COMPOSITIONS AND METHODS FOR TREATMENT OF AN ABNORMAL IMMUNE RESPONSE VIA GSK INHIBITION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of United States Provisional Application 62/537,092, entitled GSK inhibitors as a therapeutic target to alter T cell function/differentiation in auto-immune disease/colitis development, filed on July 26, 2017, the contents of which are incorporated in its entirety for all purposes.

BACKGROUND

[0002] An autoimmune disease is a condition arising from an abnormal immune response, mostly but not limited to a T cell response or antibody response against normal cells and/or tissues of the body. There are at least 80 types of autoimmune diseases, which may affect nearly any body part. While the cause may generally be unknown, some autoimmune diseases such as lupus run in families, thus involving hereditary factors or genetic defects increasing the risk of disease, and certain cases may be triggered by infections or other environmental factors. Common diseases that are generally considered autoimmune include, but are not limited to, celiac disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, auto-immune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) and systemic lupus erythematosus.

[0003] Current treatments may include nonsteroidal anti-inflammatory drugs

(NSAIDs) and immunosuppressants, and depend on the severity of the condition. While treatment may improve symptoms the disease typically is not cured, and there is a need for improved treatments for those with immune diseases. In particular, there is a need for methods that may improve T cell immunosuppressive function and immune homeostasis that may prevent or correct immune-associated sequelae observed in patients having immune disorders characterized by faulty regulatory T cell function. Further, there is a need in the art for early identification of individuals that may be likely to develop an autoimmune disease, including, but not limited to, islet autoimmunity in type I diabetes, systemic lupus erythematosus, auto-immune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or asthma. The instant invention seeks to address one or more of the aforementioned needs in the art.

BRIEF SUMMARY

[0004] Disclosed herein are glycogen synthase kinase-3 (for example, GSK or GSKa) inhibitors for use in the therapy of a T-cell mediated disease in an individual in need thereof. The T-cell mediated disease may be one associated with a loss of Treg

immunosuppressive function, for example, an immune disease or an autoimmune disease such as, for example, an immunodeficiency, primary immunodeficiency disease (PID), type I diabetes, systemic lupus erythematosus (SLE), asthma, colitis, psoriasis, bronchiectasis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or combinations thereof. The disclosed compositions and/or methods may be used to ameliorate, prevent, or reduce the severity and/or progression of any aforementioned disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0006] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0007] FIG 1A-1D: Loss of Gimap5 impairs CD4+ T cell survival and iTreg cell induction. Ten-week-old control Gimap5 ^WT/WT ;Rag2 ^/ ;OT-II and Gimap5 ^s P ^h/s P ^h ;Rag2-/-;OT- II mice received either normal water or water containing 1 mg/mL ovalbumin ad libitum during a 5-week period. At 15 weeks of age, (A) the number of splenic CD4+ T cells and (B) the percentage of naive (CD441o;CD62hi) and memory-like (CD44hi;CD621o) CD4+ T cells was determined using flow cytometry. (C) Frequency of iTregs (CD4+CD25+) in the mesenteric lymph nodes (MLN) of vehicle- and OVA-treated mice (n=4). (D) Survival of peripheral (splenic) CD4+ T cells ex vivo in the presence of IL-7 (5 ng/mL) as determined by live/dead staining and flow cytometry (n=3). Data represent mean values + SD of samples from individual mice; statistical significance is determined by Student's two-tailed test.

[0008] FIG 2A-2H: Impaired CD4+ T cell proliferation is associated with increased GSK3 activity. (A) Immunoblot analysis of c-Myc expression in total lysates of CD4+ T cells from WT and Gimap5 ^Ρ ¹¹ mice stimulated with aCD3/aCD28 or during resting conditions. (B) Myc mRNA levels in resting and aCD3/aCD28-activated (24h) CD4+ T cells. Data represents mean expression + SD relative to unstimulated WT cells (n=6). (C)

Phosphorylation c-Myc (T58) after 24h of aCD3/aCD28 stimulation. Proteasomal inhibitor MG132 was added after 20h of stimulation. Ratio of p-c-Myc (T58) to total c-Myc in MG132-treated Gimap5 ^sph/sph CD4+ T cells relative to WT (n=5). (D) C-Myc expression in WT and Gimap5 ^S P ^H/S P ^H CD4+ T cells stimulated for 24h with aCD3/aCD28 + 2.5mM LiCl. (E) Proliferation of WT and Gimap5 ^S P ^H/S P ^H CD4+ T cells in the presence/absence of aCD3/aCD28 and/or GSK3 inhibitors BIO (lOOnM) or LiCl (2.5 mM) as measured by CFSE dilution after 3 days. Experiments were repeated 5 times and representative plots from a single experiment are shown. (F) Representative images of NFATcl localization in resting and aCD3/aCD28-activated WT and Gimap5 ^S P ^H/S P ^H CD4+ T cells. (G,H) Bright detail similarity quantification of NFATcl nuclear localization upon stimulation (G) and in the in the presence/absence of BIO (H) for 4 hours (n=6). Graphs depict mean values + SEM. ImageStream data represents average values of >500 CD4+ T cells per sample; all experiments were performed at least three times. Statistical significance is determined by Student's two-tailed test. BF: Bright field

[0009] FIG 3A-3I: Loss of Gimap5 results in impaired inactivation of GSK3 . (A) Association of GSK3 with vesicles in WT and Gimap5 ^S P ^H/S P ^H CD4+ T cells as exemplified by GSK3 intensity, vesicle number, and vesicle size of GSK3 spots 6 and 24h after aCD3/aCD28 activation. (B) Bright detail similarity analysis (ImageStream) of activated CD4+T cells from WT and Gimap5 ^s P ^Ns P ^h mice show colocalization between Gimap5 and GSK3 . (C) Representative Z-stacks of WT and Gimap5 ^sph/sph CD4+ T cells stimulated for 24h with aCD3/aCD28. (D) Immunoblot analysis of WT and Gimap5 CD4+ T cells stimulated with aCD3/aCD28 depicting total and phosphorylated protein levels of GSK3 (P- Ser9 and P- Ser389), p38, and p53. (E-I) Localization of total and phospho-GSK3 (Ser389) in WT or Gimap5 CD4+ T cells after 2 days stimulation with aCD3/aCD28 (n=3). (F) Nuclear localization and (H) expression of total GSK3 . (G) Nuclear localization and (I) expression of p-GSK3 (Ser389). Hatched bars represent resting and solid bars represent CD3/CD28-activated CD4+ T cells. Graphs depict mean values + SD. ImageStream data represents average values of >500 CD4+ T cells per sample. All experiments were performed at least three times. Statistical significance is determined by Student's two-tailed test. BF: Bright field

[0010] FIG 4A-4D: Loss of Gimap5 causes increased DNA damage in activated CD4+ T cells. (A) γΗ2ΑΧ expression in live WT or Gimap5 ^sph/sph CD4+ T cells following aCD3/aCD28 stimulation. (B) Survival as determined by viability stain of WT or Gimap5 _S ph/ _S ph _{CD4+ x cells after a} CD3/aCD28 stimulation (n=4). (C,D) Effect of lithium on γΗ2ΑΧ expression in WT or Gimap5 ^s P ^h/s P ^h CD4+ T cells, after 3 days stimulation with aCD3/aCD28. Plots depict live cells (C,D), while bar graphs (D) represent mean values + SD (n=6). All experiments were performed at least three times. Statistical significance is determined by Student's two-tailed test.

[0011] FIG 5A-5G: GSK3 inhibition improves lymphocyte survival and prevents immunopathology. (A,B) LiCl treatment of Gimap5 ^s P ^h/s P ^h mice in vivo starting at 3 weeks of age and analyzed at 7-8 weeks of age, rescues CD4+ T cell (A) and B cell (B) survival. (C) Reduced frequency of CD4+ T cells undergoing lymphopenia-induced proliferation

(CD44hi;CD621o) upon LiCl treatment of Gimap5 ^s P ^h/s P ^h mice in vivo. (D) Suppressive capacity of regulatory T cells (CD4+CD25hi) isolated from vehicle or LiCl-treated WT and Gimap5 ^s P ^h/s P ^h mice. Data represents mean + SD and is representative to two independent experiments (n=3). (E,F) Reduced colitis as defined by disease score (E) and based on colon histology (F) in 7-8 week old Gimap5 ^s P ^h/s P ^h mice following LiCl treatment (3.5 weeks) in vivo. (G) Reduced liver pathology in Gimap5 ^s P ^h/s P ^h treated with LiCl. Data represent mean values + SD from 6 mice per group at 7-8 weeks of age; histology is a representative depiction of disease severity. Statistical significance is determined by ANOVA followed by Sidak's multiple comparisons test.

[0012] FIG 6A-6D: GSK3 -deletion in CD4+ T cells improves CD4+ T cell survival and prevents colitis. (A) Tamoxifen treatment of Gimap5 ^s P ^h/s P ^h ; Gsk3 fl/fl; Cd4cre-ert2 mice starting at 3 weeks of age selectively rescues splenic CD4+ T cell survival while maintaining overall CD4+ T cell quiescence (B). (C,D) Reduced colitis in Gimap5 ^sph/sph ; Gs^*™;

Cd4cre-ert2 mice treated with tamoxifen as determined by disease scores (C) based on histology (D). Data represent mean values + SEM from at least 6 mice per group at 8 weeks of age; histology is a representative depiction of disease severity. Statistical significance is determined by ANOVA followed by Sidak's multiple comparisons test.

[0013] FIG 7A-7D: A human loss-of-function mutation in GIMAP5 results in a similar T cell deficiency. (A) Whole exome sequencing uncovered a homozygous variant (SNP: rs72650695), causing an L204P amino acid change in GIMAP5, resulting in complete loss of GIMAP5 protein expression in T cells. (B) Impaired expansion of CD3+ T cells from GIMAP5-/- patient compared to heterozygous control cells following stimulation with PHA (4 days) and IL-2 (days 4-10). The proliferation capacity is restored in the presence of LiCl. Experiment is representative of three independent experiments from samples obtained several months apart. (C,D) Immunoblot analysis of control and GIMAP5-/- CD3+ T cells shows reduced c-Myc expression (C) at resting conditions and (D) after 2-day restimulation with aCD3/aCD28 +/- 5 mM LiCl. Immunoblot was repeated twice with an identical outcome.

[0014] FIG 8A-8J: Human GIMAP5-/- T cells show impaired GSK3 sequestration and increased DNA damage. (A-D) Colocalization and spot analysis of GIMAP5+, GSK3 + and CD 107+ vesicles in primary CD4+ T cells isolated from healthy controls at a resting state (day 0) or after aCD3/aCD28-stimulation for 1-2 days (representative images from day 1 shown) using ImageStream analysis. (E-H) ImageStream analysis of GSK3 -specific vesicle association in control or GIMAP5-/- patient CD4+ T cells restimulated with aCD3/aCD28 after primary expansion. Data depicts (E) GSK3 + spot number, (F) GSK3 intensity therein, and (G) spot area in live CD4+ T cells at 0, 6, or 24 hrs of aCD3/aCD28-restimulation. Data represent mean values + SD. (H) Representative images of GSK3 vesicular association in control and GIMAP5-/- CD4+ T cells taken using a 60x objective. (I,J) Analysis of DNA damage response (γΗ2ΑΧ) in control of GIMAP5 ^sph/sph CD4+ T cells after (I) 1-3 days or (J) 2 days restimulation with aCD3/aCD28. Data represents mean values of a single experiment performed in duplicate and repeated twice. ImageStream data represent average values of >500 CD4+ T cells per experiment. BF: Bright field [0015] FIG 9: Gimap5 is a critical regulator of GSK3 during T cell activation.

Gimap5 controls regulation of GSK3 in T cells through vesicular sequestration and affects both the 1) (early) transcriptional program required for T cell growth, and 2) the late stage nuclear accumulation of P- Ser389 GSK3 required for the DNA damage response during cycling. Gimap5-deficient CD4+ T cells, fail to inhibit GSK3 leading to a failed transcriptional program and increased DNA damage during T cells proliferation.

[0016] FIG 10A-B. Normal thymocyte survival and thymic emigration. (A) Number of single positive CD4+/CD8- thymocytes surviving ex vivo when cultured with IL-7 (5 ng/mL) (n=6). Data depict mean +/- SD. (B) Frequency of peripheral CD4+ T cells (n=4). (C) Frequency of recent thymic emigrants (RTEs) (CD24hi) and naive (CD62LhiCD441o) cells among peripheral CD4+ T cells from three- week-old mice (n=6). Data depict mean +/- SEM. Statistical significance is determined by Student' s two-tailed test.

