Inducing MAIT cells for treating inflammatory skin and systemic disorders

Abstract

Disclosed herein are methods for using 5-OP-RU to treat or prevent disorders characterized by reduced mucosal-associated invariant T (MAIT) cells, such as cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE). The methods include administration of 5-OP-RU in topical or systemic formulations. Also disclosed are liposome formulations comprising the 5-OP-RU.

Description

Atty. Dkt. No. 178981.00043INDUCING MAIT CELLS FOR TREATING INFLAMMATORY SKIN AND SYSTEMIC DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 770, 185filed on March 11, 2025, the content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01AR080641-02 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Systemic lupus erythematosus (SLE) is a leading cause of death in young females, and 70-85% of SLE patients experience skin disease (cutaneous LE, CLE). The mechanisms of CLE pathogenesis remain unknown, and current treatments rely on broad immunosuppressive agents. More targeted and effective strategies for treating CLE and SLE are of interest.SUMMARY

[0004] In an aspect, provided herein is a method for treating or preventing a disorder characterized by reduced mucosal-associated invariant T (MAIT) cells in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). In embodiments, disorder comprises a skin disorder. In embodiments, the skin disorder is an autoimmune skin disorder. In embodiments, the skin disorder comprises cutaneous lupus erythematosus (CLE).

[0005] In embodiments, the 5-OP-RU is administered topically. The 5-OP-RU may be provided in a topical formulation at a concentration of between about 0.5 mM and about 5 mM. The 5-OP-RU may be provided at a concentration of about 1 mM.

[0006] In embodiments, the 5-OP-RU is applied to at least a portion of skin on the subject. The 5-OP-RU may be applied at least one time every seven days. The 5-OP-RU may be applied at least one time every two days.Atty. Dkt. No. 178981.00043

[0007] In embodiments, the subject is expressing CLE lesions. The 5-OP-RU may be applied to the CLE lesions.

[0008] The 5-OP-RU may be administered within two days prior to exposure to UV light.

[0009] The method may further comprise administering an additional topical skin therapy to the subject. The additional topical skin therapy may comprise a topical steroid. The additional topical skin therapy may comprise a UV protectant

[0010] The disorder may comprise a systemic autoimmune disorder. The systemic autoimmune disorder may comprise systemic lupus erythematosus (SLE).

[0011] In embodiments, the 5-OP-RU is administered systemically. In embodiments, 5-OP-RU is administered by intraperitoneal injection. The 5-OP-RU may be administered at a concentration of between about 0.5 mM and about 5 mM. The 5-OP-RU may be administered at a concentration of between about 1 mM. The 5-OP-RU may be administered at least one time every 1 to 7 days.

[0012] The method may further comprise administering an additional systemic autoimmune disorder therapy. The additional systemic autoimmune disorder therapy may comprise an antiinterferon receptor antibody.

[0013] The 5-OP-RU may be in a liposome. The liposome may comprise: a) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-lOOO) (DSPE-PEG1000), and cholesterol; b) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-2000) (DSPE-PEG2000), and cholesterol; or c) l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP), and 1,2-dimyristoyl-rac-glycero-3 -methoxy (poly (ethylene glycol) (DMG-PEG2000), and cholesterol.

[0014] In another aspect, provided herein is a liposome comprising 5-OP-RU, cholesterol, a phospholipid, and a PEGylated lipid. The phospholipid may be selected from DOPE and DPPC. The PEGylated lipid may be selected from DSPE-PEG1000, DSPE-PEG2000, and DMG-PEG2000. The liposome may further comprise DOTAP.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1C. MAIT cells are reduced but activated in CLE-like skin. A) Gating strategy for identification of MAIT cells from total lymphocytes using 5-OP-RU loaded MR1-Atty. Dkt. No. 178981.00043tetramer. B) Frequency and number of MAIT cells in CD3+TCR0+ T cells in B6 and MRL-lpr ears identified as in A. Student’s t-test, n = 21-30 per group, ***p<0.001, ****p<0.0001. C) Frequency of CD69+PD1+ MAIT cells in B6 and MRL-lpr ears. Significance was determined by Student’ s t-test (n = 5 per group, **p<0.01).

[0016] FIGS. 2A-2D. Topical expansion of MAIT cells resolves active CLE skin lesions. A) Experimental design for lesion treatment via topical 5-OP-RU in female MRL-lpr mice beginning when lesions score >10. B) Representative images of the vehicle (water) and 5-OP-RU treated mice throughout the treatment course. C) Lesion score over time quantified by extent of skin erythema, scaling, thickness, and alopecia. D) Duration of sustained lesion score reduction in vehicle versus 5-OP-RU treated mice. Significance was determined by (C) Chi-squared test and (D) Student's t-test( n=3, *p<0.05, **p<0.01; Tx = Treatment cessation).

