two-tailed unpaired t-test. The gray histogram represents unstimulated cells. (Figure 9C) CTV-labeled PBMCs from SARS-CoV-2 unexposed healthy individuals were cultured with SARS- CoV-2 peptide megapools for CD4 and CD8 cells for 8 days. VPS39 silencing FANA ASO or scramble control were added to the culture on day 0. Representative flow plots of the frequencies of CTV low CD4 and CD8 T cells for indicated conditions and summary data from six individuals; two-tailed paired t test. (Figures 9D and 9E) Pertussis peptide megapool responses of PBMC from six healthy individuals under the conditions of VPS39 genetic silencing (Figure 9D) and PD-1 blockade (Figure 9E). Two-tailed paired t test. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Figures 10A- 10O. Inhibition of late endosomal mTORCl promotes primary CD4 T cell responses after LCMV infection in vivo. (Figures 10 A- IOC) 1 x 10 ⁴ If ^' eb shRNA or control shRNA retrovirally transduced Amcyan ⁺ LCMV-specific naive SMARTA TCR transgenic CD4 T cells were adoptively transferred into CD45.2 ⁺ naive recipients followed by infection with LCMV Armstrong. On day 8 post infection, spleens were harvested and analyzed. FACS plots are gated on CD4 ⁺ Amcyan ⁺ SMARTA cells. (Figure 10 A) Tfeb protein expression in transduced cells before adoptive transfer. (Figure 10B) PD-1 expression on day 8 p.i. (Figure IOC) SMARTA CD4 T cell numbers per spleen on day 8 p.i. (Figures 10D-10K) Analysis of T cells responses as described in (Figures 10A-10C) after adoptive transfer of Vps39 shRNA transduced SMARTA CD4 T cells. (Figure 10D) Vps39 protein expression in transduced cells before adoptive transfer. (Figure 10E) Phosphorylation of S6RP on day 8 p.i. (Figure 10F) PD-1 expression on day 8 p.i. (Figure 10G) SMARTA CD4 T cell numbers per spleen on day 8 p.i. (Figure 10H) Cell apoptosis and proliferation of transferred SMARTA CD4 T cells on day 8 p.i.

(Figure 101) Mice infected with LCMV after adoptive transfer of transduced cells were additionally treated with anti-PD-1 antibody (29F.1A12) or control IgG on days 0, 3 and 6 post infection. SMARTA CD4 T cell numbers were determined on day 8 after LCMV infection. (Figure 10J) SMARTA CD4 T cell numbers in the spleen at day 30 after infection. (Figure 10K) Longitudinal analysis of SMARTA cells in the spleen of LCMV- infected B6 mice. (Figures 10L-10O) 1 x 10 ⁴ Slc7a5 shRNA or control shRNA retrovirally transduced Amcyan ⁺ LCMV-specific naive SMARTA CD4 T cells were adoptively transferred into CD45.2 ⁺ naive recipients as in (Figures 10A-10C). Phosphorylation of S6RP (Figure 10L), PD-1 expression (Figure 10M), SMARTA cell numbers per spleen (Figure 10N), and cell apoptosis and proliferation (Figure 10O) of transduced SMARTA CD4 T cells on day 8 post LCMV infection. Data are pooled from two independent experiments with 3-4 mice per group (Figures 10B- IOC and Figures 10E-10G), representative of two independent experiments with 3-5 mice per group (Figures 10A, 10D, and 10H-10K) or one experiment with 5 mice per group (Figures 10L-10O). Statistical significance by two-tailed unpaired t test (Figures lOB-lOC, 10E- 10H, and 10J-10O) or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (Figure 101). The gray histograms represent naive cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS, not significant. Figures 11 A- 11G. Inhibition of late endosomal mTORCl promotes virus- specific CD4 T cell expansion without affecting their cytokine production. (Figures 11 A- 11C) Naive SMARTA transgenic CD4 T cells were activated in plates coated with 8 pg/ml anti-CD3 Ab and 8 pg/ml anti-CD28 Ab. Cells were transduced with a retroviral vector expressing either scrambled control shRNA, Vps39 or Slc7a5 shRNAon days 1 and 2 after activation. At day 6 after activation, retrovirus-transduced Amcyan positive SMARTA cells were purified and effects of silencing on protein expression were confirmed by Western blotting. 1 x 10 Vps39 shRNA or control shRNA retrovirally transduced Amcyan ⁺ LCMV-specific naive SMARTA CD4 T cells were adoptively transferred into CD45.2 ⁺ naive recipients. Mice were infected with 2 x 10 PFU of LCMV Armstrong. On day 8 post infection, spleens were harvested and analyzed. (Figure 11 A) Lysosomal activities of SMARTA cells were determined by flow cytometry-based analysis of splenocytes treated with 5 pg/ml of DQ=BSAfor 6 hours. (Figure 11B) Cytokine production of SMARTA cells after LMC V gp66 peptide restimulation of splenocytes ex vivo. (Figure 11C) CTLA-4 expression by SMARTA cells on day 8 after LCMV infection. (Figure 11D) Vps39 silencing by a second sh Vps39 clone (#2, 5’- CCAC ACTCTCTGGTGCTGAAC-3 ’ (SEQ ID NO:l)). (Figure 11E) Numbers of SMARTA cells after shVps39 #2 silencing on day 30 after LCMV infection. (Figure 1 IF) Numbers of Db LCMV GP33-41 (GP33) tetramer+ cells gated on endogenous CD8 T cells in the spleen of host mice at day 30 after infection. (Figure 11G) Slc7a5 silencing by three different shSlc7a5 clones. Clone 1: 5’-GGCATTGGCTTCGCCATCATC-3’ (SEQ ID NO:2); Clone #2: 5’- : CGCAATATC ACGCTGCTCAAC-3 ’ (SEQ ID NO:3); Clone #3: 5’ AGCAGAAGTTGTCCTTTGAAG-3 ’ (SEQ ID NO:4); based on its partial inhibition resembling the age-associated difference in humans, Clone #2 was chosen for subsequent studies. Data are representative of two independent experiments with 3-4 mice per group (Figures 11A-11C) or one experiment (Figures 11D-11G). Comparison by two-tailed unpaired t test. *p < 0.05, **p < 0.01; NS, not significant.

Figures 12A- 12H. Inhibition of late endosomal mTORCl augments CD4 T cell helper responses in vivo. 1 x 10 ⁴ Vps39 shRNA or control shRNA retrovirally transduced Amcyan ⁺ naive SMARTA CD4 T cells were adoptively transferred into CD45.2 ⁺ naive recipient followed by LCMV infection. On days 8 (Figures 12A-12C) and 30 (Figure 12E) post infection, spleens were harvested and analyzed. Alternatively, on day 30, immune mice were rechallenged with Lm-gp33 for 6 days (Figures 12F-12H). (Figure 12A) Number of germinal center (GC) SMARTA TFH cells on day 8 after infection. (Figures 12B and 12C) Number of endogenous Fas ⁺ GL-7 ⁺ GC B cells (Figure 12B) and CD138 ⁺ IgD plasma cells (Figure 12C) in host mice on day 8 after infection. Representative contour plots gated on B220 ⁺ CD4 B cells (left) and summary data (right). (Figure 12D) Anti-LCMV nucleoprotein IgG titers in serum on day 14 after infection n = 5 mice per group. (Figure 12E) Numbers of D ^b LCMV GP33-41 (GP33) tetrameC cells gated on endogenous CD8 T cells in the spleen of host mice on day 30 after infection. (Figure 12F) Numbers of endogenous D ^b LCMV GP33 tetrameC CD8 T cells in the spleen on day 6 after Lm-GP33 challenge. (Figure 12G) Fold expansion of D ^b GP33 tetramer+ memory CD8 T cells upon Lm-GP33 on day 6. (Figure 12H) Cytokine production by CD8 T cells harvested on day 6 after infection and restimulated with GP33 peptide in vitro. Data are representative of two independent experiments with 4-5 mice per group (Figures 12A-12E) or one experiment with 5 mice per group (Figures 12F- 12H). Statistical significance by two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS, not significant.

Figures 13A- 13C. Improved CD4 T cell helper responses after Vps39 silencing depends on reduced PD-1 expression. 1 x 10 ⁴ Vps39 shRNAor control shRNA retrovirally transduced Amcyan ⁺ LCMV-specific naive SMARTA CD4 T cells were adoptively transferred into CD45.2 ⁺ naive recipients followed by infection with LCMV Armstrong. Mice were treated with anti-PD-1 antibody (29F.1 A12) or control IgG at days 0, 3, and 6 post infection. On day 8 post infection, spleens were harvested and analyzed. (Figure 13 A) Number of CXCRS^PD-l ¹ * SMARTA GC TFH cells. (Figure 13B) Number of endogenous Fas ⁺ IgD plasma cells in host mice (Figure 13C). Data are from one experiment with 4 mice per group. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. *p < 0.05; NS, not significant.

Figures 14A - 14G. (Figure 14A) Schematic of experimental design to prevent melanoma growth by adoptively transferred Vps39- silenced CAR-T cells. (Figure 14B) Tumor growth curves for B 16-OVA tumors following transfer of naive OT-1 control or Vps39- silenced CD8 ⁺ T cells separately into wild-type recipients. n=8 mice. (Figure 14C) Survival curves of mice in B. (Figure 14D) Cell surface PD-1 expression of tumor- infiltrating Amcyan ⁺ OT-I cells at day 9 after tumor implant. (Figure 14E) Numbers of tumor-infiltrating Amcyan ⁺ OT-I cells at day 9 after tumor implant. (Figure 14F) Cytokine production of tumor-infiltrating Amcyan ⁺ OT-I cells restimulated with OVA257-264 peptide ex vivo at day 9 after tumor implant. (Figure 14G) Alternatively, at day 18 after tumor implant, Amcyan ⁺ tumor-infiltrating OT-I cells were sorted and injected into a new group of wild-type C57BL/6 mice followed by B 16-OVA tumor implant. Tumor growth curves for B 16-OVA tumors following transfer of OT-1 control or Vps39- silenced CD8 ⁺ T cells that had been challenged with the same tumor cell line for 18 days separately into wild-type recipients. n=6 mice. Data are one experiment with 6 to 8 mice per group (Figures 14B, 14C, and 14G) or one experiment with four mice per group (Figures 14D-14F). Statistical significance by two-tailed unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

DETAILED DESCRIPTION

This document provides methods and materials for engineering immune cells (e.g., T cells such as chimeric antigen receptor (CAR) T cells (CAR-T cells) or tumor- infiltrating lymphocytes (TILs)) to have reduced expression of a VPS39 polypeptide. VPS39 is part of the complex that mediates fusion of autophagosomes with lysosomes (HOPS complex). In some embodiments, expression of VPSll, VPS16, VPS18, VPS33, or VPS41 can be reduced in an immune cell.