[0017] FIG 11A-11G. Reduced T cell survival is independent of Bim and Bax/Bak. (A-C) Mean frequency +/- SD of CD4+ T cells (A), CD 8+ T cells (B), and NK cells (C) in the spleen of 5-233k-old Bim deficient Gimap5 ^s P ^h/s P ^h mice (n>3 mice/group). (D) Analysis of CD4+ T cells undergoing lymphopenia-induced proliferation (CD621oCD44hi) in the spleen of 5 -week-old Gimap 5 ^s P ^h/s P ^h mice deficient for Bim. (E) Mcl-1 expression in CD4+ T cells stimulated for 24 h with alphaCD3/alphaCD28 (n=4); statistical significance is determined by Student's two-tailed test. (F) Frequency of CD4+ and CD8+ T cells in the spleen of 5-week- old Gimap5 ^s P ^h/s P ^h mice lacking pro-apoptotic factors Bax and Bak. Data is representative of two mice/group. (G) Frequence of dead + apoptotic CD4+ T cells after 8 h stimulation with alpha CD3/alphaCD28 +/- activating alpha CD95 (anti-Fas). Bars depict mean +/- SD (n=4).

[0018] FIG 12A-D. Normal proximal CD4+ T cell signaling in 3-week-old Gimap5 sph/sph mice (A) Analysis of the percentage of naive CD4+ T cells from 3 -week old WT and Gimap5 ^s P ^h/s P ^h spleens (n=4). (B) Phosphorylation of ERK (Thr202/Tyr204), JNK

(Thrl83/Tyrl85), AKT (Ser473), and p38 (Thyl80/Tyrl82) in CD4+ T cells from WT and Gimap5 ^s P ^h/s P ^h mice stimulated with PMA/Ionomycin for the indicated number of minutes. Values measured by flow cytometry and are relative to unstimulated samples and depict mean + SD (n=3). (C,D) Immunoblot analyses of mTOR and S6K phosphorylation in CD4+ T cells stimulated with aCD3/aCD28 for (C) lh and (D) 24h. Immunoblot data depicts representative immunoblots/graphs of at least 3 independent experiments performed on pooled CD4+ T cells.

[0019] FIG 13A-13F. Reduced β-catenin but normal NFATcl expression and calcium flux. Analysis of f3-catenin (A) protein and (B) mRNA in WT and Gimap5 ^S P ^H/S P ^H CD4+ T cells after 24h of stimulation with aCD3/aCD28 (n=6). (C) Immunoblot analysis of total f3- catenin protein levels after stimulation with aCD3/aCD28 + 2.5mM LiCl for 24h.

Immunoblot data depicts representative immunoblots of at least 3 independent experiments performed on pooled CD4+ T cells. (D) Proliferation of WT CD4+ T cells after 3 days of aCD3/aCD28 stimulation + calcineurin inhibitor cyclosporin A (200 ng/mL). Plots are representative of 4 independent samples. (E) Expression of NFATcl in WT and Gimap5 ^P ¹¹ CD4+ T cells stimulated for 4h with aCD3/aCD28 (n=3). (F) Representative flow plots detailing calcium flux upon stimulation with aCD3/aCD28 or Ionomycin. Comparison of calcium flux in WT and Gimap5 ^sph/sph CD4+ T cells upon stimulation with aCD3/aCD28 or Ionomycin. Plots are representative of 4 independent samples. Bars represent mean + SD. Statistical significance is determined by Student' s two-tailed test.

[0020] FIG 14A-14E. Vesicular localization of Gimap5 and GSK3p. (A)

Representative images of WT CD4 ⁺ T cells resting or stimulated 24h with aCD3/aCD28. (B) Number of Gimap5 ⁺ and Gimap5 ⁺ Lampl ⁺ vesicles. (C) Colocalization of GSK3 with Gimap5 or Lampl in activated CD4 ⁺ T cells. Colocalization of Gimap5 and GSK3 with Rab5, Rab7, and Lampl by (D) spot area. (E) Intensity of Rab5, Rab7, or Lampl within Gimap5 ⁺ GSK3 ⁺ vesicles. Bars depict mean + SD (n=4). Each ImageStream data point represents average values of >500 CD4 ⁺ T cells. BF: Bright field

[0021] FIG 15A-E. GSK3 vesicular localization is Wnt independent. (A-C)

Vesicular localization of GSK3 in WT CD4+ T cells stimulated 24h with 200 ng/mL Wnt3a or aCD3/aCD28 + 2 μΜ IWP-2 as measured by (A) number of GSK3 + spots, (B) GSK3 vesicular intensity, and (C) size of GSK3 + spots (n=3). Each ImageStream data point represents average values of >500 CD4+ T cells. (D) Proliferation of WT CD4+ T cells after 3d stimulation with aCD3/aCD28 + Wnt3a or rWP-2 (n=4). Data represent mean + SD. (E) Wnt3a mRNA levels in resting and aCD3/aCD28-activated (24h) CD4+ T cells (n=9). Bars depict mean + SEM. Statistical significance is determined by Student's two-tailed test. [0022] FIG 16A-16F. TCR-induced P-Ser9 GSK3 is unaffected. (A) Immunoblot analysis of P-Ser9 GSK3 in WT and Gimap5 ^sph/sph CD4+ T cells during 24 hours activation with aCD3/aCD28. Phosphorylation of (B) Akt (S473), (C) p38 (T180/Y182), and (D) GSK3 (S9) after stimulation with aCD3/aCD28 for the indicated times. Values depicted are relative to unstimulated samples and depict mean + SD (n=5). (E) Representative flow plots of live WT and Gimap5 ^ ⁵ ? ¹¹ CD4+ T cells after stimulation with aCD3/aCD28 for 3 days. (F) Percent apoptotic (Annexin V+) cells of live stimulated CD4+ T cells (n=3). (G) Survival of WT and Gimap5 CD4+ T cells after 3d stimulation with aCD3/aCD28 + 2.5 mM LiCl. Bars represent mean + SD (n=6). Immunoblot data depicts representative

immunoblots/graphs of at least 3 independent experiments performed on pooled CD4+ T cells. Statistical significance is determined by ANOVA followed by Sidak' s multiple comparisons test.

[0023] FIG 17A-17B. Lithium chloride prevents colitis. (A,B) Gross morphology of the colon (A) and liver (B) of Wildtype and Gimap5 ^S P ^H/S P ^H mice treated with either LiCl or vehicle from 3 weeks of age to 8 weeks of age. Images representative of 6 mice per group at 7-8 weeks of age. Scale bar shown is in cm. (C) Frequency of regulatory T cells

(CD25hiFoxp3+) within the CD4+ T cells population (n=6). Bars represent mean + SD. Statistical significance is determined by ANOVA followed by Sidak' s multiple comparisons test.

[0024] FIG 18A-18D. Genetic targeting of GSK3 in CD4+ T cells improves T cell survival. (A) Loss of GSK3 protein in splenic CD4+ T cells from Gsk3 fl/flCd4-cre/ert2 mice treated with tamoxifen from 3 to 8 weeks of age. Representative flow plot is shown. (B,C) Absolute number of (B) B cells and (C) CD4+ T cells in the spleen of WT and Gimap5 ^sph/sph mice either GSK3 -sufficient or insufficient CD4+ T cells. (D) Representative H&E stained liver sections of tamoxifen- treated Wildtype, Gimap5 ^ ⁵ ? ¹¹ Gsk3 WT/WTCd4- cre/ert2, and Gimap5 ^P ¹¹ Gsk3 fl/flCd4-cre/ert2 mice at 8 weeks old. Scale bar is 50 μιη. Data represent mean values + SEM from at least 6 mice per group at 8-9 weeks of age; statistical significance is determined by ANOVA followed by Sidak's multiple comparisons test.

[0025] FIG 19. Structure of C66H11ON15O20P. DETAILED DESCRIPTION

[0026] DEFINITIONS

[0027] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0028] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0029] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5- fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0030] As used herein, the term "effective amount" means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0031] The terms "individual," "host," "subject," and "patient" are used

interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.

[0032] Applicant has discovered that administration of certain compounds may be useful in the treatment of disease states associated with immune dysfunction. In one aspect, an inhibitor of glycogen synthase kinase 3, for example glycogen synthase 3β (GSK3 ) or glycogen synthase 3a (GSKa) for use in the therapy of a T-cell mediated disease in an individual in need thereof is disclosed. The T-cell mediated disease may be one associated with a loss of Treg immunosuppressive function.

[0033] In one aspect, the T-cell mediated disease may be an immune disease or an autoimmune disease. In certain aspects, the T cell mediated disease may be selected from an immunodeficiency, primary immunodeficiency disease (PID), type I diabetes, systemic lupus erythematosus (SLE), asthma, colitis, psoriasis, bronchiectasis, auto-immune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or combinations thereof.

[0034] In one aspect, the treatment step may ameliorate, prevent, or reduce the severity and/or progression of any aforementioned disease.

[0035] In one aspect, the individual may have a loss of function (LOF) mutation in one Gimap5 allele, or in both Gimap5 alleles. A version of Gimap5 is described in, for example, GIMAP5 Acc.#: HGNC: 18005. The LOF mutation may be a heterozygous mutation or a homozygous mutation. One example of a single nucleotide polymorphism in GIMAP5 that results in a complete loss of GIMAP5 protein expression in T cells is SNP: rs72650695 (FIG 7A). In other aspects, the LOF mutation may be one in which a mutation causes one or more of a loss of GIMAP5 protein expression or function, impairment of RNA stability, impairment of transcription factor (TF) binding sites, or a combination thereof. The TF may be one that is a direct target of GSK3 β regulation.

[0036] In one aspect, the inhibitor of GSK3 or GSK3a may be administered to an individual at a time period selected from within a day, within a week, within two weeks, within three weeks, within a month, within two months, within three months, within four months, within five months, within six months, or within a year of diagnosis. In one aspect, the administration step may occur prior to said disease state being characterized as late-stage.

[0037] In one aspect, the inhibitor of GSK3 or GSK3a described above may be selected from an inhibitor listed in Table 1. In one aspect, the inhibitor is LiCl. In one aspect, the inhibitor may be LiCl and may be administered in a dose of about 100 to about 150 mg/kg/day to the individual. Prescription Tablets currently available include 300 mg and are generally prescribed three times daily (900mg/adult/day). Each 5 mL of Lithium Oral Solution contains 8 mEq of Lithium ion (Li+) which is equivalent to the amount of Lithium in 300 mg of Lithium carbonate. In other aspects, the GSK3 inhibitor may be selected from a metal cation, for example, Lithium Chloride, an ATP-competitive GSK inhibitor, a marine organism-derived compound (6-BIO, Dibromocantharelline, Hymenialdesine, Indirubins, Meridianins) an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an

arylindolemaleimide (SB-216763, SB-41528), a thiazole (AR-A014418, AZD-1080), a paullone (Alsterpaullone, Cazpaullone, Kenpaullone) an aloisine, a non-ATP competitive Marine organism-derived compound (Manzamine A, Palinurine, Tricantine), a

thiadiazolidindione (TDZD-8, NP00111, NP031115, Tideglusib) a halomethylketone (HMK- 32), a non-ATP competitive peptide (L803-mts). See Eldar-Finkelman H, Martinez A (2011). "GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS". Frontiers in Molecular

Neuroscience. 4: 32. doi: 10.3389/fnmol.2011.00032. PMC 3204427 Freely accessible. PMID 22065134. The active agent may form salts, which are also within the scope of the preferred embodiments. Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a _i C _t aons

carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps, which may be employed during preparation. Salts of the compounds of the active agent may be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. When the compounds are in the forms of salts, they may comprise pharmaceutically acceptable salts. Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates,

hydroxynaphthoates, glycerophosphates, ketoglutarates and the like. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium,

butylammonium, tetramethylammonium salts and the like. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like.

[0038] Table 1. GSK3 Inhibitors

Type Inhibitor Structure Proposed Refs.

Target

Lithium GSK3 / 1, 2

Zinc GSK3 3

Tungstate GSK3 4 - 13 -

- 17 -

[0039] References for Table 1 above are as follows:

[0040] Table 1-1. Klein, P.S. & Melton, D.A. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci U S A 93, 8455-8459 (1996). [0041] Table 1-2. Phiel, C.J. & Klein, P.S. Molecular targets of lithium action. Annu Rev Pharmacol Toxicol 41, 789-813 (2001).

[0042] Table 1-3. Chanoit, G. et al. Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3beta. Am J Physiol Heart Circ Physiol 295, H1227- H1233 (2008).

[0043] Table 1-4. Gomez-Ramos, A. et al. Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation. J Neurosci Res 83, 264-273 (2006).

[0044] Table 1-5. Meijer, L. et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 10, 1255-1266 (2003).

[0045] Table 1-6. Meijer, L. et al. Inhibition of cyclin-dependent kinases, GSK- 3beta and CK1 by hymenialdisine, a marine sponge constituent. Chem Biol 7, 51-63 (2000).

[0046] Table 1-7. Gompel, M. et al. Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum. Bioorg Med Chem Lett 14, 1703- 1707 (2004).

[0047] Table 1-8. Naujok, O., Lentes, J., Diekmann, U., Davenport, C. & Lenzen, S. Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes 7, 273 (2014).

[0048] Table 1-9. Ring, D.B. et al. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes 52, 588-595 (2003).