[0017] FIGS. 3A-3C. Topical expansion of MAIT cells prevents the development of severe CLE lesions. A) MRL-lpr mice were treated once weekly with topical 5-OP-RU (ImM) beginning at 8 weeks of age. B) Prevalence of skin lesions (score > / = 5) during treatment (n=3-4 / group; *p<0.05). Lesions were scored based on criteria of erythema, thickness, scaling, and alopecia. C) Time to 50% reduction in initial lesion score of mice in which lesions reached 50% score reduction. Significance was determined by (B) Chi-squared test and (C) Student's t-test (n=3, *p<0.05).

[0018] FIGS. 4A-4E. MAIT cell expansion leads to Tregexpansion and reduction in inflammatory mediators in CLE skin. A) Experimental design for the expansion of MAIT cells by topical 5-OP-RU application. (B) Flow cytometry contour plots and corresponding quantification of MAIT cells (live CD45+ ySTCR- CD3+ TCRP+MR1: op’RUTetramer+) in MRL / lpr skin with (5-OP-RU) and without (Veh) topical antigen (n=10). (C) Flow cytometry contour plots and corresponding quantification of Treg (live CD45+ ybTCR- CD3+ TCR0+ CD4+ Foxp3+) in MRL / lpr skin with (5-OP-RU) and without (Veh) topical antigen (n=10). (D) Quantification of CD8 and CD4 T cell cytokine production in MRL / lpr skin with (5-OP-RU) and without (Veh) MAIT cell expansion determined by flow cytometry (n=5). (E) Expression of cytotoxic Qfng, Gzmb) and inflammatory (116, Tnf) genes determined by qPCR relative to Gapdh in B6 and MRL / lpr skin with (5-OP-RU) and without (Veh) MAIT cell expansion (n=10).

[0019] FIGS. 5A-5D. MAIT cell activation drives local MAIT cell and Treg expansion. (A-B) Flow cytometry contour plots and corresponding quantification of skin MAIT cells after (A)Atty. Dkt. No. 178981.00043UVB (n=4) or (B) 5-OP-RU (n=7) in the presence or absence of SIP receptor blocker FTY720 (FTY). (C-D) Flow cytometry contour plots and corresponding quantification of skin Treg after (C) UVB (n=4) or (D) 5-OP-RU (n=7) in the presence or absence of S IP receptor blocker FTY720 (FTY). Data analyzed with (A-D) one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. ns, not significant.

[0020] FIG.6. MAIT cells are required for Treg expansion with 5OPRU and in an inducible model of lupus skin disease. 5-OP-RU was administered topically (ImM) as in FIG. 4A on the ears of B6 and MAIT-deficient (B6.A7 / 7- / -) mice. Treg were quantified in the treated ears by flow cytometry. Imiquimod (TLR7 agonist, 5% cream) was administered topically on one ear of B6 and B6.A / 7- / - mice 3x per week for 4 weeks and Treg levels were quantified by flow cytometry in the contralateral ear (lupus model). Representative flow cytometry plots (left) and the number of Treg per ear quantification summary (right). Significance was determined by two-way ANOVA with Bonferroni post-hoc: n=3-10; *p< 0.05; **p< 0.01.

[0021] FIGS. 7A-7C. Expanded MAIT cells in healthy skin are II17a+IU3+Areg+ and Treg are tissue-resident IU0+Cxcr6+ effector cells. A) Single-cell RNAseq on sorted MAIT and Treg from B6.Foxp3-GFP+ mice treated with vehicle (PBS) or 5-OP-RU, as in FIG. 4A. B) Differential gene expression (DGE) analysis of MAIT cells (Travl+) and feature plots of selected genes. C) DGE analysis of Treg (Foxp3+) and feature plots of selected genes.

[0022] FIGS. 8A-8D. Systemic administration of 5-OP-RU expands skin MAIT cells and Treg in CLE mice. A) Experimental design for systemic administration of 5-OP-RU to OLE mice (female, 17 weeks with proteinuria). B-C) Number of MR-1- 5-OP-RU-tetramer+ (B) MAIT cells and (C) CD4+Foxp3+ Treg in the skin on day 21 after the start of treatment administration. D) Change in the concentration of serum cytokines in vehicle (water) and 5-OP-RU treated mice. Significance was determined by (B-C) Student’s t-test and (D) Two-way ANOVA: n=5-9; *p< 0.05.