In some cases, a T cell (e.g., CAR T cells) can be engineered to knock out (KO) a nucleic acid encoding a VPS39 polypeptide to reduce VPS39 polypeptide expression in that T cell (e.g, as compared to a T cell that is not engineered to KO a nucleic acid encoding a VPS39 polypeptide). AT cell that is engineered to KO a nucleic acid encoding a VPS39 polypeptide can also be referred to herein as a VPS39 KO T cell. In some cases, the methods and materials provided herein can be used to treat cancer or improve the efficacy of adoptive transfer of immune cells. The term “reduced level” as used herein with respect to an expression level of VPS39 refers to any level that is lower than a reference expression level of VPS39. The term “reference level” as used herein with respect to VP S39 refers to the level of VP S39 typically observed in a sample (e.g., a control sample) from one or more mammals (e.g., humans) not engineered to have a reduced expression level of VPS39 as described herein. Control samples can include, without limitation, T cells that are wild-type T cells (e.g., T cells that are not VPS39 KO T cells). In some cases, a reduced expression level of a VPS39 polypeptide can be an undetectable level of VPS39. In some cases, a reduced expression level of VPS39 polypeptides can be an eliminated level of VPS39.

In some cases, a T cell having (e.g., engineered to have) a reduced level of a VPS39 polypeptide (e.g, a VPS39 KO T cell) can have enhanced CAR T cell function such as antitumor activity, enhanced T cell expansion, proliferation, cell killing, cytokine production, exhaustion susceptibility, antigen specific effector functions, persistence, and differentiation (e.g, as compared to a CAR T cell that is not engineered to have a reduced level of VPS39 polypeptides as described herein).

AT cell having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide such as a VPS39 KO T cell can be any appropriate T cell. AT cell can be a naive T cell. Examples of T cells that can be designed to have a reduced expression level of VPS39 as described herein include, without limitation, cytotoxic T cells (e.g, CD4+ CTLs and/or CD8+ CTLs). For example, a T cell that can be engineered to have a reduced level of a VPS39 polypeptide as described herein can be a CAR T cell. In some cases, one or more T cells can be obtained from a mammal (e.g, a mammal having cancer). For example, T cells can be obtained from a mammal to be treated with the materials and method described herein.

AT cell having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide can be generated using any appropriate method. In some cases, a T cell (e.g, a CAR T cell) can be engineered to KO a nucleic acid encoding a VPS39 polypeptide to reduce VPS39 polypeptide expression in that T cell. Examples of techniques that can be used to knock out a nucleic acid sequence encoding a VPS39 polypeptide include, without limitation, gene editing, homologous recombination, non-homologous end joining, and microhomology end joining. For example, gene editing ( e.g ., with engineered nucleases) can be used to KO a nucleic acid encoding a VPS39 polypeptide. Nucleases useful for genome editing include, without limitation, CRISPR-associated (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, and homing endonucleases (HE; also referred to as meganucleases).

In some cases, a clustered regularly interspaced short palindromic repeat (CRISPR) / Cas system can be used (e.g., can be introduced into one or more T cells) to KO a nucleic acid encoding a VPS39 polypeptide. A CRISPR/Cas system used to KO a nucleic acid encoding a VP S39 polypeptide can include any appropriate guide RNA (gRNA). In some cases, a gRNA can be complementary to a nucleic acid encoding a VPS39 polypeptide.

A CRISPR/Cas system used to KO a nucleic acid encoding a VPS39 polypeptide can include any appropriate Cas nuclease. Examples of Cas nucleases include, without limitation, Casl, Cas2, Cas3, Cas9, CaslO, and Cpfl. In some cases, a Cas component of a CRISPR/Cas system designed to KO a nucleic acid encoding a VPS39 polypeptide can be a Cas9 nuclease. For example, the Cas9 nuclease of a CRISPR/Cas9 system described herein can be a lentiCRISPRv2 (see, e.g., Shalem et al, 2014 Science 343:84-87; and Sanjana et al, 2014 Nature methods 11 : 783-784).

Components of a CRISPR/Cas system (e.g, a gRNA and a Cas nuclease) used to KO a nucleic acid encoding a VP S39 polypeptide can be introduced into one or more T cells (e.g, CAR T cells) in any appropriate format. In some cases, a component of a CRISPR/Cas system can be introduced into one or more T cells as a nucleic acid encoding a gRNA and/or a nucleic acid encoding a Cas nuclease. For example, a nucleic acid encoding at least one gRNA (e.g, a gRNA sequence specific to a nucleic acid encoding a VPS39 polypeptide) and a nucleic acid at least one Cas nuclease (e.g, a Cas9 nuclease) can be introduced into one or more T cells. In some cases, a component of a CRISPR/Cas system can be introduced into one or more T cells as a gRNA and/or as a Cas nuclease. For example, at least one gRNA (e.g., a gRNA sequence specific to a nucleic acid encoding a VPS39 polypeptide) and at least one Cas nuclease (e.g, a Cas9 nuclease) can be introduced into one or more T cells. In some cases, when components of a CRISPR/Cas system (e.g., a gRNAand a Cas nuclease) are introduced into one or more T cells as nucleic acid encoding the components (e.g, nucleic acid encoding a gRNA and nucleic acid encoding a Cas nuclease), the nucleic acid can be any appropriate form. For example, a nucleic acid can be a construct (e.g, an expression construct). A nucleic acid encoding at least one gRNA and a nucleic acid encoding at least one Cas nuclease can be on separate nucleic acid constructs or on the same nucleic acid construct. In some cases, a nucleic acid encoding at least one gRNA and a nucleic acid encoding at least one Cas nuclease can be on a single nucleic acid construct. A nucleic acid construct can be any appropriate type of nucleic acid construct. Examples of nucleic acid constructs that can be used to express at least one gRNA and/or at least one Cas nuclease include, without limitation, expression plasmids and viral vectors (e.g, lentiviral vectors). In cases where a nucleic acid encoding at least one gRNA and a nucleic acid encoding at least one Cas nuclease are on separate nucleic acid constructs, the nucleic acid constructs can be the same type of construct or different types of constructs. In some cases, a nucleic acid encoding at least one gRNA sequence specific to a nucleic acid encoding a VPS39 polypeptide and a nucleic acid encoding at least one Cas nuclease can be on a single lentiviral vector and used in ex vivo engineering of T cells to have a reduced expression level of VPS39.

In some cases, components of a CRISPR/Cas system (e.g, a gRNAand a Cas nuclease) can be introduced directly into one or more T cells (e.g, as a gRNA and/or as Cas nuclease). A gRNA and a Cas nuclease can be introduced into the one or more T cells separately or together. In cases where a gRNA and a Cas nuclease are introduced into the one or more T cells together, the gRNA and the Cas nuclease can be in a complex. When a gRNA and a Cas nuclease are in a complex, the gRNA and the Cas nuclease can be covalently or non-covalently attached. In some cases, a complex including a gRNA and a Cas nuclease also can include one or more additional components. Examples of complexes that can include components of a CRISPR/Cas system (e.g, a gRNAand a Cas nuclease) include, without limitation, ribonucleoproteins (RNPs) and effector complexes (e.g., containing a CRISPR RNAs (crRNAs) a Cas nuclease). For example, at least one gRNA and at least one Cas nuclease can be included in a RNP. In some cases, a RNP can include gRNAs and Cas nucleases at a ratio of about 1 : 1 to about 10:1 (e.g., about 1:1 to about 10:1, about 2:1 to about 10:1, about 3:1 to about 10:1, about 5:1 to about 10:1, about 8:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 7:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 2:1 to about 8:1, about 3:1 to about 6:1, about 4:1 to about 5:1, or about 5:1 to about 7:1). For example, a RNP can include gRNAs and Cas nucleases at about a 1 : 1 ratio. For example, a RNP can include gRNAs and Cas nucleases at about a 2: 1 ratio. In some cases, a RNP including at least one gRNA sequence specific to a nucleic acid encoding a VPS39 polypeptide and at least one Cas9 nuclease can be used in ex vivo engineering of T cells to have a reduced level of a VPS39 polypeptide.

Components of a CRISPR/Cas system (e.g., a gRNA and a Cas nuclease) used to KO a nucleic acid encoding a VP S39 polypeptide can be introduced into one or more T cells (e.g, CAR T cells) using any appropriate method. A method of introducing components of a CRISPR/Cas system into a T cell can be a physical method. A method of introducing components of a CRISPR/Cas system into a T cell can be a chemical method. A method of introducing components of a CRISPR/Cas system into a T cell can be a particle-based method. Examples of methods that can be used to introduce components of a CRISPR/Cas system into one or more T cells include, without limitation, electroporation, transfection (e.g, lipofection), transduction (e.g, viral vector mediated transduction), microinjection, and nucleofection. In some cases, when components of a CRISPR/Cas system are introduced into one or more T cells as nucleic acid encoding the components, the nucleic acid encoding the components can be transduced into the one or more T cells. For example, a lentiviral vector encoding at least one gRNA sequence specific to a nucleic acid encoding a VPS39 polypeptide and at least one Cas9 nuclease can be transduced into T cells (e.g, ex vivo T cells). In some cases, when components of a CRISPR/Cas system are introduced directly into one or more T cells, the components can be electroporated into the one or more T cells. For example, a RNP including at least one gRNA sequence specific to a nucleic acid encoding a VPS39 polypeptide and at least one Cas9 nuclease can be electroporated into T cells (e.g, ex vivo T cells). In some cases, components of a CRISPR/Cas system can be introduced ex vivo into one or more T cells. For example, ex vivo engineering of T cells to have a reduced level of a VPS39 polypeptide can include transducing isolated T cells with a lentiviral vector encoding components of a CRISPR/Cas system. For example, ex vivo engineering of T cells having reduced levels of a VPS39 polypeptide can include electroporating isolated T cells with a complex including components of a CRISPR/Cas system. In cases where T cells are engineered ex vivo to have a reduced level of VPS39 polypeptide, the T cells can be obtained from any appropriate source ( e.g ., a mammal such as the mammal to be treated or a donor mammal, or a cell line).