[0049] Table 1-10. Muralidharan, S. et al. Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes. J Immunol 187, 5221-5232 (2011).

[0050] Table 1-11. Coghlan, M.P. et al. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 7, 793-803 (2000). [0051] Table 1-12. Cross, D.A. et al. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77, 94-102 (2001).

[0052] Table 1-13. Gaisina, I.N. et al. From a natural product lead to the identification of potent and selective benzofuran-3-yl-(indol-3-yl)maleimides as glycogen synthase kinase 3beta inhibitors that suppress proliferation and survival of pancreatic cancer cells. J Med Chem 52, 1853-1863 (2009).

[0053] Table 1-14. Kozikowski, A.P. et al. Highly potent and specific GSK-3beta inhibitors that block tau phosphorylation and decrease alpha- synuclein protein expression in a cellular model of Parkinson's disease. ChemMedChem 1, 256-266 (2006).

[0054] Table 1-15. Gray, J.E. et al. A first- in-human phase I dose-escalation, pharmacokinetic, and pharmacodynamic evaluation of intravenous LY2090314, a glycogen synthase kinase 3 inhibitor, administered in combination with pemetrexed and carboplatin. Invest New Drugs 33, 1187-1196 (2015).

[0055] Table 1-16. Kunnimalaiyaan, S., Schwartz, V.K., Jackson, I.A., Clark Gamblin, T. & Kunnimalaiyaan, M. Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro. BMC Cancer 18, 560 (2018).

[0056] Table 1-17. Zamek-Gliszczynski, M.J. et al. Pharmacokinetics, metabolism, and excretion of the glycogen synthase kinase-3 inhibitor LY2090314 in rats, dogs, and humans: a case study in rapid clearance by extensive metabolism with low circulating metabolite exposure. Drug Metab Dispos 41, 714-726 (2013).

[0057] Table 1-18. Bhat, R. et al. Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418. J Biol Chem 278, 45937-45945 (2003).

[0058] Table 1-19. Gould, T.D., Einat, H., Bhat, R. & Manji, H.K. AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. Int J Neuropsychopharmacol 7, 387-390 (2004). [0059] Table 1-20. Chen, S., Sun, K.X., Liu, B.L., Zong, Z.H. & Zhao, Y. The role of glycogen synthase kinase-3beta (GSK-3beta) in endometrial carcinoma: A carcinogenesis, progression, prognosis, and target therapy marker. Oncotarget 7, 27538-27551 (2016).

[0060] Table 1-21. Knockaert, M. et al. Intracellular Targets of Paullones.

Identification following affinity purification on immobilized inhibitor. J Biol Chem 277, 25493-25501 (2002).

[0061] Table 1-22. Leost, M. et al. Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25. Eur J Biochem 267, 5983-5994 (2000).

[0062] Table 1-23. Tolle, N. & Kunick, C. Paullones as inhibitors of protein kinases. Curr Top Med Chem 11, 1320-1332 (2011).

[0063] Table 1-24. Guzman, E.A., Johnson, J.D., Linley, P.A., Gunasekera, S.E. & Wright, A.E. A novel activity from an old compound: Manzamine A reduces the metastatic potential of AsPC-1 pancreatic cancer cells and sensitizes them to TRAIL-induced apoptosis. Invest New Drugs 29, 777-785 (2011).

[0064] Table 1-25. Hamann, M. et al. Glycogen synthase kinase-3 (GSK-3) inhibitory activity and structure- activity relationship (SAR) studies of the manzamine alkaloids. Potential for Alzheimer's disease. J Nat Prod 70, 1397-1405 (2007).

[0065] Table 1-26. del Ser, T. et al. Treatment of Alzheimer's disease with the GSK-3 inhibitor tideglusib: a pilot study. J Alzheimers Dis 33, 205-215 (2013).

[0066] Table 1-27. Lovestone, S. et al. A phase II trial of tideglusib in Alzheimer's disease. J Alzheimers Dis 45, 75-88 (2015).

[0067] Table 1-28. Sereno, L. et al. A novel GSK-3beta inhibitor reduces

Alzheimer's pathology and rescues neuronal loss in vivo. Neurobiol Dis 35, 359-367 (2009).

[0068] Table 1-29. Zhao, K. et al. Inhibition of glycogen synthase kinase-3beta attenuates acute kidney injury in sodium taurocholateinduced severe acute pancreatitis in rats. Mol Med Rep 10, 3185-3192 (2014). [0069] Table 1-30. Perez, D.I. et al. Thienylhalomethylketones: Irreversible glycogen synthase kinase 3 inhibitors as useful pharmacological tools. Bioorg Med Chem 17, 6914-6925 (2009).

[0070] Table 1-31. Liang, Z. & Li, Q.X. Discovery of Selective, Substrate- Competitive, and Passive Membrane Permeable Glycogen Synthase Kinase-3beta Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling of New C-Glycosylflavones. ACS Chem Neurosci 9, 1166-1183 (2018).

[0071] Table 1-32. Chen, G. et al. Glycogen synthase kinase 3beta (GSK3beta) mediates 6-hydroxydopamine-induced neuronal death. FASEB J 18, 1162-1164 (2004).

[0072] Table 1-33. Kaidanovich-Beilin, O. & Eldar-Finkelman, H. Long-term treatment with novel glycogen synthase kinase-3 inhibitor improves glucose homeostasis in ob/ob mice: molecular characterization in liver and muscle. J Pharmacol Exp Ther 316, 17-24 (2006).

[0073] Table 1-34. Kaidanovich-Beilin, O., Milman, A., Weizman, A., Pick, C.G. & Eldar-Finkelman, H. Rapid antidepressive-like activity of specific glycogen synthase kinase-3 inhibitor and its effect on beta-catenin in mouse hippocampus. Biol Psychiatry 55, 781-784 (2004).

[0074] In one aspect, a method of treating an individual having a T-cell mediated disease is disclosed. The method may comprise the step of administering to an individual in need thereof, an effective amount of an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a). In one aspect, the T-cell mediated disease may be associated with a loss of Treg immunosuppressive function. In one aspect, the T-cell mediated disease may be an immune disease or an autoimmune disease. In certain aspects, the T cell mediated disease may be selected from an immunodeficiency, primary immunodeficiency disease (PID), type I diabetes, systemic lupus erythematosus (SLE), asthma, colitis, psoriasis, bronchiectasis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or combinations thereof. The disclosed methods may be used to ameliorate, prevent, or reduce the severity and/or progression of a disease as described herein. [0075] In one aspect, the methods may further include the step of identifying an individual having a loss of function (LOF) mutation in at least one Gimap5 allele. In certain instances, the LOF mutation may be a heterozygous mutation, while in other instances the LOF mutation may be a homozygous mutation. In one aspect, the LOF mutation may be a mutation that causes one or more of a loss of GIMAP5 protein expression, impairment of RNA stability, impairment of transcription factor (TF) binding sites, or a combination thereof. The TF may be one that is a direct target of GSK3 β or GSK3a regulation.

[0076] In one aspect, disclosed is a method of identifying an individual likely to benefit from administration of an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a). In this aspect, the method may comprise the step of determining whether a LOF mutation is present in the genome of the individual, wherein detection of an LOF may be performed on a sample isolated from the individual. If a LOF mutation is present, the individual may be administered an effective amount of an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a), for example, any inhibitor as described herein.

[0077] In one aspect, a method of maintaining immune homeostasis in an individual having, or suspected of having, a T-cell mediated disease associated with a loss of Treg immunosuppressive function is disclosed. In this aspect, the method may comprise the step of administering an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a), for example, a GSK3 inhibitor of Table 1 or as otherwise described herein. In one aspect, the inhibitor may be LiCl and may be administered in a dose of about 100 to about 150 mg/kg/day to the individual. In one aspect, the administration step may occur at a time period selected from within a day, within a week, within two weeks, within three weeks, within a month, within two months, within three months, within four months, within five months, within six months, or within a year of diagnosis. In one aspect, the administration step may occur prior to the disease state being characterized as late-stage, such that late-stage disease may be delayed or avoided entirely.

[0078] PHARMACEUTICAL COMPOSITIONS

[0079] In one aspect, active agents provided herein may be administered in a dosage form selected from intravenous or subcutaneous unit dosage form, oral, parenteral, intravenous, and subcutaneous. In some embodiments, active agents provided herein may be formulated into liquid preparations for, e.g., oral administration. Suitable forms include suspensions, syrups, elixirs, and the like. In some embodiments, unit dosage forms for oral administration include tablets and capsules. Unit dosage forms configured for administration once a day; however, in certain embodiments it may be desirable to configure the unit dosage form for administration twice a day, or more.

[0080] In one aspect, pharmaceutical compositions are isotonic with the blood or other body fluid of the recipient. The isotonicity of the compositions may be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes. An example includes sodium chloride. Buffering agents may be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. Parenteral vehicles include sodium chloride solution, Ringer' s dextrose, dextrose and sodium chloride, lactated Ringer' s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[0081] Viscosity of the pharmaceutical compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is useful because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose,

hydroxypropyl cellulose, carbomer, and the like. In some embodiments, the concentration of the thickener will depend upon the thickening agent selected. An amount may be used that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0082] A pharmaceutically acceptable preservative may be employed to increase the shelf life of the pharmaceutical compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative is typically from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts may be desirable depending upon the agent selected. Reducing agents, as described above, may be advantageously used to maintain good shelf life of the formulation. [0083] In one aspect, active agents provided herein may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. See, e.g., "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins; 20th edition (June 1, 2003) and "Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18th and 19th editions (December 1985, and June 1990, respectively). Such preparations may include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.

[0084] For oral administration, the pharmaceutical compositions may be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and may include one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives. Aqueous suspensions may contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions.

[0085] Formulations for oral use may also be provided as hard gelatin capsules, wherein the active ingredient(s) are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers and microspheres formulated for oral administration may also be used. Capsules may include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubrimayts such as talc or magnesium stearate and, optionally, stabilizers.

[0086] Tablets may be uncoated or coated by known methods to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate may be used. When administered in solid form, such as tablet form, the solid form typically comprises from about 0.001 wt. % or less to about 50 wt. % or more of active ingredient(s), for example, from about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.

[0087] Tablets may contain the active ingredients in admixture with non-toxic pharmaceutically acceptable excipients including inert materials. For example, a tablet may be prepared by compression or molding, optionally, with one or more additional ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubrimayt, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered active agent moistened with an inert liquid diluent.

[0088] In some embodiments, each tablet or capsule contains from about 1 mg or less to about 1,000 mg or more of an active agent provided herein, for example, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg. In some embodiments, tablets or capsules are provided in a range of dosages to permit divided dosages to be administered. A dosage appropriate to the patient and the number of doses to be administered daily may thus be conveniently selected. In certain embodiments two or more of the therapeutic agents may be incorporated to be administered into a single tablet or other dosage form (e.g., in a combination therapy);

however, in other embodiments the therapeutic agents may be provided in separate dosage forms. [0089] Suitable inert materials include diluents, such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like, or inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride. Disintegrants or granulating agents may be included in the formulation, for example, starches such as corn starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, karaya or tragamayth, or alginic acid or salts thereof.

[0090] Binders may be used to form a hard tablet. Binders include materials from natural products such as acacia, tragamayth, starch and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, and the like.

[0091] Lubrimayts, such as stearic acid or magnesium or calcium salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like, may be included in tablet formulations.

[0092] Surfactants may also be employed, for example, anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, cationic such as benzalkonium chloride or benzethonium chloride, or nonionic detergents such as polyoxy ethylene hydrogenated castor oil, glycerol monostearate, polysorbates, sucrose fatty acid ester, methyl cellulose, or carboxymethyl cellulose.

[0093] Controlled release formulations may be employed wherein the active agent or analog(s) thereof is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Other delivery systems may include timed release, delayed release, or sustained release delivery systems.

[0094] Coatings may be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters. Dyestuffs or pigments may be added for identification or to characterize different combinations of active agent doses.

[0095] When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added to the active ingredient(s). Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers. The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragamayth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as

polyoxyethylene sorbitan mono-oleate. The emulsions may also contain sweetening and flavoring agents.

[0096] Pulmonary delivery of the active agent may also be employed. The active agent may be delivered to the lungs while inhaling and traverses across the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products may be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. These devices employ formulations suitable for the dispensing of active agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.

[0097] The active ingredients may be prepared for pulmonary delivery in particulate form with an average particle size of from 0.1 um or less to 10 um or more, for example, from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μιη to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 μιη. Pharmaceutically acceptable carriers for pulmonary delivery of active agent include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC, and DOPC. Natural or synthetic surfactants may be used, including polyethylene glycol and dextrans, such as cyclodextran. Bile salts and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids may also be used. Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers may also be employed.

[0098] Pharmaceutical formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise the active agent dissolved or suspended in water at a concentration of about 0.01 or less to 100 mg or more of active agent per mL of solution, for example, from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the active agent caused by atomization of the solution in forming the aerosol.

[0099] Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the active ingredients suspended in a propellant with the aid of a surfactant. The propellant may include conventional propellants, such as

chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons. Example propellants include trichlorofluoromethane, dichlorodifluoromethane,

dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, and combinations thereof. Suitable surfactants include sorbitan trioleate, soya lecithin, and oleic acid.

[00100] Formulations for dispensing from a powder inhaler device typically comprise a finely divided dry powder containing active agent, optionally including a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, for example, from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation. [00101] In some embodiments, an active agent provided herein may be administered by intravenous, parenteral, or other injection, in the form of a pyrogen-free, parenterally acceptable aqueous solution or oleaginous suspension. Suspensions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The preparation of acceptable aqueous solutions with suitable pH, isotonicity, stability, and the like, is within the skill in the art. In some embodiments, a pharmaceutical composition for injection may include an isotonic vehicle such as 1,3- butanediol, water, isotonic sodium chloride solution, Ringer' s solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or other vehicles as are known in the art. In addition, sterile fixed oils may be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the formation of injectable preparations. The pharmaceutical compositions may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

[00102] The duration of the injection may be adjusted depending upon various factors, and may comprise a single injection administered over the course of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more of continuous intravenous administration.

[00103] In some embodiments, the active agents provided herein may be provided to an administering physician or other health care professional in the form of a kit. The kit is a package which houses a container which contains the active agent(s) in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject. The kit may optionally also contain one or more additional therapeutic agents currently employed for treating one or more disease states as described herein. For example, a kit containing one or more compositions comprising active agents provided herein in combination with one or more additional active agents may be provided, or separate pharmaceutical compositions containing an active agent as provided herein and additional therapeutic agents may be provided. The kit may also contain separate doses of an active agent provided herein for serial or sequential administration. The kit may optionally contain one or more diagnostic tools and instructions for use. The kit may contain suitable delivery devices, e.g., syringes, and the like, along with instructions for administering the active agent(s) and any other therapeutic agent. The kit may optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits may include a plurality of containers reflecting the number of

administrations to be given to a subject.

EXAMPLES

[00104] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[00105] GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, autoimmunity and colitis, but its mechanisms of action are unclear. Here Applicant has shown that Gimap5 is essential for inactivation of glycogen synthase kinase-3 (GSK3 ) following T cell activation. In the absence of Gimap5, constitutive GSK3 activity constrains c-Myc induction and NFATcl nuclear import, thereby limiting CD4 ⁺ T cells to complete cell cycle. Additionally, Gimap5 facilitates Ser389 phosphorylation and nuclear translocation of GSK3 thereby limiting DNA damage in CD4 ⁺ T cells.

Importantly, pharmacological inhibition and genetic targeting of GSK3 can override Gimap5 -deficiency in CD4 ⁺ T cells and ameliorates immune-pathology in mice. Finally, Applicant has shown that a human patient with a GIMAPS loss-of-function mutation has lymphopenia and impaired T cell proliferation in vitro that can be rescued with GSK3 inhibitors. Given that expression of Gimap5 is lymphocyte-restricted, without intended to be limited by theory, it is believed that its control of GSK3 is an important checkpoint in lymphocyte proliferation. [00106] GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, immune homeostasis and (auto-)immune disease. Specifically, polymorphisms in human GIMAP5 are associated with increased risk of islet autoimmunity in type 1 diabetes (T1D), systemic lupus erythematosus (SLE) ^{1, 2, 3} , and asthma ⁴ . Mice and rats with complete loss-of-function (LOF) mutations have reduced lymphocyte survival, loss of immunological tolerance predisposing to autoimmunity and colitis, and abnormal liver pathology resulting from persistent post-natal extramedullary hematopoiesis ^{5, 6} · ⁷ · ⁸ · ⁹ · ^{10, η} · ^{12, 13} ' ¹⁴ . Despite this critical role of Gimap5 in lymphocyte survival and peripheral tolerance, the underlying mechanism(s) are unclear.

[00107] Gimap proteins are predominantly expressed in lymphocytes and regulate lymphocyte survival during development, selection, and homeostasis ¹⁵ . Members of this family share a GTP-binding AIG1 homology domain ^{16, 17} and seem to be localized to different subcellular compartments, with Gimap5 localizing in multivesicular bodies (MVB) and lysosomes ¹⁸ . Overall, a function for Gimap5 in maintaining T-cell homeostasis is not clearly defined. Applicant previously generated Gimap5-deficient mice, so-called "sphinx" mice, which have a missense mutation in Gimap5 that results in what is essentially a null allele ⁶ . Gimap5 ^sph/sph mice progressively lose CD4+ T cells and B cells, an effect that is associated with reduced regulatory T (Treg) cell function, while remaining CD4+ T cells have an activated phenotype, but have an impaired capacity to proliferate ^{5, 6} . These immunologic defects result in spontaneous and lethal colitis that is preventable with CD4+ T cell depletion, Treg cell transplantation, or antibiotic therapy ^{5, 6} . Despite these effective therapies, the cell-intrinsic defects in Gimap5 ^sph/sph CD4+ T cells, including their reduced survival, persist. In addition to colitis, livers from Gimap5 ^sph/sph mice have an abnormal morphology with extramedullary hematopoiesis and associated foci of hematopoietic cells and hepatocyte apoptosis ^{6, 7} · ⁸ .

[00108] The family of glycogen synthase kinases-3 (GSK3) includes constitutively active protein serine/threonine kinases encoded by two genes, Gsk3a and Gsk3b. Studies have shown that GSK3 has an essential function in T cell differentiation and proliferation ^{19, 20, 21, 22} . GSK3 phosphorylates a variety of substrates, often within the phosphodegron domains, thereby regulating protein ubiquitination/degradation and their activity ²³ . Among the substrates targeted directly by GSK3 are c-Myc, NFATcl, Mcl-1, and β-catenin ²⁴ . Studies have established that upon antigen- specific activation of T cells, GSK3 activity is inhibited ¹⁹ ' ²⁰ ' ²¹ ' ²² , thereby facilitating T cell activation and proliferation.

Phosphorylation of GSK3 at residues Ser9 or Ser389 and physical separation of GSK3 from its target proteins through vesicle association have been proposed as mechanisms of GSK3 inhibition ²⁵ ' ²⁶ . Inhibition of GSK3 by phosphorylation of Ser389 is essential for lymphocyte viability upon double-stranded DNA breaks (DSB) evident during V(D)J recombination in thymic T cell development or in B cells undergoing immunoglobulin class switch recombination (CSR) ²⁷ . Further reports have demonstrated that GSK3 is involved in the regulation of nonhomologous end-joining repair of DSB, with pharmacological inhibition and shRNA knockdown of GSK3 promoting DNA repair ²⁸ . Nonetheless, the exact mechanism by which inhibition of GSK3 activity is regulated in T cells is unclear.

[00109] In the current study, Applicant provides important insight into the functional role of Gimap5. Gimap5 is a critical inhibitor of GSK3 in both human and mouse CD4+ T cells, affecting c-Myc, and Mcl-lexpression, NFATcl nuclear translocation, and T cell fitness by controlling the DNA damage response occurring during T cell proliferation.

[00110] Results

[00111] Gimap5 ^sph/sph mice have normal thymic output of CD4+ T cells

[00112] Studies implicate a loss of peripheral CD4+ T cells in both Gimap5- deficient mice and rats ^{6, 8} · ¹² ' ¹⁵ ' ²⁹ ' ³ °· ³¹ . To determine whether the observed reduction in peripheral CD4+ T cells might stem from abnormal thymic CD4+ T cell development, Applicant investigated whether the survival and/or output of thymic CD4+ T cells in Gimap5 ^sph/sph mice was affected. To assess survival of thymocytes, Applicant isolated thymic CD4+ T cells and cultured them in the presence of IL-7 for 1 week. Subsequently, the number of live single positive (SP) CD4+/CD8- T cells was quantified at various incubation times. Notably, Applicant's data indicate no differences in the survival ex vivo between SP CD4+ thymocytes isolated from WT and Gimap5 ^sph/sph mice (FIG 10A).

[00113] Applicant next assessed if reduced thymic output of CD4+ T cells might contribute to lymphopenia in Gimap5 ^sph/sph mice, and quantified the presence of recent thymic emigrants (RTE) ³² in spleen of WT and Gimap5 ^sph/sph mice. Importantly, Applicant found no marked differences in the frequency of splenic RTE as defined by CD24hi CD4+ T cells between 3-week-old WT or Gimap5 ^sph/sph mice (FIG 10B). These data are in line with Applicant's previous studies showing Gimap5 ^sph/sph mice have a relatively normal thymic development of CD4+ T cells ⁶ .

[00114] Activation-induced cell death of peripheral CD4+ T cells

[00115] Applicant next focused on the peripheral survival of CD4+ T cells in Gimap5 ^sph/sph mice. Applicant considered that either post-thymic survival of Gimap5 ^sph/sph CD4+ T cells or TCR-induced activation contributes to the loss of CD4+ T cells in the periphery. The latter would be consistent with Applicant' s previous studies showing that Gimap5 ^sph/sph T cells failed to proliferate after TCR stimulation with aCD3/aCD28 +/- IL-2 ⁶ . Moreover, a progressive loss of CD4+ T cells is observed post-weaning— a period in which the CD4+ T cell compartment has to cope with marked changes in gut microbial antigens. To directly test the role of TCR activation in vivo, Applicant generated Gimap5 ^sph/sph ;Rag2 ^~/~ ;OT- II TCR transgenic mice. These mice contain CD4+ T cells that only recognize the OVA323- 339 epitope presented by I-Ab in C57BL/6 mice and lack endogenous CD4+ T cell repertoires and CD8+ T cells and B cells. Interestingly, their CD4+ T cell compartment is largely maintained in the absence of antigen and predominantly display a naive

(CD44 ^lo ;CD62 ^hi ) phenotype (Fig.lA,B). In contrast, Gimap5 ^sph/sph ;Rag2 ^/ ;OT-II mice exposed to ovalbumin in drinking water have a reduced CD4+ T cell population with an increased proportion of remaining CD4+ T cells displaying a memory-like phenotype (CD44 ^hi ;CD62L ^lD ) compared to WT controls (Fig.lA,B). Moreover, these mice failed to induce antigen- specific Treg cells (Fig.lC). These data show that antigen-specific activation of Gimap5 ^sph/sph CD4+ T cells directly contributes to the loss of these cells in vivo. To assess the potential contribution of reduced homeostatic survival of peripheral CD4+ T cells, Applicant isolated CD4+ T cells from the spleen of WT and Gimap5 ^sph/sph mice and cultured them in the presence of IL-7. The number of live CD4+ T cells was quantified at various time points; in contrast to the thymic SP CD4 T cells, splenic Gimap5 ^sph/sph CD4+ T cell numbers were significantly reduced compared to WT (Fig. ID). These data suggest that peripheral Gimap5 ^sph/sph CD4+ T cells have a reduced peripheral survival compared to WT CD4+ T cells.

[00116] Applicant next sought to identify whether the cell-intrinsic apoptosis pathways contributed to the reduced CD4+ T cell survival in Gimap5 ^sph/sph mice. Studies suggested that Gimap5 directly binds anti-apoptotic proteins Bcl-2 and Bcl-xL15, while a recent study suggests that loss of Gimap5 was associated with increased Mcl- 1 degradation and compromised mitochondrial integrity in hematopoietic progenitor cells ⁷ . To test the potential role of Bcl-2 and Bcl-xL in the reduced survival of Gimap5sph sph CD4+ T cells, Applicant crossed the Gimap5 ^sph/sph allele onto a Bim ^_/~ deficient background. Importantly, Applicant observed no rescue of peripheral T cell survival (FIG 11A-11D). Applicant next assessed Mcl- 1 expression in resting and activated CD4+ T cells and observed that accumulation of Mcl-1, similar to hematopoietic progenitor cells7, was reduced in

Gimap5 ^sph/sph CD4+ T cells compared to WT controls after 24h of stimulation with aCD3/aCD28 (FIG HE). Studies have shown that Bax/Bak deletion can prevent CD4+ T cell death in the complete absence of Mcl- 133. However, crossing the Gimap5 ^sph/sph allele onto a Bax/Bak double-deficient background failed to rescue CD4+ T cell survival (FIG 11F), thus the impact of the reduced Mcl- 1 expression is unclear. Finally, Applicant sought to investigate if Gimap5-deficient CD4+ T cells were more susceptible to cell-extrinsic pathways. Stimulation of CD4+ T cells with aCD3/aCD28 in the absence/presence of activating Fas antibodies for 8h, however, resulted in similar frequencies of apoptotic and dead cells between WT and Gimap5 ^sph/sph CD4+ T cells (FIG 11G).

[00117] Together these data indicate that the reduced survival of peripheral CD4+ T cells in Gimap5 ^sph/sph mice occurs independently of the cell-intrinsic apoptosis pathways.