In some cases, a T cell (e.g., a CAR T cell) can be treated with one or more inhibitors of VPS39 polypeptide expression to reduce VPS39 polypeptide expression in that T cell (e.g, as compared to a T cell that was not treated with one or more inhibitors of VPS39 polypeptide expression). An inhibitor of VPS39 polypeptide expression can be any appropriate inhibitor, including without limitation, nucleic acid molecules designed to induce RNA interference (e.g, a siRNA molecule or a shRNA molecule), antisense molecules, miRNAs, receptor blockade, and antibodies (e.g, antagonistic antibodies and neutralizing antibodies).

AT cell having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide can express (e.g, can be engineered to express) any appropriate antigen receptor. In some cases, an antigen receptor can be a heterologous antigen receptor. In some cases, an antigen receptor can be a CAR. In some cases, an antigen receptor can be a tumor antigen (e.g, tumor-specific antigen) receptor. For example, a T cell can be engineered to express a tumor-specific antigen receptor that targets a tumor-specific antigen (e.g, a cell surface tumor-specific antigen) expressed by a cancer cell in a mammal having cancer. Examples of antigens that can be recognized by an antigen receptor expressed in a T cell having reduced expression of a VPS39 polypeptide as described herein include, without limitation, cluster of differentiation 19 (CD 19), mucin 1 (MUC-1), human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), epidermal growth factor receptor (EGFR), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), CD33, CD123, CLL-1, E-Cadherin, folate receptor alpha, folate receptor beta, IL13R, EGFRviii, CD22, CD20, kappa light chain, lambda light chain, desmopressin, CD44v, CD45, CD30, CD5, CD7, CD2, CD38, BCMA, CD 138, FAP, CS-1, and C-met.

Any appropriate method can be used to express an antigen receptor on a T cell having ( e.g ., engineered to have) a reduced expression level of a VPS39 polypeptide. For example, a nucleic acid encoding an antigen receptor can be introduced into one or more T cells. In some cases, viral transduction can be used to introduce a nucleic acid encoding an antigen receptor into a non-dividing a cell. A nucleic acid encoding an antigen receptor can be introduced in a T cell using any appropriate method. In some cases, a nucleic acid encoding an antigen receptor can be introduced into a T cell by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection. In some cases, a nucleic acid encoding an antigen receptor can be introduced ex vivo into one or more T cells. For example, ex vivo engineering of T cells expressing an antigen receptor can include transducing isolated T cells with a lentiviral vector encoding an antigen receptor. In cases where T cells are engineered ex vivo to express an antigen receptor, the T cells can be obtained from any appropriate source (e.g, a mammal such as the mammal to be treated or a donor mammal, or a cell line).

In some cases, when a T cell having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide also expresses (e.g, is engineered to express) an antigen receptor, that T cell can be engineered to have a reduced expression level of VPS39 and engineered to express an antigen receptor using any appropriate method. In some cases, a T cell can be engineered to have a reduced expression level of a VPS39 polypeptide first and engineered to express an antigen receptor second, or vice versa. In some cases, a T cell can be simultaneously engineered to have a reduced expression level of a VPS39 polypeptide and to express an antigen receptor. For example, one or more nucleic acids used to reduce expression of a VPS39 polypeptide (e.g, a lentiviral vector encoding at least one gRNA sequence specific to a nucleic acid encoding VPS39 and at least one Cas9 nuclease or a nucleic acid encoding at least one oligonucleotide that is complementary to the VPS39 mRNA) and one or more nucleic acids encoding an antigen receptor (e.g, a CAR) can be simultaneously introduced into one or more T cells. One or more nucleic acids used to reduce expression of a VPS39 polypeptide and one or more nucleic acids encoding an antigen receptor can be introduced into one or more T cells on separate nucleic acid constructs or on a single nucleic acid construct. In some cases, one or more nucleic acids used to reduce expression of a VPS39 polypeptide and one or more nucleic acids encoding an antigen receptor can be introduced ex vivo into one or more T cells. In cases where T cells are engineered ex vivo to have a reduced expression levels of VPS39 and to express an antigen receptor, the T cells can be obtained from any appropriate source ( e.g ., a mammal such as the mammal to be treated or a donor mammal, or a cell line).

In some cases, a T cell having (e.g., engineered to have) a reduced expression level of a VPS39 polypeptide can be stimulated. AT cell can be stimulated at the same time as being engineered to have a reduced level of a VPS39 polypeptide or independently of being engineered to have a reduced level of a VPS39 polypeptide. For example, one or more T cells having a reduced level of a VPS39 polypeptide used in an adoptive cell therapy can be stimulated first, and can be engineered to have a reduced expression level of a VPS39 polypeptide second, or vice versa. In some cases, one or more T cells having a reduced expression level of a VPS39 polypeptide used in an adoptive cell therapy can be stimulated first, and can be engineered to have a reduced level of VPS39 polypeptide second. AT cell can be stimulated using any appropriate method. For example, a T cell can be stimulated by contacting the T cell with one or more CD polypeptides. Examples of CD polypeptides that can be used to stimulate a T cell include, without limitation, CD3, CD28, inducible T cell co-stimulator (ICOS), CD137, CD2, 0X40, and CD27. In some cases, a T cell can be stimulated with CD3 and CD28 prior to introducing components of a CRISPR/Cas system (e.g, a gRNA and/or a Cas nuclease) to the T cell to KO a nucleic acid encoding a VPS39 polypeptide.

This document also provides methods and materials involved in treating cancer. For example, one or more T cells having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide can be administered (e.g, in an adoptive cell therapy such as a CART therapy) to a mammal (e.g, a human) having cancer to treat the mammal. In some cases, methods of treating a mammal having cancer as described herein can reduce the number of cancer cells (e.g, cancer cells expressing a tumor antigen) within a mammal. In some cases, methods of treating a mammal having cancer as described herein can reduce the size of one or more tumors ( e.g ., tumors expressing a tumor antigen) within a mammal.

Any appropriate mammal (e.g., a human) having a cancer can be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, primates (such as monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a human having a cancer can be treated with one or more T cells having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide in, for example, an adoptive T cell therapy such as a CART cell therapy or TIL therapy using the methods and materials described herein.

When treating a mammal (e.g, a human) having a cancer as described herein, the cancer can be any appropriate cancer. In some cases, a cancer treated as described herein can be a solid tumor. In some cases, a cancer treated as described herein can be a hematological cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can be a relapsed cancer. In some cases, a cancer treated as described herein can express a tumor-associated antigen (e.g, an antigenic substance produced by a cancer cell). Examples of cancers that can be treated as described herein include, without limitation, B cell cancers (e.g., diffuse large B cell lymphoma (DLBCL) and B cell leukemias), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, non- Hodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia (AML), multiple myeloma, head and neck cancers, sarcomas, breast cancer, gastrointestinal malignancies, bladder cancers, urothelial cancers, kidney cancers, lung cancers, prostate cancers, ovarian cancers, cervical cancers, genital cancers (e.g., male genital cancers and female genital cancers), and bone cancers.

Any appropriate method can be used to identify a mammal having cancer. For example, imaging techniques and biopsy techniques can be used to identify mammals (e.g, humans) having cancer. Once identified as having a cancer, a mammal can be administered one or more T cells having ( e.g ., engineered to have) a reduced expression level of a VPS39 polypeptide as described herein.

For example, one or more T cells having (e.g., engineered to have) a reduced expression level of a VPS39 polypeptide can be used in an adoptive T cell therapy (e.g, a CAR T cell therapy) to treat a mammal having a cancer. For example, one or more T cells having a reduced level of a VPS39 polypeptide can be used in an adoptive T cell therapy (e.g, a CAR T cell therapy) targeting any appropriate antigen within a mammal (e.g, a mammal having cancer). In some cases, an antigen can be a tumor-associated antigen (e.g, an antigenic substance produced by a cancer cell). Examples of tumor-associated antigens that can be targeted by an adoptive T cell therapy provided herein include, without limitation, CD 19 (associated with DLBCL, ALL, and CLL), AFP (associated with germ cell tumors and/or hepatocellular carcinoma), CEA (associated with bowel cancer, lung cancer, and/or breast cancer), CA-125 (associated with ovarian cancer), MUC-1 (associated with breast cancer), ETA (associated with breast cancer), MAGE (associated with malignant melanoma), CD33 (associated with AML), CD 123 (associated with AML), CLL-1 (associated with AML), E-Cadherin (associated with epithelial tumors), folate receptor alpha (associated with ovarian cancers), folate receptor feta (associated with ovarian cancers and AML), IL13R (associated with brain cancers), EGFRviii (associated with brain cancers), CD22 (associated with B cell cancers), CD20 (associated with B cell cancers), kappa light chain (associated with B cell cancers), lambda light chain (associated with B cell cancers), CD44v (associated with AML),

CD45 (associated with hematological cancers), CD30 (associated with Hodgkin lymphomas and T cell lymphomas), CD5 (associated with T cell lymphomas), CD7 (associated with T cell lymphomas), CD2 (associated with T cell lymphomas), CD38 (associated with multiple myelomas and AML), BCMA (associated with multiple myelomas), CD138 (associated with multiple myelomas and AML), FAP (associated with solid tumors), CS-1 (associated with multiple myeloma), and c-Met (associated with breast cancer).

In some cases, one or more T cells having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide can be used in an adoptive T cell therapy (e.g, a CAR T cell therapy) to treat a mammal having a disease or disorder other than cancer.

For example, one or more T cells having a reduced level of VPS39 polypeptide can be used in an adoptive T cell therapy ( e.g ., a CAR T cell therapy) targeting any appropriate disease-associated antigen (e.g., an antigenic substance produced by cell affected by a particular disease) within a mammal. Examples of disease-associated antigens that can be targeted by an adoptive T cell therapy provided herein include, without limitation desmopressin (associated with auto immune skin diseases).

In some cases, one or more T cells having (e.g, engineered to have) a reduced expression level of a VPS39 polypeptide used in an adoptive T cell therapy (e.g, a CAR T cell therapy) can be administered to a mammal having a cancer as a combination therapy with one or more additional agents used to treat a cancer. For example, one or more T cells having a reduced level of a VPS39 polypeptide used in an adoptive cell therapy can be administered to a mammal in combination with one or more anti-cancer treatments (e.g, surgery, radiation therapy, chemotherapy (e.g, alkylating agents such as busulfan), targeted therapies, hormonal therapy, angiogenesis inhibitors, and/or immunosuppressants (e.g, interleukin-6 inhibiting agents such as tocilizumab). In cases where one or more T cells having a reduced level of a VPS39 polypeptide used in an adoptive cell therapy are used with additional agents treat a cancer, the one or more additional agents can be administered at the same time or independently. In some cases, one or more T cells having a reduced level of a VPS39 polypeptide used in an adoptive cell therapy can be administered first, and the one or more additional agents administered second, or vice versa.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1 - Materials and Methods

Aging is associated with a decline in adaptive immunity, resulting in decreased efficacy of vaccination and increased morbidity from infections with newly as well as previously encountered pathogens. Most noticeable are the increased susceptibility during the annual influenza epidemic and, most recently, the infection with the SARS-Cov-2 virus. Although the increased morbidity is clearly multifactorial, age-related changes in CD4 T cell survival and differentiation have been identified that contribute to the immune decline in older individuals. CD4 T helper cells are pivotal for mounting a protective immune response after vaccination or infections. Generation of CD4 T follicular helper (TFH) cells is essential for germinal center (GC) formation, class switching and affinity maturation. Moreover, CD4 T cells are required for effective CD8 T cell memory and recall responses. Recent studies have shown a T cell-intrinsic bias towards short-lived effector cells differentiation in CD4 T cell responses of older adults at the expense of TFH and memory precursor cells. One important mechanism causing this bias is a sustained activation of the mechanistic target of rapamycin complex 1 (mTORCl). mTORCl plays an important role in regulating T cell responses by coordinating cell growth and cellular metabolism with environmental inputs, in particular nutrient resources, and facilitating the switch toward anabolic metabolism that is required for cell proliferation and effector cell differentiation. Complete block of mTORCl activities by either a treatment with high dose of rapamycin or a genetic depletion of RHEB (an upstream activator of mTORCl) dramatically reduced the number of antigen-specific effector and memory precursor T cells at peak responses after primary infection RHEB- deficient memory CD8 T cells even failed to respond to secondary immunization. However, overactivation of mTORCl by genetic depletion of TSC1 (an upstream inhibitor of mTORCl) reduced the numbers of memory precursor CD8 T cells at peak responses after primary infection instead of promoting them. Indeed, moderate inhibition of mTORCl by pharmacological compounds produced increased clonal expansion of antigen-specific T cells after primary responses and enhanced memory recall responses after antigen rechallenge in several infection models. These data led to the conclusion that mTORCl inhibition promotes long-term memory over short-lived effector CD8 T cell differentiation. Taken together, fine-tuning of mTORCl activity is important for generating protective primary and recall T cell responses, in particular for older adults, who have sustained mTORCl activity after T cell activation resulting in the preferential generation of short-lived effector T cells. mTORCl translocates to the lysosomal membrane, where it is activated in response to amino acid signaling. In turn, mTORCl suppresses lysosomal activity by phosphorylating TFEB. T cells from old adults had lower lysosomal gene expression and proteolytic activities due to reduced TFEB transcription. In parallel, mTORCl activity was more sustained in activated T cells from older adults. How lysosome-deficient aged T cells maintain mTORCl activity remains unresolved. The late endosome is an alternative platform for mTORCl activation. Compared to the lysosome, late endosomes have no proteolytic activity and therefore no amino acid efflux. They originate by homotypic fusion and vacuole protein sorting (HOPS) complex-mediated conversion from early endosomes. Inhibiting early to late endosomal conversion by silencing Vam6/Vps39-like protein (VPS39, a key member of the HOPS complex) led to the formation of hybrid endosomal compartments and attenuated mTORCl activity. Conversely, inhibition of lysosomal activities induced an intracellular expansion of late endosomes. Consistent with this observation, an increased number of late endosomes is seen in responses of T cells from older individuals that fail to replenish their lysosomes.

Study design

The aim of this study was to examine the mechanism how activated naive CD4 T cells from older adults exhibit increased and sustained mTORCl activation in spite of lysosomal dysfunction, and to identify targets for intervention other than direct mTORCl inhibition to improve T follicular helper cell responses and memory T cell generation. Purified naive CD4 T cells collected from young and old healthy individuals were used to perform in vitro signaling and functional studies after polyclonal activation. Pharmacological inhibition as well as gene expression silencing were applied to interrogate lysosomal and endosomal pathways in the context of age. In vitro human data were validated in vivo in an infection mouse model by adoptively transferring antigen- specific CD4 T cells that had been genetically manipulated. Mice were randomly assigned to control versus sample groups. Date analysis was conducted in an unblinded manner. Sampling and experimental replicates were specified in figure legends. No outliers were removed. Study population and cell isolation

PBMC were collected from 53 young (20-35 years old) and 64 old (65-85 years old) healthy individuals with no history of autoimmune disease or cancer and no uncontrolled renal disease, diabetes mellitus, or cardiovascular disease. 98 of them were de-identified samples purchased from the Stanford Blood Center (Palo Alto, CA, USA) from donors younger than 35 years (43 donors) or older than 65 years (55 donors). 19 samples were from individuals recruited from the local area. The study was in accordance with the Declaration of Helsinki, approved by the Stanford Institutional Review Board, and all participants gave informed written consent. Naive CD4 T cells were purified with human CD4 T cell enrichment cocktail (15062, STEMCELL Technologies), followed by negative selection with anti-CD45RO magnetic beads (19555, STEMCELL Technologies).

Cell culture

Isolated human naive CD4 T cells were activated with Dynabeads Human T- Activator CD3/CD28 (11132D, Thermo Fisher Scientific) in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin (Thermo Fisher Scientific). Mouse T cells were activated in plates coated with 8 pg/mL anti-CD3 Ab (16-0032-82, eBioscience) and 8 pg/mL anti-CD28 Ab (16- 0281-82, eBioscience) in culture medium supplemented with 10 ng/mL IL-2 (21212, Peprotech).

Western blotting

Cells were lysed in RIPA buffer containing PMSF and protease and phosphatase inhibitors (sc-24948, Santa Cruz Biotechnology) for 30 minutes on ice. Proteins were separated on denaturing 4%— 15% SDS-PAGE (4561086, Bio-Rad), transferred onto nitrocellulose membrane (1704270, Bio-Rad) and probed with antibodies to SLC7A5 (5347S), c-MYC (18583S), b-actin (4970S), S6K1 (2708S), pS6Kl Kinase (Thr389, 9234S), EEA1 (3288S), RAB7 (9367S), Cathepsin D (2284S), Na/K ATPase ( 3010S), Tubulin (2125S), mTOR (2983S), Raptor (2280S), RHEB (13879S), PD-1 (86163S, all Cell Signaling Technology), CD63 (ab68418), CathepsinH (abl85935) and VPS39 (ab224671, all Abeam). Membranes were developed using HRP-conjugated secondary antibodies (Cell Signaling Technology) and Chemiluminescent Western Blot Detection Substrate (Thermo Fisher Scientific).

Flow cytometry

For cell surface staining, cells were incubated with fluorescently conjugated antibodies in PBS containing 2% FBS at 4 °C for 30 minutes. For intracellular cytokine assays, cells were stimulated with 10 mM LCMV GP66-77 DIYKGVYQFKSV (SEQ ID NO:5) or 0.2 pM LCMV GP33-41 KAVYNFATC (SEQ ID NO:6) (Anaspec) in the presence of Brefeldin A (GolgiPlug, BD Biosciences) for 4 hours at 37 °C. Cells were then incubated with antibodies to cell surface molecules, permeabilized with Cytofix/Cytoperm kit (BD Biosciences) and stained with fluorescently labeled antibodies specific for the indicated cytokines. For pS6RP (S235/S236) staining, cells were treated with FOXP3 Fix/Perm Buffer Set (421403, Biolegend) before incubation with fluorescently conjugated antibodies at room temperature for 60 minutes. Dead cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (eBioscience). Staining for flow cytometry was done with monoclonal antibodies against: CD4 (anti-human: RPA- T4; anti-mouse: RM4-5), CD8 (anti-human: RPA-T8; anti-mouse: 53-6.7), CD44 (IM7), B220 (RA3-6B2), Fas (Jo2), GL-7 (GL7), CD138 (281-2), IgD (11-26), CD62L (MEL- 14), CD 127 (SB/199), KLRG-1 (2F1), pS6RP (S235/S236, N7-548, all BD Bioscience), PD-1 (anti-human: A17188B; anti-mouse: 29F.1A12), CTLA-4 (UC10-4B9), IFN-g (XMG1.2), TNF-a (MP6-XT22) and IL-2 (JES6-5H4, all Biolegend). Mouse CXCR5 was stained with biotin-conjugated anti-CXCR5 (2G8, BD Bioscience) followed by APC-streptavidin binding (BD Bioscience). D ^b GP33-41 KAVYNFATC (GP33) tetramer was obtained from the NIH tetramer core facility (Atlanta, GA). To stain LCMV-specific CD8 T cells, cells were incubated with D ^b GP33 tetramers along with cell surface antibodies at 4 °C for 30 minutes in antibody staining buffer. Cells were analyzed on an LSRII or LSR Fortessa (BD Biosciences). Flow cytometry data were analyzed using FlowJo (TreeStar). RNA isolation and quantitative RT-PCR

Total RNA was isolated using the RNeasy Plus Mini Kit (74134, QIAGEN) and converted to cDNA using the Superscript VILO cDNA Synthesis Kit (11754, Invitrogen). Quantitative RT-PCR was performed on the ABI 7900HT system (Applied Biosystems) using Power SYBR Green PCR Master Mix (4368706, Thermo Fisher

Scientific), according to the manufacturer’s instructions. Oligonucleotide primer sets are shown in Table 1.