[00118] Gimap5 ^sph/sph CD4+ T cells have abnormal GSK3 activity

[00119] To identify the molecular defects that mediate this decreased proliferation, Applicant assessed the activation of proximal signaling pathways in CD4+ T cells from 3-week-old Gimap5 ^sph/sph mice. At this age, CD4+ T cell numbers are comparable to WT with a normal frequency of naive CD4+ T cells that have a quiescent phenotype (FIG 12A). Importantly, Applicant's data indicated no abnormalities in the activation of proximal signaling pathways (ΙκΒ, ERK, JNK, AKT, p38, mTORCl, and p70 S6K) (FIG 12B-D). However, Applicant observed a marked reduction in protein levels of the transcription factor (TF) c-Myc in activated Gimap5sph/sph CD4+ T cells (Fig.2A). C-Myc is a TF necessary for the metabolic programming of T cells following activation and is required for optimal T cell proliferation ³⁴ ' ³⁵ . Notably, myc mRNA levels in resting or aCD3/aCD28-stimulated

Gimap5 ^sph/sph CD4+ T cells were comparable to WT controls, suggesting that reduction in protein levels was the result of changes in post-translational regulation (Fig.2B). Post- translational regulation of c-Myc is mediated in part by the family of GSK3 proteins, which is comprised of constitutively active protein serine/threonine kinase paralogs GSK3a and GSK3 . GSIGa/β phosphorylate c-Myc at Thr58, priming it for ubiquitination and subsequent proteasomal degradation ³⁶ .

[00120] Studies suggest GSK3 plays an essential role in T cell differentiation and proliferation. Specifically, upon antigen-specific activation of T cells, GSK3 activity is inhibited ^{19, 20, 21} ' ^{22, 37} , thereby facilitating T cell activation and proliferation. GSK3 phosphorylates a variety of substrates, regulating protein ubiquitination/degradation and/or their activity ²³ . Among the substrates targeted by GSK3 are c-Myc, Mcl-1, NFATcl, and β- catenin ²⁴ ' ³⁸ ' ³⁹ ' ⁴⁰ - ⁴¹ . Elevated GSK3 activity in stimulated Gimap5 ^s P ^h/s P ^h CD4+ T cells would account for the reduced protein levels of Mcl-1(FIG 1 IE), which is targeted for proteasomal degradation upon phosphorylation by GSK3 ³⁹ ' ⁴⁰ . Moreover, aCD3/aCD28-stimulated Gimap5 ^sph/sph CD4+ T cells also showed a slight reduction in β-catenin protein expression, but normal β-catenin mRNA levels (FIG 13A, 13B), consistent with an elevated GSK3 activity.

[00121] To further investigate whether GSK3 activity was elevated in the absence of Gimap5, Applicant stimulated WT and Gimap5 ^sph/sph CD4+ T cells with aCD3/aCD28 for 24 hours. During the final four hours, proteasomal degradation was blocked with the inhibitor MG132 to allow the evaluation of GSK3 phosphorylation of c-Myc at Thr58. Strikingly, Gimap5 ^sph/sph CD4+ T cells had a higher proportion of c-Myc

phosphorylation than WT CD4+ T cells (Fig.2C). Moreover, the GSK3-inhibitor LiCl increased c-Myc and β-catenin accumulation in Gimap5 ^sph/sph CD4+ T cells following TCR stimulation (Fig.2D and FIG 13C). Applicant thus hypothesized that impaired suppression of GSK3 activity compromises productive T cell proliferation in Gimap5 ^sph/sph mice. To test this, CD4+ T cells from WT and Gimap5sph/sph mice were stimulated with aCD3/aCD28 in the presence/absence of GSK3-inhibitors, LiCl or 6-bromoindirubin-3'-oxime (BIO). Strikingly, in the presence of GSK3-inhibitors, CD4+ T cells from Gimap5 ^sph/sph mice completely regained their proliferative capacity in vitro and showed comparable survival to WT CD4+ T cells (Fig.2E).

[00122] Finally, Applicant assessed nuclear translocation of NFATcl, a transcription factor necessary for productive T cell activation (FIG 13D) and regulated by GSIGa/β ⁴² · ⁴³ . Unlike c-Myc and β-catenin, however, GSK3 phosphorylation of NFATcl regulates its localization rather than stability ^{38 41} . To test if activation of NFATcl was impaired in the absence of Gimap5, Applicant stimulated WT and Gimap5 ^sph/sph CD4+ T cells with aCD3/aCD28 and examined NFATcl nuclear localization by ImageStream analysis using the similarity dilate algorithm (see Methods section). Notably, while expression of NFATcl is normal (FIG 13E), its nuclear translocation is significantly impaired in stimulated Gimap5 ^sph/sph CD4+ T cells, and can be restored with BlO-treatment (Fig.2F-H). Furthermore, accumulation of intracellular calcium, a key step in calcineurin activation and NFATcl translocation, was normal in Gimap5 ^sph/sph CD4+ T cells stimulated with either aCD3/aCD28 or Ionomycin (FIG 13F). Together these data suggest that the absence of Gimap5 causes impaired inactivation of GSK3, ultimately resulting in a failure to accumulate TFs and nuclear localization that are critically required for CD4+ T cell survival/proliferation.

[00123] Loss of Gimap5 results in impaired sequestration of GSK3

[00124] As mentioned, antigen-specific activation of T cells requires inactivation of GSK3 activity ²² ; Applicant therefore sought to determine how inactivation of GSK3 in Gimap5 ^sph/sph CD4+ T cells was impaired. The exact mechanism by which inhibition of GSK3 activity is regulated is not well defined. However, a variety of mechanisms have been proposed, including phosphorylation at residue Ser9 ²⁶ and Ser389 ²⁷ as well as changes in localization— i.e. physical sequestration of GSK3 in MVB25. Given that Gimap5 expression is observed in lysosomes and MVB ¹⁸ , Applicant first tested whether vesicular sequestration of GSK3 is affected. Specifically, Applicant quantified vesicular GSK3 intensity, the number of vesicles, and the size of GSK3 -associating vesicles in WT and Gimap5 ^sph/sph CD4+ T cells at 0, 6, and 24 hours after CD4+ T cell stimulation. Notably, GSK3 became associated with punctate vesicles in WT cells and GSK3 intensity, vesicle number, and vesicle size were markedly decreased in Gimap5 ^s P ^h/s P ^h CD4+ T cells after 24- hours stimulation (Fig.3A), indicating that physical segregation of GSK3 from cytosolic target proteins ²⁵ ' ^{44 45} may provide a mechanism of inhibition. Next, Applicant sought to identify if GSK3 + vesicles associate with Gimap5 expression following CD4+ T cell activation. Interestingly, ImageStream analysis of resting/activated WT and Gimap5 ^sph/sph CD4+ T cells indeed showed colocalization between Gimap5 and GSK3 in a subset of punctate cytosolic vesicles of activated WT CD4+ T cells (Fig.3B) with -54% of Gimap5+ vesicles also positive for GSK3 . Given that Gimap5 is expressed in lysosomes (Lampl+), but also Lampl ^neg vesicles (FIG 14B), Applicant sought to further characterize

Gimap5/GSK3 double positive (DP) vesicles using various endosomal/lysosomal markers. Interestingly, studies showed limited association between Rab5, Rab7 or Lampl expression and Gimap5/GSK3 DP vesicles (FIG 14A-14E). To confirm colocalized expression of Gimap5 and GSK3 in vesicles, Applicant examined WT and Gimap5 ^sph/sph CD4+ T cells by high-resolution, three-dimensional confocal microscopy, enabling us to visualize Gimap5- GSK3 colocalization in the axial as well as xy planes. Consistent with the ImageStream analysis, GSK3 colocalized with Gimap5 in stimulated WT CD4+ T cells in punctate vesicles (Fig.3C).

[00125] GSK3 vesicular sequestration has predominantly been described in the context of Wnt signaling ²⁵ . To test the potential involvement of Wnt signaling Applicant incubated WT CD4+ T cells either directly with Wnt3a or examined blocking of Wnt secretion by incubating activated CD4+ T cells in the presence/absence of rWP-2 (a Wnt secretion inhibitor ^{46 47} ). Subsequently, Applicant assessed GSK3 sequestration using ImageStream and determined the proliferation capacity of WT CD4+ T cells. Neither Wnt3a nor IWP-2 affected the vesicular sequestration in WT CD4+ T cells or impacted T cell proliferation (FIG 15A-15D). Moreover, no significant differences in Wnt3A mRNA expression was observed between WT and Gimap5 ^sph/sph CD4+ T cells at resting or activated conditions (FIG 15E). Together, Applicant's data indicate that following activation of CD4+ T cells, GSK3 is sequestered in vesicles and that this vesicular accumulation of GSK3 is markedly impaired in Gimap5 -deficient CD4+ T cells following activation.

[00126] Impaired GSK3 phosphorylation and increased DNA damage

[00127] Phosphorylation at residue Ser9 ²⁶ and Ser389 ²⁷ have been proposed as mechanisms of GSK3 inhibition. Phosphorylation of GSK3 at Ser9 is mediated by AKT (and possibly other kinases) following TCR signaling ⁴⁸ , while inhibition of GSK3 by phosphorylation of Ser389 is linked with lymphocyte fitness in the context of DNA DSB observed either during V(D)J recombination in thymic T cells or in B cells undergoing immunoglobulin class switch recombination (CSR) ²⁷ . Furthermore, reports have shown that pharmacological inhibition and shRNA knockdown of GSK3 promote the repair of DSB, suggesting a role for GSK3 inhibition during the DNA damage response ²⁸ . Applicant thus next considered that GSK3 inhibition by phosphorylation— i.e. P- Ser9 or P-Ser389— during T cell activation was impaired in mutant cells. Interestingly, analysis of

aCD3/aCD28-stimulated WT and Gimap5 ^sph/sph CD4+ T cells showed normal

phosphorylation of Ser9 within the first 30 minutes (FIG 16A, 16D), 24 hours (FIG 16A), and after 2 days (Fig.3D). In contrast, phosphorylation of GSK3 at position Ser389 (primarily observed 2 days after T cell activation), was impaired in Gimap5 ^sph/sph T cells (Fig.3D). Phosphorylation of Ser389 is thought to be mediated by p38 MAPK ²⁷ . However, Applicant observed no differences in the activation of p38 MAPK between WT and Gimap5 ^sph/sph CD4+ T cells (Fig.3D, FIGs 16C and 3B). To further define whether the reduced phosphorylation of GSK3 at position Ser389 was due to the result of an overall reduction in GSK3 nuclear expression, Applicant performed ImageStream studies to define the level of both total GSK3 and P- Ser389 GSK3 in the nucleus. Interestingly, both total and phosphorylated GSK3 levels were markedly reduced in the nucleus of activated Gimap5 ^sph/sph CD4+ T cells (day 2) (Fig.3E-H), while some P-Ser389 GSK3 could be observed in Gimap5 ^sph/sph CD4+ T cells predominantly in punctate vesicles outside of the nucleus (Fig.3E), revealing an impaired distribution of P- Ser389 GSK3 in activated Gimap5 ^sph/sph CD4+ T cells.

[00128] Importantly, the reduction of P- Ser389 was associated with a marked increase in DNA damage (γΗ2ΑΧ expression; Fig.4A) in Gimap5 ^sph/sph CD4+ T cells undergoing cell cycle, leading to increased p53 expression and phosphorylation (Fig.3D), and ultimately reduced cell survival (Fig.4B and FIG 16G). Notably, Gimap5 ^sph/sph CD4+ T cells with DNA damage (γΗ2ΑΧ+) were not actively undergoing apoptosis (FIG 16E, 16F).

Importantly, the significant increase in DNA damage observed in Gimap5 ^sph/sph CD4+ T cells was reduced in the presence of GSK3 inhibitors (Fig.4C,D), confirming the critical function of GSK3 inactivation in limiting DNA damage and promoting productive T cell proliferation.

[00129] Pharmacological targeting of GSK3 prevents immune pathology

[00130] Applicant next tested the potential of GSK3 inhibitors to maintain T cell survival and prevent colitis/liver pathology in vivo. Applicant treated WT and

Gimap5 ^sph/sph mice with LiCl (150 mg/kg in drinking water, ad libitum) starting at 3 weeks of age until 8 weeks of age, by which time severe colitis can be observed. LiCl treatment of Gimap5 ^sph/sph mice effectively maintained the CD4+ T cell and B cell populations in

Gimap5 ^sph/sph mice (Fig.5A,B), while the proportion of CD4+ T cells displaying the memorylike phenotype (CD44 ^hl ;CD62 ^lD ) was reduced compared to untreated Gimap5 ^sph/sph mice (Fig.5C).

[00131] Notably, while the Treg cell frequency within the CD4+ T cell population of Gimap5 ^sph/sph mice is not affected (FIG 17C), their progressive loss of suppressive function is thought to be a key factor in driving colitis in Gimap5 ^sph/sph mice ⁵ . Applicant next asked if pharmacological inhibition of GSK3 could prevent this defect.

Specifically, Applicant purified regulatory T cells from the spleens of vehicle- and LiCl- treated WT and Gimap5 ^sph/sph mice and cocultured them with aCD3-stimulated WT CD8+ T cells. Strikingly, while Treg cells from vehicle-treated Gimap5 ^sph/sph mice were incapable of suppressing CD8+ T cell proliferation in vitro, Treg cells from LiCl-treated Gimap5 ^sph/sph mice demonstrated normal suppressive capacity (Fig.5D). Importantly, LiCl treatment completely prevented colitis and significantly reduced crypt loss and leukocyte infiltration (Fig.5E-5F and FIG 17A). Finally, LiCl treatment corrected the regenerative liver phenotype with limited hemorrhage and hematopoietic stem cell presence observed in the liver of LiCl- treated Gimap5 ^sph/sph mice (Fig.5G and FIG 17B). Overall, these data provide a causal link between GSK3 activity and reduced T cells and B cell survival in Gimap5 ^sph/sph mice and the development of regenerative liver disease and colitis. [00132] Genetic targeting of GSK3 in CD4+ T cells prevents colitis

[00133] While Applicant observed comparable results in rescuing effects of loss of Gimap5 with two mechanistically distinct inhibitors of GSK3, each of these molecules has other effects on cellular processes and proteins. GSK3 consists of two ubiquitously expressed paralogs, GSK3a and GSK3 ; to assess selectivity and importance of GSK3 specifically in the CD4+ T cell-mediated pathology, Applicant next examined whether genetic ablation of GSK3 in CD4+ T cells was sufficient to prevent loss of CD4+ T cells and immunopathology in Gimap5 ^sph/sph mice. To test this hypothesis, Applicant generated

WT- or Gimap5 ^sph/sph~ ; Cd4-cre/ert2; Gsk3 ^fl/fl mice ⁴⁹ — allowing for the conditional ablation of GSK3 specifically in CD4+ T cells upon treatment with tamoxifen. WT and Gimap5 ^sph/sph on a GskS ™ or Gsk3 ^WT/WT background were treated with tamoxifen starting at 3 weeks of age. At 8 weeks of age, mice were characterized for CD4+ T cell survival and their phenotype. Tamoxifen treatment resulted in an effective ablation of GSK3 expression in

CD4+ T cells (FIG 18 A) and increased survival of CD4+ T (but not B) cells in the periphery

(Fig.6A and FIG 18B.18C). Moreover, in the absence of GSK3 , Gimap5 ^sph/sph CD4+ T cells maintained a naive phenotype comparable to WT peripheral CD4+ T cells (Fig.6B).

Importantly, conditional deletion of Gsk3 in CD4+ T cells was sufficient to prevent the development of colitis (Fig.6C,D). In contrast, no effect on the liver damage with a similar presence of hematopoietic foci observed in tamoxifen-treated Gimap5 ^sph/sph ; Gsk3 ^fl/fl mice was observed (FIG 18D). These findings confirm the critical function of GSK3

expression/activity in CD4+ T cell survival and T cell-mediated gut pathology in Gimap5 _S ph/ _S ph mic^

[00134] A human GIMAP5 loss-of-function mutation results in similar T cell deficiency

[00135] During the course of Applicant's studies, Applicant identified a 16- year-old patient who was referred to Applicant's institution for evaluation of an immune deficiency. The patient presented with splenomegaly, lymphopenia with low CD4+ and CD8+ T cells as well as NK cells, and was found to have decreased frequency of naive CD4+ T cells and increased CD4+ T-effector memory cells (see supplemental patient description). Using whole exome sequencing the patient was found to be homozygous for a rare non- synonymous SNP (rs72650695, position 7: 150742750) in the coding region of GIMAP5 causing a Leu→Pro amino acid change at residue 204 (Fig.7A). Immunoblot analysis showed that the missense mutation resulted in undetectable protein expression of both GIMAP5 isoforms. (Fig.7A). The rs72650695 SNP is a rare variant (minor allele frequency -0.002076 in ExAC database) and both parents were found to be heterozygous carriers. Given that the lymphopenia and overall clinical profile were strikingly similar to Gimap5 -deficient mice, and no other mutations in candidate genes were identified, Applicant considered that the immune deficiency was caused by a LOF mutation in GIMAP5.

[00136] To define the T cell deficiency in the GIMAP5 ^"7" patient, Applicant compared T cell proliferation from the patient with his heterozygous mother. Similar to CD4+ T cell proliferation in Gimap5-deficient mice, a marked reduction in T cell proliferation was observed that could be rescued in the presence of LiCl (Fig.7B). After expansion of T cells in vitro using PHA followed by incubation with IL-2, rested T cells were examined for c-Myc expression. Immunoblot analysis showed a marked reduction of c-Myc levels in both resting (Fig.7C), and aCD3/aCD28 re-stimulated patient cells as compared to the heterozygous mother (Fig.7D). Importantly, similar to T cell proliferation, c-Myc levels could be restored upon treatment of cells with LiCl, suggesting that in human GIMAP5 ^7" T cells, GSK3-inhibitors offer therapeutic potential to correct the immunodeficiency and prevent immune pathology.

[00137] Applicant next assessed colocalization of GIMAP5 and GSK3 in healthy control cells activated with aCD3/aCD28. Similar to previous studies, ImageStream analysis of primary CD4+ T cells from healthy individuals showed that GIMAP5 was selectively expressed in vesicles, including but not limited to lysosomal vesicles (Fig.8A,B). Moreover, Applicant observed colocalization between GIMAP5 and GSK3 that was significantly increased 1-2 days after CD4+ T cell activation with -66% of GIMAP5+ spots in CD4+ T cells positive for GSK3 (Fig.8A,C-D). Similar to murine CD4+ T cells, accumulation of GSK3 was predominantly observed in GIMAP5+ vesicles that were CD107 negative (Fig.8C,D). Furthermore, vesicular association of GSK3 in GIMAP5 ^"7" CD4+ T cells restimulated with aCD3/aCD28 after expansion was markedly reduced as determined by number of GSK3 spots, GSK3 spot size, and overall GSK3 intensity within spots (Fig.8E- H). Notably, overall GSK3 expression and vesicular localization was also enhanced in CD4+ T cells that had first been expanded compared to primary CD4+ T cells both before and after aCD3/aCD28 stimulation (Fig.8A,H-G). Applicant next determined whether activation of GIMAP5 ^"7" CD4+ T cells was associated with increased DNA damage, by comparing the level of γΗ2ΑΧ in CD4+ T cells from control and patient cells. After 2 days of re- stimulation with aCD3/aCD28, a significant increase in intensity of γΗ2ΑΧ was observed in patient CD4+ T cells compared to control T cells upon activation (Fig.8I,J). Together these data reveal a striking similarity between the molecular pathways in mouse and human T cells that are affected by loss of GIMAP5 function.

[00138] Discussion

[00139] Here Applicant reports a critical role for Gimap5 in inactivating GSK3 during CD4+ T cell activation that affects two key regulatory events required for T cell proliferation. In the absence of Gimap5, impaired inactivation of GSK3 through a reduced vesicular association results in a failure to accumulate or induce nuclear translocation of TFs necessary for productive T cell proliferation that occurs at an early stage (Fig.9). Moreover, when T cells are cycling (i.e. at day 2) Applicant observed an impaired nuclear accumulation of P- Ser389 GSK3 that was associated with increased DNA damage and a reduced lymphocyte fitness. Importantly, Applicant showed that pharmacological targeting of GSK3 or genetic deletion of Gsk3 corrects T lymphocyte survival and prevents severe early- onset colitis in Gimap5 -deficient mice. Moreover, Applicant describes a human patient with a GIMAP5 LOF mutation. The patient presents with an immunodeficiency strikingly similar to Gimap5 -deficient mice, including the development of lymphopenia, reduction in TFs such as c-Myc, as well as increased DNA damage and reduced survival upon T cell activation.

Notably, GSK3 inhibitors can restore accumulation of c-Myc protein and improve T cell survival during activation in vitro.

[00140] GSK3 is constitutive active kinase in resting T cells and has a large number of reported targets (>100) that include important signaling components and TFs important for growth and suvival ⁵⁰ . These include but are not limited to c-Myc, β-catenin and NFATcl, key TFs required for growth and activation of the transcriptional program in T cells ^{34, 51} ' ⁵² . In addition, Applicant's studies identify an important function for GSK3 in limiting DNA damage during CD4+ T cell proliferation. The latter could very well be mediated by direct effect of GSK3 β on components such as p53, Mcll but also Mdm2, a GSK3 β target that is an important negative regulator of p53 ⁵³ . Applicant tested the contribution of the pro-survival protein Mcl-1 as one potentially important survival factor, but Applicant's studies showed little rescue of CD4+ T cells in Bax/Bak-deficient mice, suggesting that a combination of pathways are likely underlying the failure of CD4+ T cells to survive and undergo proliferation.

[00141] Until recently, AKT-mediated phosphorylation of the N-terminal tail of GSK3 was believed to be the primary mechanism of its inhibition. However, the generation of phospho-insensitive forms of GSK3 (GSK3a ^S21A /GSK3 ^S9A ) and the characterization of GSK3 's active site has clearly demonstrated that GSK3 N-terminal phosphorylation neither completely inhibits activity nor is involved in every GSK3 signaling axis ⁴⁴ ' ⁵⁴ ' ^{55 56} . More recently, phosphorylation of the Ser389 site in GSK3 has been implicated as a key step in GSK3 inactivation and maintaining lymphocyte fitness during the DNA damage response to DSB ²⁷ . Reports by Yang et al. indicate that reduction of GSK3 activity through inhibition or knockdown limits the accumulation of these DSB ²⁸ . Other reports link GSK3 inhibition to increased cell survival upon induction of DNA damage through its action on p53; active GSK3 binds to and promotes the actions of p53 ^{57, 58, 59} . In addition, evidence suggests that the GSK3-mediated regulation of the transcriptional program (e.g. c-Myc, NFATcl) contributes to the optimal DNA repair response. For instance, consistent with Applicant's observations, inhibition of NFAT nuclear translocation impairs DNA repair upon UV irradiation ^{60, 61} . Moreover, the report by Thornton et al. demonstrates the importance of phosphorylation of GSK3 at position Ser389 by using Ser389Ala knockin mice that have a reduced fitness of T cells and B cells undergoing V(D)J recombination and peripheral B cells undergoing activation ²⁷ . However, P- Ser389 of GSK3 can also readily be observed following γ-radiation or upon treatment with doxorubicin ^{27, 28} , suggesting a broader context for this mechanism of GSK3 inactivation in limiting DNA damage. While the study by Thornton et al. suggests limited P- Ser389 after 18 hours of stimulation in peripheral CD4+ T cells27, Applicant's studies show significant Ser389 phosphorylation at 48 hours during the peak of the proliferation-associated DNA damage response in WT CD4+ T cells— phosphorylation that is notably absent in Gimap5 -deficient T cells. Currently, it is unclear why Gimap5-deficient CD4+ T cells fail to phosphorylate Ser389— a process thought to involve activation of Ataxia telangiectasia mutated (ATM) and phosphorylation by p38. Applicant's data suggest phosphorylation of p38 is unaffected in Gimap5 -deficient T cells, indicating an alternative pathway is involved. Notably, at 24 hours of activation, Applicant did observe a marked decrease in the association of GSK3 with cytosolic vesicles— a mechanism proposed for GSK3 inhibition through the Wnt signaling pathway. This was followed by an overall reduced P- Ser389 and nuclear translocation of GSK3 , while remnant P- Ser389 GSK3 could be observed in punctate vesicles outside of the nucleus of

Gimap5 ^sph/sph CD4+ T cells after 2 days stimulation. These observations suggest that Gimap5 facilitates the subcellular localization of GSK3 and imply that the nuclear translocation of GSK3 is required for a productive T cell response.

[00142] While dysregulation of GSK3 may be the most striking phenotype observed in the absence of Gimap5, it may not be the only effect of Gimap5 deficiency. Crystallographic studies revealed that Gimap proteins manifest a nucleotide coordination and dimerization mode similar to dynamin GTPase— a component essential for the scission and fusion of cellular vesicular compartments such as endosomes ⁶² . Thus Gimap5's homology to dynamin raises the possibility that it may be more broadly involved in vesicular transport, rather than restricted solely to the sequestration of GSK3 . Interestingly, while Gimap5 is expressed in lysosomes and MVB, the data demonstrates that the increased vesicular association of GSK3 following T cell activation occurs predominantly in Lamp 1/CD 107 negative vesicles, suggesting lysosomal-independent functions for Gimap5.