Table 1. Oligonucleotide primer sets used in this study.

Gene set enrichment analysis

Gene set enrichment analysis (GSEA) software from the Broad Institute (software.broadinstitute.org/gsea/index.jsp) was used to determine the enrichment of gene sets in T cells from young (20-35 years) or old (65-85 years) adults. The datasets describing age-associated differences in activated CD4 T cells were obtained from SRA database under accession number SRP158502. See, e.g., Hu, et al, Aging Cell 18, el2957 (2019).

Transfection

Naive CD4 T cells were transfected with either SMARTpool negative control siRNA, SMARTpool TFEB siRNA, SMARTpool MYC siRNA or SMARTpool VPS39 siRNA (all from Dharmacon) using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). Cells were rested for 2 hours, washed and activated by dynabeads for 5 days. Cells were then harvested and analyzed.

Pharmacologic inhibition

The SLC7A5 inhibitor JPH203 (S8667, Selleckchem) was used at a dose of 5 mM for all experiments. Lysosome inhibitors Chloroquine (PHR1258-1G, Sigma- Aldrich) and Bafilomycin A1 (11038, Cayman CHEMICAL) were used at doses of 20 pM and 10 nM, respectively. The mTORCl inhibitor Torin 1 (4247, Tocris) was used at a dose of 100 nM. The protein synthesis inhibitor cycloheximide (C4859, Sigma- Aldrich) was used at a dose of 5 pg/mL.

Endosome isolation and in vitro mTORCl kinase activity assay

Endosomes were isolated from naive CD4 T cells on day 3 after activation when mTORCl activity peaked, by using an endosome isolation kit (ED-028, Invent Biotechnologies) according to manufacturer’s instructions. In some experiments, cells were pre-treated for 2 hours with 1 pM AKT inhibitor (MK-22062HC1, Selleckchem) prior to harvesting. Briefly, 2 x 10 ⁷ cells were suspended in lysis buffer A (supplemented with protease inhibitor cocktail) and cell extracts were filtered to remove intact cells, larger organelles and plasma membranes. The flow-through cell lysates were mixed with the supplied precipitation buffer B followed by centrifugation. After centrifugation, endosomes were enriched in pellets. Supernatants were discarded, and the endosome pellets were resuspended in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology). Protein concentrations were measured using BCA Protein Assay Kit (23227, Pierce) and equal amounts of protein were loaded for immunoblotting analysis of indicated markers. Alternatively, the endosome pellets were resuspended in mTORCl kinase buffer (25 mM HEPES, 50 mM KC1, 10 mM MgCk, 20% glycerol, 4 mM MnCk and 250 pM ATP) prepared according to a previous study (Kim, et al. , Cell 110, 163-175 (2002)) and divided into aliquots of 10 pL each containing 10 pg endosomes. 100 ng of S6K1 Human Recombinant Protein (TP317324, Origene) diluted in 5 pL of mTORCl kinase buffer was added as the substrate, and the Kinase assays were performed at 30°C for 20 minutes in a final volume of 15 pL. Reactions were stopped by adding 5 pL of 4 x sample buffer and loaded for immunoblotting analysis.

Confocal microscopy

Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X- 100, and incubated with primary antibodies to LAMP1 (#9091), mTOR (#2983), pS6RP (S235/236, #4858) and EEA1 (#48453, all Cell Signaling Technology) at 4°C overnight. Incubation with secondary antibodies was performed at room temperature for 2 hours using Alexa Fluor 488-conjugated AffmiPure Donkey anti-Rabbit immunoglobulin G (IgG) or Cy3 -conjugated AffmiPure Donkey anti-Mouse IgG (Jackson Immuno Research Laboratories). The images were analyzed using an LSM 710 microscope system with ZEN 2010 software (Carl Zeiss) and a 63 ^c oil immersion objective (Carl Zeiss).

DQ-BSA lysosomal activity assay

Cells were treated with 5 pg/mL of DQ-BSA (D12050, Thermo Fisher Scientific) diluted in prewarmed medium and incubated at 37 °C for 6 hours. Cells were then briefly washed once with ice-cold PBS containing 2% FBS and kept on ice. Fluorescence of cleaved BSA-DQ was analyzed by flow cytometry.

Cell proliferation assay in vitro

Freshly purified naive CD4 T cells were labeled with CellTrace Violet (CTV; Thermo Fisher Scientific) and stimulated with anti-CD3/anti-CD28 beads in the presence of recombinant human PD-L1/B7-H1 Fc chimera (156-B7, R&D systems; 5 pg/mL) crosslinked by goat anti-human IgGFc polyclonal antibody (G-102-C, R&D systems; 5 pg/ml). At day 5-6, cells were harvested and analyzed by flow cytometry.

SARS-CoV-2 and pertussis-reactive CD 4 and CD8 T cell responses

PBMCs were isolated by density gradient centrifugation using Lymphoprep

(STEMCELL Technologies) from pheresis samples of SARS-CoV-2 unexposed healthy donors. The HLA class II peptide megapool covering the entire SARS-CoV-2 orfeome and the HLA class I peptide megapool covering half of the entire orfeome were described previously. See, Grifoni, et al. , Cell Host Microbe, 27:671-680-e672 (2020); Grifoni, et al., Cell, 181:1489-1501 el415 (2020). PBMCs were labeled with CellTrace Violet (CTV; Thermo Fisher Scientific) and cultured with either the mixture of SARS-CoV-2 class II megapool (1 pg/mL) and class I megapool (1 pg/mL) or pertussis megapool (1 pg/mL) in RPMI 1640 media (Sigma) supplemented with 5% human AB serum (Sigma),

1 pg/mL anti-CD28 (ebioscience) and 100 U/mL penicillin and streptomycin (Thermo Fisher Scientific). VPS39- silencing FANA ASO (FANA antisense oligonucleotide, 1 pM, AUM Biotech), scrambled control FANA ASO (1 pM, AUM Biotech), anti-PD-1 blocking antibody (A17188B, 1 pg/mL) or control IgG (1 pg/mL) were added to the culture on day 0. Peptide-reactive CD4 and CD8 T cell responses were measured on day 8

Mice, adoptive transfers and LCMV infection

Naive CD4 T cells specific to the GP66-77 epitope of LCMV obtained from 5- to 8-week-old SMARTATCR transgenic mice (CD45.1, provided by Rafi Ahmed at Emory University) were activated in plates coated with anti-CD3 Ab and anti-CD28 Ab. Cells were transduced with a retroviral vector expressing either scrambled control RNA, Tfeb shRNA (5 ’ -CGGC AGTACTATGACTATGAT-3 ’ (SEQ ID NO:25)), Vps39 shRNA(5’- AGTGAGCATGTGCTGAAGAAG-3 ’ (SEQ ID NO:26)) or Slc7a5 shRNA(5’- CGCAATATCACGCTGCTCAAC-3 ’ (SEQ ID NO:3)) on days 1 and 2 after activation. On day 6 after activation, retrovirus-transduced Amcyan-positive SMARTA cells were isolated and 1 ^c 10 ⁴ transduced cells were intravenously (i.v.) transferred to 5- to 8-week- old naive C57BL/6 (CD45.2) mice (Jackson Laboratory). On day 3 post-transfer, mice were infected intraperitoneally (i.p.) with 2 x 10 ⁵ PFU of LCMV Armstrong. On day 8 post-infection, spleens were harvested and analyzed. For PD-1 Blockade: anti-PD-1 (29F.1A12, #BE0273, Bio X Cell; 200 pg, i.p.) or control IgG (2A3, #BE0089, Bio X Cell; 200 pg, i.p.) was administered to LCMV-infected B6 mice on days 0, 3 and 6 post infection. On day 8 post-infection, spleens were harvested and analyzed. For recall responses of CD8 memory T cells, recombinant Listeria monocytogenes expressing the LCMV glycoprotein 33-41 epitope (Lm-gp33) were grown to log phase in BHI broth. Concentrations were determined by measuring the O.D. at 600 nm (O.D. of 1 = 1 x 10 ⁹ CFU/ml). LCMV-immune mice were injected i.v. with 2 x 10 ⁵ colony forming units (CFU) for recall responses. All animal experiments were approved by the Stanford Administrative Panel on Laboratory Animal Care Committee.

ELISA

Serum was collected from mice on day 14 after LCMV (Armstrong) infection. LCMV-specific IgG was measured using an anti-LCMV-NP IgG ELISA kit (AE-300200- 1, Alpha Diagnostic International) according to the manual. Briefly, ELISA plates coated with LCMV-VP1 antigen were pre-washed and incubated with 5-fold serial diluted serum for 60 min. Plates were then washed and incubated with anti-mouse IgG HRP for 30 minutes. After washing, bound antibody was detected by adding TMB substrate and the reaction was stopped with stop solution. Plates were read for absorbance at 450 nm.

Western blotting

Cells were lysed in RIPA buffer containing PMSF and protease and phosphatase inhibitors (sc-24948, Santa Cruz Biotechnology) for 30 minutes on ice. Proteins were separated on denaturing 4%— 15% SDS-PAGE (4561086, Bio-Rad), transferred onto nitrocellulose membrane (1704270, Bio-Rad) and probed with antibodies to ART (4685S), pAKT (Ser473, 4060S), anti-mouse TFEB (32361 S), anti-human p-TFEB (Ser211, 37681S), FOXOl (2880T, all Cell Signaling Technology), anti-human TFEB (ab220695, Abeam) and VPS39 (ab224671, Abeam). Membranes were developed using HRP-conjugated secondary antibodies (Cell Signaling Technology) and Chemiluminescent Western Blot Detection Substrate (Thermo Fisher Scientific).

TGF-fi/SMAD reporter assays

TGF-p/SMAD signaling activity were determined by using the SBE Reporter Kit (60654, BPSbioscience) according to manufacturer's instructions. Briefly, 1 x 10 ⁶ naive CD4 T cells were cotransfected with 500 ng of reporter (component A) and either 0.4 nmol of control siRNA or 0.4 nmol of VPS39 siRNA. Alternatively, cells were cotransfected with 500 ng of negative control reporter (component B) and either 0.4 nmol of control siRNAor 0.4 nmol of VPS39 siRNA. Each condition was set up triplicates. After transfection, cells were stimulated with anti-CD3/SMAD signaling activity were determined by using the SBE Reporter Kitanti-CD28 dynabeads in the presence or absence of 10 ng/SMAD signaling activity were determined by using the SBE Reporter Kitml TGF-b. On day 3 after activation, cells were analyzed for luciferase activities by using the Dual-Luciferase® Reporter Assay System (Promega). The normalized luciferase activity was obtained by subtracting background luminescence from the negative control followed by calculating the ratio of firefly luminescence from the SBE reporter to Renilla luminescence from the control Renilla luciferase vector.

Statistical analysis

Statistical analysis was performed using the Prism 7.0 software (GraphPad Software Inc.). Paired or unpaired two-tailed Student’s t-tests were used for comparing two groups. One-way ANOVA with Tukey’s post hoc test was used for multi-group comparisons p < 0.05 was considered statistically significant. Statistical details and significance levels can be found in the figure legends.

Example 2 - Lysosome-independent activation ofmTORCl in naive CD4 T cell responses. mTORCl is recruited to lysosomal membranes where it is activated by released amino acids. However, day 5-activated naive CD4 T cells from old adults have impaired lysosomal activity in parallel to enhanced mTORCl activity as shown by reduced fluorescence of cleaved de-quenched bovine serum albumin (DQ-BSA) and increased S6K1 protein phosphorylation, the downstream effector of mTORCl activity (Fig. 1A and Fig. 2 A and B). To examine this apparent conundrum, the effects of lysosome inhibition was investigated on mTORCl activity; chloroquine and Bafilomycin Al, two lysosomal acidification inhibitors, were used for pharmacological inhibition at nontoxic doses (Fig. 2C); TFEB silencing was used as a genetic intervention. All interventions reduced lysosomal activities but surprisingly neither reduced S6K1 (Fig. IB and C and Fig. 2D to F) nor AKT phosphorylation (Fig. 2G), the downstream effector of the growth- factor signaling arm of mTORCl pathway. See, for example, Liu, et al., Nat Rev Mol Cell Biol 21, 183-203 (2020).

The mechanism was investigated of how mTORCl activities is even increased under lysosome-deficient conditions in T cell responses of old adults. Because lysosome deficiency likely affects lysosomal efflux of amino acids, expression of genes encoding the amino acid signaling arm of the mTORCl pathway was examined using data from a recent RNA-seq study comparing naive CD4 T cell responses from young and old adults after activation. See, Hu, et al. , 2019, supra. SLC7A5 , which encodes a plasma membrane leucine transporter, is selectively more expressed in T cells from old adults while other genes involved in the nutrient arm do not change with age (Fig. ID). Increased SLC7A5 transcription was confirmed in an independent cohort of day 5-activated naive CD4 T cells from twelve young and twelve old individuals. In contrast, no difference was seen for SLC7A1 that encodes the arginine transporter (Fig. IE). SLC7A5 expression increased with activation in naive CD4 T cells from young and old donors, but expression was more sustained beyond day 3 in old naive CD4 T cells (Fig. 3 A-B). This difference persisted and was still seen for memory CD4 T cells (Fig. 3C). Accordingly, ATAC-seq studies showed increased chromatin accessibility of SLC7A5 in old memory CD4 T cells in three virus-specific (EBV, VZV and influenza) systems compared to young cells (Fig. IF). Consistent with increased SLC7A5 expression in lysosome-deficient aged T cells, pharmacological inhibition of lysosome activity or genetic silencing of TFEB upregulated SLC7A5 transcript and protein expression (Fig. 1G and H and Fig. 2H). These data suggested that the up-regulation of SLC7A5 transcription in day 5-stimulated naive CD4 T cells from old adults was mechanistically related to their reduced lysosome activity.

Upstream of the differentially opened chromatin sites of SLC7A5 are two c-MYC binding motifs (Fig. IF). c-MYC is the major driver of SLC7A5 transcription in T cell responses. See, Marchingo, etal ., Elife 9, (2020). As shown in Fig. 1H, c-MYC protein expression was increased after lysosomal inhibition or TFEB silencing, indicating a lysosome-dependent degradation of c-MYC protein. Consistently, day 5-activated aged naive CD4 T cells had higher c-MYC protein level compared to young cells (Fig. II). Global gene expression profiles obtained by RNA-seq supported the notion that day 5- activated old naive CD4 T cells had higher c-MYC activity. Gene set enrichment analysis (GSEA) showed that age-associated transcriptional signatures of day 5-activated naive CD4 T cells were correlated with the expression pattern of genes in the Hallmark MYC target pathway (Fig. 1 J). To confirm that the increased SLC7A5 expression is related to higher c-MYC protein expression, c-MYC silencing was performed in old naive CD4 T cells and found reduced SLC7A5 protein expression (Fig. IK). Taken together, these data showed that lysosome deficiency stabilized c-MYC protein, thereby increasing SLC7A5 expression in day 5-stimulated aged naive CD4 T cells

Example 3 - SLC 7 AS -dependent late endosomal mTORCl activation in naive CD4 T cell responses.

To determine whether SLC7A5 activity accounts for the sustained mTORCl activity in day 5-stimulated old naive CD4 T cells, T cell cultures from young and older adults were treated with the specific SLC7A5 inhibitor JPH203. mTORCl activities were monitored by Western blotting of S6K1 phosphorylation or by flow cytometry of S6RP phosphorylation on days 3 and 5 after activation. In titration experiments, a dose of 5 mM JPH203 was not cytotoxic (Fig. 2C) and inhibited mTORCl activity by about 50% (Fig. 3D). On day 3, SLC7A5 inhibition reduced mTORCl activity in both young and old cells (Fig. 4A and B). On day 5, mTORCl activity was already attenuated in T cell responses of young adults. In contrast, mTORCl activity remained high in old activated T cells; the excess activity was sensitive to SLC7A5 inhibition (Fig. 4Aand B). To exclude off-target effects of the inhibitor, genetic SLC7A5 silencing and overexpression were performed. Data observed were consistent with those obtained by JPH203 treatment (Fig. 3E-H). Taken together, old activated CD4 T cells had a sustained uptake of extracellular amino acids through SLC7A5 to maintain mTORCl activity in spite of reduced lysosomal function.

Activated T cells from old adults failed to rebuilt their lysosomes and expand the late endosomal compartments. Jin, et al, SciAdv 6, eabal808 (2020). To investigate whether these late endosomes provide an alternative platform for mTORCl signaling in T cells, endosome fractions were isolated from naive CD4 T cells on day 3 after stimulation when mTORCl activity peaks in both young and old individuals. The endosome isolates were enriched for early and late endosome markers (EEA1, RAB7 and CD63) and depleted of the lysosome markers Cathepsin D and Cathepsin H, and of the plasma membrane marker Na/K ATPase, indicating the high purity of the endosome extracts (Fig. 4C). These endosome isolates contained mTOR, Regulatory-associated protein of mTOR (Raptor) and Ras homolog enriched in brain (RHEB) that are essential for substrate recruitment and kinase activity of mTORCl (Fig. 4C). In an in vitro kinase assay, these endosome isolates exhibited mTORCl kinase activity toward the substrate S6K1 that was not seen in endosomes extracted from cells treated with an ART inhibitor (Fig. 4C). Day 5-stimulated naive CD4 T cells from old individuals had reduced lysosomal cathepsins, similar early endosome contents (EEA1) and increased late endosomal compartments, with increased late endosomal mTOR, Raptor and RHEB protein levels and higher mTORCl kinase activity as shown by increased S6K1 phosphorylation in the in vitro kinase assay (Fig. 4D). Taken together, these data indicate that mTORCl is activated on late endosomes and that late endosomal compartments are expanded in stimulated naive CD4 T cells from old adults accounting for the sustained mTORCl activity.

VPS39 silencing was performed to examine whether inhibition of late endosome maturation prevents mTORCl activation. Confocal imaging confirmed that VPS39 silencing inhibited late endosome maturation and produced hybrid endosomal compartments that contain both the early endosome marker EEA1 and the late endosome/lysosome marker LAMP1 (Fig. 4E). These hybrid compartments co-localized with mTOR (Fig. 4E); however. S6K1 and S6RP phosphorylation were reduced after VPS39 silencing documenting that these hybrid structures do not support mTORCl activation (Fig. 4F and G). T cells from young and old adults exhibited reduced mTORCl activities after VPS39 silencing on day 3 after T cell stimulation. On day 5, an effect of silencing was only observed in T cells from old adults (Fig. 4F and G) suggesting that the sustained mTORCl signaling observed with age depended on the formation of late endosomes. VPS39 has also been implicated in regulating TGF-p/SMAD signaling (see, e.g., Felici, et al., EMBO J 22, 4465-4477 (2003)); however, no changes of SMAD2/3 phosphorylation or reporter activities of a SMAD reporter were observed in T cells after VPS39 silencing (Fig. 5A-C), supporting the notion that the observed reduced mTORCl activity is due to its impact on endosome maturation and not SMAD signaling.

Example 4 - Sustained activation of late endosomal mTORCl suppresses lysosomal activities in naive CD4 T cell responses.