[00143] A study suggested that Gimap5 can interact with Bcl2 family members to regulate apoptotic pathways ¹⁵ ; however, Applicant's studies show that the loss of CD4+ T cells could not be rescued when Gimap5 ^sph/sph mice were crossed to the apoptosis-resistant Bim-deficient or Bax/Bak-deficient backgrounds. Nonetheless, the Bcl2 family member Mcl- 1 is targeted for degradation by GSK3 -mediated phosphorylation, allowing the release of pro- apoptotic binding partners. A study by Chen et al. suggested that loss of Gimap5 was associated with enhanced Mcl- 1 degradation in hematopoietic stem cells (HSC) ultimately leading to compromised mitochondrial integrity and an overall reduced survival of HSC ⁷ . Applicant observed a similar reduction in Mcl-1 levels in stimulated CD4+ T cells, although the in vivo relevancy is unclear. This reduction is consistent with impaired GSK3 inhibition, providing an explanation for the increased Mcl-1 degradation. Moreover, Applicant's data indicate that treatment of Gimap5 ^sph/sph mice with GSK3 inhibitors in vivo prevents/corrects the development of hematopoietic foci in the liver. This correction was not observed when GSK3 was genetically ablated in CD4+ T cells, potentially indicating that HSC were directly targeted by LiCl treatment. This is consistent with the observation that the liver phenotype still develops in Gimap5 ^sph/sph mice on a Rag-deficient background, which lacks CD4+ T cells ⁸ .

[00144] It is feasible that GSK3 dysregulation has more subtle effects than activation-induced T cell death. Applicants have previously shown that a subset of highly pathogenic memory-like CD4+ T cells survives and drive colitis in the Gimap5 ^sph/sph mouse. These remaining cells have a limited capacity to proliferate but robustly produce ΙΡ γ and IL-17a. The accumulation of these cells and concurrent loss in number and function of regulatory T cells in Gimap5-deficient models results in an imbalance between Thl7 and Treg cells that promotes the development of autoimmune conditions, including severe colitis (mice), aggravated EAE (LEW rats), and T1D (BB rats) ⁵ - ⁶ · ¹⁰ · ^η · ⁶³ . Although the roles of Wnt signaling, GSK3 and β-catenin in CD4+ T cell polarization and activity are controversial ¹⁹ ' ² °· 64, 65, 66, 67, 68 _^ dysregulation of GSK3 activity in CD4+ T cells has been linked with skewing towards pathogenic Thl7 cells. Studies by Beurel et al. have shown that upregulation of GSK3 promotes Thl7 polarization, while its inhibition blocks this process ²⁰ . In contrast, inhibition of GSK3 activity potentiates the polarization and suppressive capacity of Treg cells ^{64, 67} · ⁶⁹ . This provides a potential mechanism as to why Gimap5 ^sph/sph Treg cells have a reduced suppressive capacity that can be restored by long-term LiCl treatment.

[00145] Applicant showed that a dysregulation of GSK3 in lymphocytes can lead to a primary immune deficiency, as observed in Gimap5 ^sph/sph mice and the GIMAP5 LOF patient. Given its importance in maintaining CD4+ T cell homeostasis and

differentiation, Applicant proposes that even a minor dysregulation of GSK3 within the T cell compartment could result in abnormal polarization of CD4+ T cells, leading to preferential differentiation of Thl7 cells over regulatory T cells. Such a dysregulation may be occurring in autoimmune diseases associated with pathogenic Thl7 cells, such as Crohn's disease, T1D, and multiple sclerosis ⁷⁰ ' ^{71 72} , or in Gimap5-associated diseases such as SLE, T1D, and allergic asthma ¹ ' ² ' ^{3 4} . In this case, selective inhibition of GSK3 activity offers a new promising therapeutic approach for treating patients with immunopathogenic T cell responses.

[00146] T cells depend on their ability to undergo clonal expansion for an efficient immune response during infection or to maintain immune homeostasis in the gut. Applicant's studies reveal a key role for Gimap5 in inactivating GSK3 during CD4+ T cell activation, a link that is critically required to maintain T cell fitness and allows for productive T cell proliferation. Applicant proposes that the Gimap5-mediated inactivation of GSK3 is an essential molecular mechanism to support productive CD4+ T cell responses. Moreover, Applicant's studies point to a remarkable therapeutic potential for GSK3 inhibitors to improve CD4+ T cell survival/proliferation and prevent immunopathology. Thus far, GSK3 inhibitors have been used to treat a variety of diseases including Alzheimer's disease, mood disorders, cancer, and diabetes mellitus (for extensive reviews see 73, 74). Applicant's current data reveal a new therapeutic application of GSK3 inhibitors specifically in the treatment of immunodeficient patients that have GIMAP5 LOF mutations. These patients present a strikingly similar phenotype to Gimap5 -deficient mice and suffer from recurrent (viral) infections most likely stemming from an overall lack of T cell fitness. Applicant posits that GSK3-inhibitors will improve overall T cell survival and function and may

prevent/correct immune-associated sequelae observed in these patients. In addition, therapeutic targeting of this pathway may be relevant for the treatment of patients with SNPs in GIMAP5 linked to development of islet autoimmunity in type I diabetes, systemic lupus erythematosus ^{1, 2} · ³ , or asthma ⁴ .

[00147] Supplementary note: Clinical profile description of patient carrying a missense mutation in GIMAP5 resulting in a Leu204Pro amino acid change

[00148] A 14-year-old Caucasian male presented with immune

thrombocytopenic purpura (ITP). The patient initially responded to intravenous

gammaglobulin (IVIG), however, the thrombocytopenia re-occurred, concomitantly with mild hemolytic anemia, as well as neutropenia and lymphopenia. The patient was positive for a direct Coombs test, but negative for anti-neutrophil or anti -platelet antibodies. A bone marrow biopsy demonstrated mild hypocellularity with low myeloid to erythroid ratio.

[00149] Past medical history includes chickenpox infection following varicella vaccine at the age of 11, and shingles at the age of 14. He has suffered and continuous to suffer from extensive warts since the age of 12, which requires frequent cryotherapy.

Otherwise, there is no history of recurrent bacterial or fungal infections, enteropathy, lymphadenopathy or other non-hematological autoimmune features.

[00150] His immune evaluation prior to referral included normal serum levels of IgG, IgA, IgM and IgG subgroups, but elevated IgE. He was found to have lymphopenia, with CD4 of 331, CD 8 of 67, CD19 of 67, and CD16/56 of 67 cells/mcL. Anti-varicella zoster virus (VZV) antibodies were positive.

[00151] At the age 15 he developed a new episode of thrombocytopenia and was given prednisone and IVIG to which he initially responded well, but subsequently became prednisone dependent.

[00152] At the age of 16, the patient was referred to Applicant's institution for a second opinion for steroid-dependent persistent thrombocytopenia, lymphopenia, and splenomegaly as part of a suspected immunodeficiency disorder. Immune evaluation at the time of referral demonstrated normal Hb/Hct, absolute neutrophil count (ANC: 3120 cells/mcL) and lymphopenia (ALC: 440 cells/mcL) with thrombocytopenia (PLT: 54 K/mcL). Direct Coombs was negative, as were anti-neutrophil and anti -platelet antibodies.

[00153] Lymphocyte subset revealed CD3: 273 (62%), CD4: 246 (56%), CD8: 30 (7%), CD19: 135 (31%), CD16/56: 20 (7%) cells/mcL. CD4 to CD8 ratio was 8.2. Serum IgG, A, E levels and IgG subgroups were normal, except low IgM (IgG: 839, IgA: 70, IgM: 54 mg/dl, IgE: 282 IU/ml). Titers to protein antigens were normal and 2/14 pneumococcal titers were protective. B cell panel demonstrated normal proportion of naive, transitional, and isotype switched CD27+ memory B cells.

[00154] He was found to have decreased naive CD4 cells (11.9, N: 33-73.5% for age) with a normal proportion of CD31+ T cells (indicative of recent thymic emigrants), increased memory CD4 cells (88.1, N: 26.3-66.3%) and increased CD4 T-effector memory cells (80.2, N: 33.4-74.1%). Naive CD8 cells were normal and CD8 TEMRA cells were slightly increased (11.2, N:0-10.3%). The proportion of TCRo$ expression was slightly low on CD 8 cells.

[00155] T-cell proliferation studies showed reduced proliferation to

phytohemagglutinin (PHA) and Candida, and borderline response to tetanus. Telomere length measurement revealed low telomere length of total lymphocytes, granulocytes, naive and memory T cells, but normal telomere length of B cells and NK cells. The patient was given a working diagnosis of immunodeficiency with features of autoimmunity. Mutations in known SCID genes, as well as LRBA, PI3KCD, CTLA-4, and STAT3 genes were excluded. Whole exome sequencing revealed a homozygous c.611T>C (p.Leu204Pro) variant in GIMAP5, where his parents were found to be heterozygous.

[00156] Methods

[00157] Study design

[00158] For the proposed experiments involving the studies of immune responses in vivo, both male and female mice were used. All strains of mice used were generated on a C57BL/6J background and confirmed by whole genome SNP analysis and Applicant anticipated limited genetic variation. To minimize confounding secondary factors arising from lymphopenia and other late-stage pathologies that develop in Gimap5 ^sph/sph mice, CD4+ T cells from 3 -week-old mice were used unless otherwise noted. At this age,

Gimap5 ^sph/sph mice have relatively normal numbers of CD4+ T cells with a normal frequency of naive T cells that have a quiescent phenotype comparable to WT mice. Since Applicants were interested in T cell proliferation/survival in WT vs Gimap5-deficient cells, Applicant expected to find strong differences in measurement (e.g. normal vs absence of T cell survival/proliferation). In cases where the differences in the observed phenomena were clear and distinct, a group size of 6 mice was suitable to give statistically significant (i.e. P<0.05) data. For these studies, Applicant estimated that, with a sample size of 6, Applicant would have a 99% power to detect at least 25% reduction in the % of proliferating cells in stimulated Gimap5 ^sph/sph cells with a significance level (alpha) of 0.05 (two-tailed).

[00159] Mice and reagents [00160] All experiments were performed according to the US National Institutes of Health guidelines and were approved by the IACUC of The Cincinnati

Children's Hospital. C57BL/6J mice were obtained from Jackson. Gimap5 ^s P ^h/s P ^h mice were generated as described6 and bred in-house to generate WT or Gimap5 ^sph/sph ;Rag2 ^_/~ ;OT-II, WT or Gimap5 ^s P ^h/s P ^h ;Tg (Cd4-cre/ert2) 1 IGnri/J; Gsk3 ^fl/fl mice in the vivarium of Cincinnati Children's Hospital. All mice were maintained under specific pathogen- free conditions. Purified a-mouse-CD3 (17A2) and a-mouse-CD28 (37.51) antibodies (Biolegend) were used for murine T cell activation. For human T cell studies, purified aCD3 (OKT3) and aCD28 (CD28.2) antibodies (Biolegend) were used. 7-AAD was purchased from BD. Ovalbumin, LiCl, 6-Bromoindirubin-3'-oxime (BIO), Ionomycin, and phytohemagglutinin-L (PHA-L) were obtained from Sigma. All antibodies used for flow cytometry were purchased from eBioscience or Biolegend unless otherwise noted. Antibodies for ΙκΒ, pERKl/2, pJNK, pAKT, p-p38, and pGSK3 were purchased from Cell Signaling Technologies. Monoclonal antibody MAC421 was used to probe for Gimap5 as described6. GSK3 was probed with monoclonal antibody Clone 7/GSK-3b from BD.

[00161] Table 2. Details of Antibodies Used. *ISFC: ImageStream Flow Cytometry, **FC: Flow Cytometry, ***WB: Western Blot

Antibody Clone Supplier Catalog Number Dilution

FC: 1:200

CD4 GK1.5 Biolegend 100437

ISFC: 1:200

CD44 IM7 BD Pharmigen 564392 FC: 1:200

CD62L MEL- 14 Biolegend 104437 FC: 1: 100

CD 19 6D5 Biolegend 115531 FC: 1: 100

B220 RA3-6B2 ThermoFisher 45-0621-82 FC: 1: 100

CD8a 53-6.7 Biolegend 100751 FC: 1: 100

NKp46 29A1.4 Biolegend 137617 FC: 1: 100

CD25 PC61 Biolegend 102035 FC: 1: 100

Foxp3 FJK-165 ThermoFisher 17-5773-82 FC: 1: 100

Annexin V n/a Biolegend 640906 FC: 1: 100

ISFC: 1 :200 WB:

Clone 7/GSK- 1:5000 Confocal:

GSK3 BD 610202

3b 0.7 μg/10 ⁶ cells

ISFC: 1: 100 pGSK3

polyclonal EMD Millipore 07-2275 WB: 1:5000 (Ser389)

[00162] T cell analyses

[00163] To characterize lymphocyte populations ex vivo, lymphocytes from spleen and mesenteric lymph node (mLN) were isolated and stained with fluorochrome- conjugated antibodies for mouse CD4, CD8, B220, CD24, CD44, CD62L, and Foxp3. In all experiments, samples were stained with a fixable viability dye; analysis was restricted to live cells unless otherwise stated.