The data thus far demonstrated that CD4 T cells from old adults sustain mTORCl activity on late endosomes that relies on the presence of SLC7A5 and VPS39. mTORCl phosphorylates TFEB, thereby preventing its transcriptional activity and inhibiting lysosome function. See, e.g., Roczniak-Ferguson, et al, Sci Signal 5, ra42 (2012). mTORCl inhibition downregulated TFEB phosphorylation while upregulating TFEB mRNA and protein levels due to reduced AKT-mediated FOXOl protein degradation (Fig. 7A-C). Consistent with this notion, mTORCl inhibition by Torin 1 of day 5- activated T cells from old adults partially restored the TFEB-dependent transcription of lysosomal cathepsin genes ( CTSB , CTSD, CTSH and CTSS) (see, e.g., Jin, etal. , Sci.

Adv. , 6 eaba 1808 (2020)) and lysosomal proteolytic activities (Fig. 6Aand B). Inhibition of late endosomal mTORCl in activated T cells from old adults by either VPS39 silencing or SLC7A5 inhibition had similar effects, while the already higher lysosomal activity in T cells from young adults could not be further increased (Fig. 6C-E). However, SLC7A5 inhibition could counteract the effects of lysosome inhibition in young T cells. As shown in Fig. 6F, lysosomal cathepsins and lysosomal activities were increased in CQ and JPH203 -cotreated young cells compared to young T cells treated with CQ alone. Together, these data demonstrate that sustained activation of mTORCl in late endosomes suppressed lysosomal activities in day 5-stimulated naive CD4 T cells from old adults.

Example 5 - Sustained activation of late endosomal mTORCl prevents PD-1 from lysosomal degradation.

It was previously observed that in the course of T cell proliferation after activation

FOXOl promoted the generation of lysosomes through induction of TFEB transcription.

See, e.g., Jin, et al. , Sci. Adv. 6, eaba 1808 (2020). Moreover, Foxol -deficient mouse CD4 T cell showed up to fourfold increase of PD-1 protein levels after antigen priming in vivo (Stone, et al, Immunity 42, 239-251 (2015)), raising the possibility that lysosomal activity regulates PD-1 protein expression. As shown in Fig. 4A, lysosome inhibition by TFEB silencing in T cells from young adults increased cell surface protein expression of PD-1 while transcript levels remained unchanged. In contrast, stimulation of lysosomal activities following late endosomal mTORCl inhibition in T cells from old adults by either Torin 1 treatment, SLC7A5 inhibition or VPS39 silencing reduced cell surface protein expression without effects on transcripts of PD-1 (Fig. 8B-D). This effect was not due to the changes of intracellular vs cell surface trafficking routes of PD-1 since VPS39 silencing equally reduced cell surface and intracellular PD-1 protein expression (Fig. 8E- G). Assessment of PD-1 half-life in vitro in day 5-stimulated old naive CD4 T cells confirmed that 171V39-silenced cells underwent enhanced PD-1 protein degradation than control-silenced cells (Fig. 8H). These data suggest that mTORCl at late endosomes stabilizes PD-1 protein by preventing lysosomal degradation of PD-1.

To determine whether PD-1 protein regulation changes with age, PD-1 expression was monitored at days 0, 3 and 5 after stimulation by flow cytometry. Consistent with the changes of the kinetics of mTORCl activity with age, PD-1 protein expression peaked at day 3 after activation in both young and old activated naive CD4 T cells to then subside in activated T cells from young but not old adults. As shown in Fig. 81 and J, there were no age-related differences of protein expression of PD-1 at days 0 or 3, but activated T cells from old adults had more PD-1 protein at day 5 while no difference in PDCD1 transcription.

Example 6 - Sustained activation of late endosomal mTORCl impairs expansion of naive CD4 T cells from old adults.

Failure to timely degrade PD-1 protein raises the possibility that lysosome- deficient T cells receive an increased inhibitory signal suppressing cell expansion. To test this hypothesis, naive CD4 T cells were stimulated with anti-CD3/anti-CD28 beads in the presence or absence of PD-Ll-Fc and anti-human IgG to cross-link PD-1 (Shi, et al ., Immunity , 49, 264-274 e264 (2018)). VPS39 silencing induced increased proliferation and cell recovery in the presence of PD-1 stimulation (Fig. 9A). In contrast, VPS39 silencing only caused a minor increase in CD4 T cell proliferation in the absence of PD-1 crosslinking that was not sufficient to increase cell numbers (Fig. 9A). These data indicate that late endosomal mTORCl activity regulates T cell expansion through lysosomal degradation of PD-1. Consistent with their sustained mTORCl activation on late endosomes and reduced lysosomal activity and therefore degradation of PD-1, naive CD4 T cells from old adults had reduced potential to proliferate and to expand in the presence of PD-1 crosslinking (Fig. 9B). In the absence of exogenous PD-Ll-Fc, age- related proliferation differences were not observed supporting the notion that the proliferative defect was related to increased PD-1 expression in T cells from old adults (Fig. 9B).

A similar effect of endosomal differentiation interference was also seen for antigen-specific T cell responses. In these experiments, CTV-labeled PBMCs from healthy individuals were stimulated with a pool containing both HLA class II peptide megapool covering the entire SARS-CoV-2 orfeome together with HLA class I peptide megapools covering half of the entire orfeome. To silence VPS39 in human T cells, FANA antisense oligonucleotide (FANA ASO) was added, which has the advantage of easy self-delivery without the need of transfection regents. Two VPS39 FANA ASO clones (#1 and #2) were added to replicate cultures at day 0. Frequencies of divided T cells were determined on day 8. Among eleven SARS-CoV-2-unexposed individuals tested, CD4 and CD8 T cell responses to SARS-CoV-2 peptide pool were observed in six individuals. VPS39 FANA ASO clone #1 and #2 significantly increased the SARS-CoV- 2-reactive CD4 and CD8 T cell expansion compared to scrambled control FANA ASO in those individuals who showed a response (Fig. 9C). A similar increase of proliferating cells was not seen for PBMCs cultured in the absence of antigenic peptides (Fig. 9C). Consistent with recent reports that SARS-CoV-2 responses in unexposed individuals derive from memory T cells, VPS39 silencing improved human T cell responses to pertussis peptide pools (Fig. 9D). A similar increase in pertussis-specific responses was seen with anti-PD-1 blockade (Fig. 9E). Example 7 - Inhibition of late endosomal mTORCl promotes primary CD4 T cell responses after LCMV infection in vivo.

To determine whether improved expansion due to lysosome dysfunction is also seen with antigen-specific T cell responses in vivo , the LCMV infection mouse model was used. SMARTA CD4 T cells were retrovirally transduced with shRNA specific for Tfeb (sh Tfeb) or control shRNA (shCtrl) and transferred the cells into 5- to 8-week-old B6 mice followed by acute LCMV infection. Tfeb silencing induced an increased PD-1 expression, leading to a reduced SMARTA CD4 T cell expansion at peak responses (Fig. 10A-C).

To determine whether LCMV-specific CD4 T cell responses can be boosted by inhibiting late endosomal mTORCl in vivo , we transferred SMARTA CD4 T cells transduced with shRNA specific for Vps39 (sh Vps39) or control shRNA (shCtrl) into B6 mice followed by acute LCMV infection. f wdP-silenced SMARTA CD4 T cells had reduced mTORCl activities, decreased PD-1 expression, enhanced lysosomal activities and up to a threefold increase in cell numbers in the spleen at peak responses on day 8 (Fig. 10D-G and Fig. 11 A). f wdP-silenced, expanded SMARTA CD4 T cells were functional and produced similar levels of cytokines as control-silenced cells after peptide restimulation ex vivo (Fig. 11B). Decrease of the Annexin V ⁺ population indicated reduced apoptosis as a mechanism of the increased SMARTA CD4 T cell recovery after Vps39 silencing, with an additional trend for increased cell proliferation as shown by Ki67 ⁺ population (Fig. 10H). To further examine whether the increased SMARTA CD4 T cell expansion is at least in part due to reduced PD-1 expression, the PD-1/PD-L1 pathway was blocked by injecting mice with anti-PD-1 blocking antibody. As shown in Fig. 101, PD-1 blockade promoted control-silenced SMARTA CD4 T cell expansion at peak responses after LCMV infection while it did not further improve l/As39-silenced SMARTA CD4 T cell expansion. At day 30 after infection, Vps39- silenced SMARTA CD4 T cells showed over 15-fold increase in cell numbers with less contraction during the effector/memory transition than control-silenced SMARTA cells (Fig. 10J and K), consistent with the previously reported role of mTORCl inhibition in promoting memory generation. See Araki, et ah, Nature 460, 108-112 (2009)). Vps39 silencing by a second shRN A reproduced the data (Fig. 11D and E), excluding the possibility of off-targets effects. Late endosomal mTORCl inhibition by partial Slc7a5 silencing in SMARTA CD4 T cells (Fig. 11G) reproduced the data with Vps39 silencing, although to a lesser extent. mTORCl activities and PD-1 protein expression were both reduced after Slc7a5 silencing, while SMARTA cell number were slightly increased (Fig. 10L-N). Again, this increase of cell number was mostly due to reduced cell apoptosis, while proliferation was even slightly decreased (Fig. 10O). Taken together, these data show that inhibition of late endosomal mTORCl promotes virus-specific CD4 T cell expansion and memory generation after primary acute LCMV infection.

Example 8 - Inhibition of late endosomal mTORCl augments CD4 T cell helper responses in vivo.

The robust expansion at primary peak responses and the reduced contraction during memory transition of f wdP-silenced SMARTA CD4 T cells prompted the further examination of whether antibody and memory CD 8 T cell responses in host mice were affected. As shown in Fig. 12A, relative frequencies of CXCR5 ^hi PD-l ^hi germinal center (GC) within adoptively transferred SMARTA CD4 T cells remained unchanged, suggesting that Vps39 silencing did not bias differentiation. However, the number of GC SMARTA TFH cells on day 8 was highly increased after Vps39 silencing (Fig. 12A). Consequently, Fas ⁺ GL-7 ⁺ GC B cell numbers (Fig. 12B), CD138 ⁺ IgD plasma cell numbers (Fig. 12C) and LCMV-specific serum IgG titer (Fig. 12D) were increased in mice receiving f wdP-silenced SMARTA CD4 T cells compared to mice receiving control-silenced cells. At day 30 after infection, the number of endogenous GP33 tetramer positive CD8 T memory cells increased by 2-fold in mice receiving Vps39- silenced SMARTA CD4 cells compared to mice receiving control-silenced cells (Fig. 12E and Fig. 1 IF). After rechallenge with Lm-GP33, these memory CD8 T cells underwent increased fold expansion and appear to have superior memory cell quality as shown by increased IFNy production on a per-cell basis (Fig. 12F-H).