[00164] For all in vitro murine CD4+ T cell experiments, unless otherwise noted, Mojo-purified (Biolegend) CD4+ T cells were stimulated with plate-bound aCD3 (1 μg/ml) + soluble aCD28 (2 μg/ml). Cells were cultured in supplemented IMDM medium containing 10% FBS, 2% penicillin/streptomycin, 1% L-glutamine, and 50 μΜ BME. T cell proliferation was quantified by incubating CD4+ T cells in 5 μΜ CFSE in 0.2% FBS for 5 min. Cells were either left unstimulated or stimulated in the presence of GSK3 inhibitors LiCl (2.5 mM) or BIO (100 nM). After 3 days incubation, proliferation was evaluated by analyzing CFSE dilution by flow cytometry. In indicated experiments, 200 ng/mL Wnt3a, 2 μΜ IWP-2, or 200 ng/mL Cyclosporin A (CsA) were added. DNA damage was evaluated by γΗ2ΑΧ staining in conjunction with 7-AAD staining for cell cycle analysis. Early phosphorylation of GSK3 (S9), Akt (S473) and p38 (T180/Y182) was evaluated by stimulating rested CD4+ T cells with aCD3 (5 μg/mL)/aCD28 (2 μg/mL) for the indicated time point before staining for flow cytometry. Early phosphorylation of AKT, p38, ERK, and JNK, and the degradation of ΙκΒ was also evaluated in CD4+ T cells upon stimulation with PMA (50 ng/mL) and Ionomycin (750 ng/mL). Ex vivo survival of CD4+ T cells was evaluated by culturing Mojo-purified peripheral CD4+ T cells or total thymocytes with 5 ng/mL IL-7 for 0-7 days. The number of surviving cells was determined by staining for CD4 and viability and counted on a flow cytometer using counting beads (Biolegend).

[00165] Immunoblotting

[00166] Protein lysates from human T cells were prepared according to standard methods from resting cells or cells stimulated with 5 μg/mL aCD3 + 2 μg/mL aCD28 + 5mM LiCl for 24h. For mouse experiments, Mojo-isolated (BioLegend) CD4+ T cells were stimulated with aCD3 and aCD28 + 2.5 mM LiCl for the indicated time periods prior to preparing protein lysates. In indicated experiments, 10 μΜ of proteasomal inhibitor MG132 was added 4h prior to lysis of the cells. Lysates were separated using 10% Bis-Tris Gels, transferred to nitrocellulose, and immunoblotted with primary antibodies to GIMAP5 (CST 14108), phospho-c-Myc (T58) (MyBioSource), c-Myc, pGSK3 (S389) (EMD

Millipore), pGSK3 (S9), total GSK3 (BD), phospho-p53 (S15), total p53, phospho-p38 (T180/Y182), total p38, p-mTOR, total mTOR, p-p70 S6K (T389), p70-S6K, β-catenin, and β-actin. All primary antibodies were obtained from Cell Signaling Technology unless otherwise noted.

[00167] AMNIS ImageStream flow cytometry

[00168] For mouse localization studies, CD4+ T cells were stimulated with aCD3 + aCD28. After 6, 24h, or 48h CD4+ T cells were stained with antibodies to CD4 (GK1.5), Gimap5 (MAC421), GSK3 (BD 610202), pGSK3 (S389) (EMD Millipore 07- 2275), DAPI, and a fixable viability dye. In indicated experiments, 200 ng/niL Wnt3a and 2 μΜ IWP-2 were added. For NFATcl localizations studies, isolated CD4+ T cells were stimulated indicated times. Cells were stained with antibodies for CD4 and NFATcl (clone 7A6), DAPI, and a fixable viability dye. Live CD4+ T cells were analyzed for NFATcl and DAPI localization by delineating regions of positive signal (i.e. a mask). Nuclear translocation was measured by similarity dilate, which represents the log transformed Pearson's Correlation Coefficient and is a measure of the degree to which two images are linearly correlated within a masked region. In human GIMAP5-localization studies, CD4+ T cells were isolated from the PBMCs of healthy donors by MACS-purification (Miltenyi Biotec) and stimulated with aCD3 (5 μg/ml) and aCD28 (2 μg/ml). After 24h and 48h, T cells were stained with antibodies to CD4, GIMAP5 (CST 14108), GSK3 (BD 610202), CD107b (Miltenyi Biotec), and a fixable viability dye. GSK3 vesicular localization in patient and control CD4+ T cells was measured upon re- stimulation of resting T cells after IL-2 expansion. Colocalization, vesicular localization, and nuclear localization was evaluated using ImageStream Data Exploration and Analysis Software (IDEAS) 6.1 as previously described ⁷⁵ . Specifically, colocalization was quantified using the Bright Detail Similarity representing the log transformed Pearson' s correlation coefficient of the localized bright spots with a radius of 3 pixels or less within the masked area in the two input images.

[00169] Confocal microscopy

[00170] To visualize the localization of GSK3 and Gimap5 at a higher resolution, CD4+ T cells isolated from the spleen and lymph nodes of WT and Gimap5 ^sph/sph mice were stimulated with aCD3/aCD28. After 24h, cells were stained with antibodies for Gimap5 (MAC421) and GSK3 (BD61202, 0.7ng / lxlO ⁶ cells). Cells were counterstained with DAPI (1 μg/mL) and mounted in Prolong Gold anti-fade reagent (Cell Signaling Technology). Samples were imaged on a Nikon Al LUN-V inverted microscope using a lOOx objective with oil immersion. For each imaged cell, Z-stacks were generated by taking images at a 0.125 μιη step. To refine localization, images were deconvolved using the Landweber algorithm (15 iterations) in NIS Elements v4.5 (Nikon). Z-stacks were assembled and GSK3 and Gimap5 localization assessed in Imaris Image Analysis software v8.3 (Bitplane).

[00171] Calcium Flux

[00172] Splenocytes isolated from individual WT or Gimap5 ^sph/sph mice were stained with Indo-1 (2 μg/mL) at lxlO ⁶ cells/mL for 30 min at 37°C. Cells were then stained for CD4 and rested for >lh in cell loading medium (HBSS + ΙμΜ CaC12 + ΙμΜ MgC12 + 1% FBS) at room temperature. Five minutes prior to analysis, cells were warmed to 37°C. To evaluate calcium flux, samples were acquired for ~30s without stimulation and an additional 270s after stimulation with aCD3/aCD28 (1.25 μg/mL) or lonomycin (800 ng/mL) for a total of 5 minutes.

[00173] Real-time PCR

[00174] Mojo-purified (Biolegend) CD4+ T cells were rested or stimulated with aCD3/aCD28 for 24h. Cells were lysed with TRIzol (Thermo Fisher Sci), mRNA isolated, and reverse transcription performed using a High-capacity cDNA Reverse

Transcription Kit (Applied Biosystems). cDNAs were amplified with LightCycler 480 SYBR Green I Master (Roche) and quantified by Light-Cycler 480-11 instrument (Roche). The following primer pairs were used: myc, forward: 5'-ATGCCCCTCAACGTGAACTTC-3' (SEQ ID NO: 1), reverse: 5 ' -GTCGCAGATGAAATAGGGCTG-3 ' (SEQ ID NO: 2); wnt3a, forward: 5 ' -CTCCTCTCGGATACCTCTTAGTG-3 ' (SEQ ID NO: 3), reverse: 5'- CCAAGGACCACCAGATCGG-3 ' (SEQ ID NO: 4); ctnnbl (β-catenin), forward: 5'- ATGGAGCCGGACAG AAAAGC-3 '(SEQ ID NO: 5), reverse: 5'- CTTGCCACTCAGGGAAGGA-3 ' (SEQ ID NO: 6); L32, forward: 5'- GAAACTGGCGGAAACCCA-3'(SEQ ID NO: 7), reverse: 5'-

GGATCTGGCCCTTGAACCTT-3 ' (SEQ ID NO: 8). Expression of myc, wnt3a, and ctnnbl was normalized to L32 and set relative to unstimulated WT samples.

[00175] In vivo OVA administration

[00176] The role of TCR signaling in Gimap5 ^s P ^h/s P ^h CD4+ T cell survival in vivo was evaluated by crossing Gimap5 ^s P ^h/s P ^h mice with Rag2 ^_/~ ; OT-II mice. OVA (1 mg/niL) was administered to 10-week-old mice in drinking water ad libitum. After 5 weeks, T cell survival and Treg cell-induction were evaluated by flow cytometry.

[00177] In vivo lithium treatment

[00178] To evaluate if in vivo inhibition of GSK3 activity could prevent the development of Gimap5 ^sph/sph -associated pathologies, 3-week-old mice were administered 150 mg/kg LiCl in drinking water. After 4-5 weeks, liver damage, colitis, and lymphocyte populations were evaluated by histology and flow cytometry, respectively. Animals were assigned to treatment and vehicle groups randomly, with equal numbers of male and female mice assigned to each group. For these experiments, WT and Gimap5 ^sph/sph genotypes were co-housed during treatment. Investigators were blinded to mouse genotype and treatment status during analysis.

[00179] Genetic deletion of GSK3

[00180] GSK3 hyperactivation in Gimap5 ^sph/sph CD4+ T cells was evaluated by crossing Gimap5 ^sph/sph ; Gsk3 ^fl/fl mice to Cd4cre-ert2 mice, allowing for the tamoxifen- inducible genetic deletion of Gsk3 in CD4+ T cells. Tamoxifen was administered in food to 3-week-old mice (40mg/kg body weight; Harlan Laboratories Teklad Diets). At 8 weeks, liver damage, colitis, and lymphocyte populations were evaluated by histology and flow cytometry respectively.

[00181] Histology

[00182] Colon tissue was collected and immediately fixed in 10% buffered formalin solution overnight, followed by routine paraffin embedding. Hematoxylin and eosin staining were performed on 4 μιη sections from the paraffin-embedded tissue blocks for conventional light microscopy analysis. Histological scoring was performed double-blind as described before (5, 6). Briefly, scoring parameters included quantitation of the area of distal colon involved, edema, erosion/ulceration of the epithelial monolayer, crypt loss/damage, and infiltration of immune cells into the mucosa. Severity for the area involved

(erosion/ulceration and crypt loss) was graded on a scale from 0 (normal), 1 (0-10%), 2 (10- 25%), 3 (25-50%), and 4 (>50%). Immune cell infiltration was scored as 0, absent; 1, weak; 2, moderate; and 3, severe. Total disease score was expressed as the mean of all combined scores per genotype.

[00183] In vitro regulatory T cell suppression assay

[00184] Treg cell suppression assays were performed as described using Treg cells isolated from the spleens of WT and Gimap5 ^sph/sph mice treated with either LiCl or vehicle (5,6). In brief, CD4+ T cells were enriched by magnetic separation (Mojo, Biolegend) and stained for viability, CD4, and CD25. Live CD4+CD25+ regulatory T cells were isolated by FACS using a Beckman Coulter MoFlo XDP cell sorter. Sorted Treg cells were cocultured with the indicated ratios with 5xl0 ⁴ CTV-labeled CD8+ T cells isolated from a naive mouse. An additional lxlO ⁵ T cell depleted, gamma-irradiated (1500 rad) splenocytes were also cocultured as bystander cells. CD8+ T cells were stimulated with 0.5 μg/mL aCD3;

proliferation was assessed by CTV dilution after three days of coculture.

[00185] Expansion of human T cells

[00186] For all studies concerning human cells, informed consent was obtained and studies were approved by the CCHMC institutional IRB. PBMCs were isolated from whole blood of a GIMAP5-deficient patient and a healthy parent by Ficoll-Paque Plus density gradient centrifugation. Isolated PBMCs were cultured in RPMI, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2mM L-glutamine, and 10 mM HEPES and were stimulated with 5 μg/mL phytohemagglutinin (PHA-L) + 5mM LiCl. After four days, T cells were selectively expanded in 150 U/mL rhIL-2 (Miltenyi) + 5mM LiCl for 8 days. Expanded T cells were rested for 2d in the absence of IL-2 and LiCl before use in reactivation experiments. Prior to expansion, patient's PBMCs were stained for CD 19, CD3, CD4, CD8, CD16, CD56, and Foxp3 and analyzed by flow cytometry for evaluation of circulating lymphocyte populations.

[00187] Analysis of DNA damage response in human CD4+ T cells

[00188] Rested patient T cells were restimulated with 5 μg/mL aCD3 and 2 μg/mL aCD28. After 24, 48, and 72h stimulation, cells were stained for viability, CD3, CD4, CD8, and γΗ2ΑΧ and analyzed by flow cytometry. At 24 and 48h, cells were stained with a- CD4, α-γΗ2ΑΧ, DRAQ5, and a fixable viability dye. Analysis was performed on live CD4+ T cells using ImageStream flow cytometry as detailed above.

[00189] Statistical analysis

[00190] All data were analyzed using GraphPad Prism4® software (GraphPad Software, San Diego, CA). For studies comparing T cells from C57BL/6J, Gimap5 ^sph/sph ; and/or Gsk3 ^flox/flox mice or involving chemical treatment of T cells, Student's two-tailed test or ANOVA followed by Sidak's multiple comparisons test for three or more groups were used. Data were considered statistically significant if P values were <0.05. Data were normally distributed.

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[00267] All percentages and ratios are calculated by weight unless otherwise indicated. [00268] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[00269] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[00270] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm."

[00271] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[00272] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.