To examine whether these changes were due to reduced PD-1 expression in the adoptively transferred cells, the PD-1/PD-L1 pathway was blocked by injecting mice with anti-PD-1 blocking antibody. As shown in Fig. 13A-C, PD-1 blockade increased the numbers of transferred GC SMARTATFH cells, endogenous GC B cells and plasma cells in mice receiving control-silenced cells while it did not further increase these numbers in mice receiving Vps39- silenced cells.

In summary, it was shown that mTORCl signaling occurs at the late endosome and not at the lysosome in old T cells. This has important consequences for the regulation of mTORCl activity as it changes a negative regulatory feedback to a forward loop. mTORCl phosphorylates TFEB, thereby inhibiting transcription of lysosomal genes and impairing proteolytic activity that is required for further activation of mTORCl at the lysosome. In contrast, reduced lysosomal activity in T cells from older adults induces the expansion of late endosomes and the expression of the leucine transporter SLC7A5, the latter by failing to degrade c-MYC. Together, increased late endosome mass and increased SLC7A5 activity support further mTORCl activation leading to further lysosome dysfunction. Progressively dysfunctional lysosomes fail to degrade PD-1, resulting in its increased and sustained cell surface expression and inhibition of proliferation. This cycle can be broken by inhibiting or silencing SLC7A5 or VPS39 that restore TFH generation in T cells from older adults and augments the generation of germinal center and CD8 memory response in the LCMV model of infection.

Lysosomal activation of mTORCl is controlled during cell proliferation by the cross-regulation of lysosomal and mTORCl activities. When lysosomal activity is deficient and amino acid efflux is low, mTORCl -dependent phosphorylation of TFEB is reduced, resulting in the translocation of TFEB into the nucleus, where it stimulates lysosomal gene transcription and restores lysosomal activities; Adequate lysosomal activities trigger the mTORCl -dependent phosphorylation and cytoplasm retention of TFEB and the termination of its transcriptional induction of lysosomal genes. This feedback loop appears to be dysregulated in lysosome-deficient aged T cells as they fail to down-regulate mTORCl to restore TFEB-dependent lysosomal genes expression; instead they show enhanced late endosomal mTORCl activity with even more reduced lysosomal genes expression compared to the young cells.

Late endosomes are acidic organelles that, in contrast to lysosomes, do not have proteolytic activity and amino acid recycling ability. They function as temporary storage tanks for proteins that are sorted into the lysosomal degradation pathway after fusion with lysosomes or secreted as exosomes. Late endosome turnover is regulated downstream by lysosomal degradation and upstream by VPS39-mediated biogenesis from early endosomes. Inhibition of lysosomal activities induces an intracellular expansion of late endosomes. Activated T cells from older adults are deficient in rebuilding functional lysosomes as consequence of FOXOl degradation and therefore expand this late endosomal compartment, which provides an alternative platform for mTORCl activation. Although late endosomes do not degrade protein to recycle amino acids for mTORCl signaling, the increased plasma membrane leucine transporter SLC7A5 induced by lysosome inhibition facilitated the uptake of extracellular amino acids as an alternative amino acid source for late endosomal mTORCl activation. In addition to late endosomes, mTORCl can be activated on the surface of Golgi via recruitment to the Golgi membrane, involving RAB1 A and Golgi-resident RHEB. Recruitment of mTORCl in mitochondria membrane by co-targeting Raptor and RHEB to mitochondria can also allow its activation. These lysosome-independent mTORCl signaling may contribute to a sustained high level of mTORCl activities as we saw in T cells from old adults in spite of impaired lysosomes, but they lack the positive feedback loop seen with late endosomal mTORCl.

One additional component in this network is activated AKT that is more sustained in naive T cell responses of old adults due to the increased expression of miR-21 and the repression of its target phosphatase and tensin homolog (PTEN). In addition to phosphorylating tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity, AKT also phosphorylates and inactivates FOXOl that is needed for generation of new lysosomes. FOXOl promotes lysosomal activities through induction of TFEB transcription in CD4 T cells. This function was impaired in T cell responses of older adults due to increased degradation of FOXOl after T cell stimulation. Sustained mTORCl activation at the late endosomes further inhibits lysosome activity that play an important role in promoting effector T cell survival, mainly through the autophagy/lysosome pathway. One possible pathway how lysosome dysfunction in older adults affects T cell responses is a failure to curtail activation-induced PD-1 expression. FOXOl -deficient mouse CD4 T cells, in part recapitulating FOXOl -deficiency in activated T cells from old adults, had an up to fourfold increase of PD-1 protein levels and a major defect in T cell expansion after antigen priming in vivo. Moreover, PD-1 is degraded by lysosomes. Indeed, proliferation of T cells from old adults, which was intact after polyclonal stimulation, was impaired by increased cross-linking of PD-1 with PD-L1 compared to that of T cells from young adults.

Silencing of VPS39 to inhibit late endosome generation and activation of mTORCl promoted T cell expansion to anti-CD3 bead stimulation in the presence of PD-L1 stimulating PD-1. A similarly enhanced expansion of VPS39-deficient T cells to stimulation of SARS-CoV-2 and pertussis peptides in the presence of antigen-specific cells was seen in vitro. The increased responses resembled those achieved by PD-1 blocking. In the acute LCMV infection model, adoptive transfer of VPS39-deficient LCMV-specific CD4 T cells resulted in increased clonal expansion, germinal center formation and improved CD8 recall responses. These results with 17fV39-silenced T cells resemble results where the PD-1/PD-L1 pathway blockade enhanced both effector function and frequency of memory precursor of antigen-specific CD8 T cells and reduced viral load in acute LCMV infection. In an immunization model, PD- 1 blockade promoted the generation of follicular helper T cells that are specialized in promoting cognate B cell responses. Thus, targeting VPS39 could serve as a strategy to conquer different types of virus infections and to establish the improved immune memory in older individuals. In addition to PD-1, several molecules of functional relevance are targets of lysosomal degradation. CTLA-4, another inhibitory receptor, was also reduced after enhancing lysosomal activities by late endosomal mTORCl inhibition. However, CTLA-4 expression occurred early in a T cell response and before the observed age-related differences in mTORCl activity.

Inhibition of late endosomal mTORCl may serve as a strategy in compensating for age-related defects in T cell differentiation and the generation of TFH and memory cells. In addition to VPS39, there are five other protein components of HOPS complex (VPS11, VPS16, VPS18, VPS33 and VPS41) that also play a role in mediating early to late endosome conversion and could be targeted. In addition, partially inhibition of SLC7A5 to compensate for the physiological differences in expression in CD4 T cells from young and old adults could also be explored. However, complete inhibition will likely be detrimental because knockout of Slc7a5 profoundly impaired T cell activation and expansion. Based on the early studies in the mouse that mTORCl inhibition by low dose of rapamycin produced an enhanced primary and memory recall CD8 T cell response as well as promoted follicular helper cell over Thl cell generation, clinical studies have been started. Treatment with low dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor enhanced the immune response to the influenza vaccine and reduced the percentage of CD4 and CD8 T lymphocytes expressing PD-1 in older individuals. However, a subsequent study by resTORbio was not successful, missing the primary endpoint (www.ClinicalTrials.gov; Identifier: NCT04139915). It should be noted that the design of the study was to show an overall improvement in immune health. The vaccine response was not specifically targeted, in fact, there was a wash out period before the vaccination. Targeting mTORCl activation on late endosomes in the days subsequent to the initiation of the T cell activation rather than mTORCl globally may be more advantageous to stimulate T cell responses and enhance vaccine responses functions in general and in particular in older individuals.

Example 9 Preventing Melanoma Growth Using Adoptively Transferred Vps39- silenced CART cells.

FIG. 14A provides a schematic of an experiment to prevent melanoma growth using adoptively transferred Vps39- silenced CAR-T cells. Naive OT-I cells were activated and transduced with a retroviral vector expressing either scrambled control RNA or Vps39 shRNA. At day 6 after activation, retrovirus-transduced Amcyan ⁺ OT-I cells were sorted, and 5 ^c 10 ⁴ transduced cells were intravenously transferred to wild- type C57BL/6 (CD45.2) mice (The Jackson Laboratory). At day 2 after transfer, mice were injected in the flank subcutaneously with 5 x 10 ⁵ B 16-OVA cells (mouse melanoma cells expressing ovalbumin peptide residues 200-290, a gift from Matthew Williams at University of Utah). FIG. 14B shows the tumor growth curves for B 16-OVA tumors following transfer of naive OT-1 control or Vps39- silenced CD8 ⁺ T cells separately into wild-type recipients. FIG. 14C shows the survival curves of the same mice. Tumor volume was significantly lower in the mice containing the adoptively transferred Vps39- silenced CD8 ⁺ T cells, and such mice had a significantly increased percent survival. FIG. 14D shows cell surface PD-1 expression of tumor- infiltrating Amcyan ⁺ OT-I cells at day 9 after tumor implant and FIG. 14E shows the numbers of tumor-infiltrating Amcyan ⁺ OT-I cells at day 9 after tumor implant. FIG. 14F shows cytokine production (TNFa and IFN-g) of tumor-infiltrating Amcyan ⁺ OT-I cells restimulated with OVA257- 264 peptide ex vivo at day 9 after tumor implant. Alternatively, at day 18 after tumor implant, Amcyan ⁺ tumor-infiltrating OT-I cells were sorted and injected into a new group of wild-type C57BL/6 mice followed by B16-OVA tumor implant. FIG. 14G shows tumor growth curves for B 16-OVA tumors following transfer of OT-1 control or Vps39- silenced CD8 ⁺ T cells that had been challenged with the same tumor cell line for 18 days separately into wild-type recipients.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.