[0023] FIGS.9A-9I. UV light stimulates Treg and MAIT expansion in healthy but not lupus-prone non-lesional skin. (A) Illustration of UVB exposure (lx, 250mJ / cm2) in healthy human skin. (B) Flow cytometry contour plots of human skin Treg (Live CD45+ CD3+ TCR[3+ CD4+ CD25+ Foxp3+) with (UV 48h) and without (No UV) UVB exposure and quantification of Treg number fold change over No UV (n=8). (C) Flow cytometry histogram of human skin Treg KI67 expression and quantification of KI67+ Treg frequency with (UV 48h) and without (No UV) UVBAtty. Dkt. No. 178981.00043exposure (n=8). (D) Illustration of chronic, low-dose UVB exposure (6x, 50mJ / cm2) in B6 and MRL / lpr mice (female, 8wks). (E) Flow cytometry contour plots and corresponding quantification of skin Treg (live CD45+ y6TCR- CD3+ TCR0+ CD4+ Foxp3+) at baseline (No UV) and after UV exposure (UVD6) in B6 and MRL / lpr mice (female, 8wks; n=14-16). (F) Flow cytometry contour plots and corresponding quantification of skin MAIT cells (live CD45+ ySTCR- CD3+ TCRP+MR1:5'op'RUTetramer+) in B6 and MRL / lpr mice with (UVD6) and without (No UV) UV exposure (n=14-16). Data derived from (E and F) three experiments, represented as means + / -SEM. Data analyzed with two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant. (G) Uniform manifold approximation and projection of skin T cells generated from concatenated flow cytometric data of representative baseline (No UV) and UVB-exposed (UVD6) B6 and MRL / lpr mice (n=2 per group). Stacked bar plot quantifying mean composition per T cell cluster. (H) Expression of Treg-associated genes in healthy and lupus skin before (No UV) and 24 hours after acute UVB (2 x MED; n=3-4). (1) Expression of MAIT cell-associated genes in healthy and lupus skin before (No UV) and 24 hours after acute UVB (2 x MED; n=3-4). Data derived from (B and C) five and (H-I) one independent experiment(s), represented as means + / - SEM. Data analyzed with (B and C) paired t-test or (H-I) two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

[0024] FIGS. 10A-10C. Topical MAIT expansion in CLE-like skin attenuates UV-light-induced immune activation. A) Experimental design for MAIT expansion prior to chronic skin UVB exposure in 8-10 week old female MRL-lpr mice. B) Quantification of skin MAIT cells determined by flow cytometry using 5 -OP-RU -loaded MR1 tetramer. C) Quantification of skin CD8 T cells by flow cytometry and expression. Significance was determined by student’s t-test (n=3-4, *p < 0.05).

[0025] FIG. 11. Type I interferon suppresses antigen-mediated expansion of human MAIT cells. Representative flow cytometry plots of MAIT cell levels, MR1:5-OP-RU-tetramer+TCRVa7.2+, after treatment of healthy human PBMCs with 5-OP-RU (luM), 5-OP-RU + IL-2 (50 U / ml), or unstimulated for 7 days. The bar graph combines quantification of percent MAIT cells, including the condition in which cells were treated with 5-OP-RU+IL-2+IFNa (lOOU / ml). Significance was determined by One-way ANOVA (n = 5-7 per group, *p<0.05, ***p < 0.001, ns = not significant).

[0026] FIGS. 12A-12J. MAIT cells are required for UVB-induced Treg response in healthyAtty. Dkt. No. 178981.00043skin. (A) Illustration of chronic, low-dose UVB exposure (6x, 50mJ / cm2) in B6 and Mrl- / - mice (female, 8wks). (B) Flow cytometry contour plots and quantification of skin Treg (live CD45+ y6TCR- CD3+ TCR0+ CD4+ Foxp3+) in B6 and Mrl- / - mice with (UVD6) and without UVB (No UV; n=17-24). (C) Flow cytometry contour plots and corresponding quantification of KI67+ Treg frequency in B6 and Mrl- / - skin with (UVD6) and without (No UV) UVB exposure (n=14-17). (D) Uniform manifold approximation and projection of skin Treg and corresponding stacked bar plot generated from concatenated flow cytometric data of representative baseline (No UV) and UVB-exposed (UVD6) B6 and Mrl- / - mice (n=5). (E) Flow cytometry histograms and corresponding quantification of skin Treg activation (ICOS) and suppression (CTLA4) markers with (UVD6) and without (No UV) UVB exposure in B6 &Mrl- / - mice (n=9-10). (F) Illustration of UVB exposure (as in A) before induction of delayed contact hypersensitivity (CHS) reactions using the hapten, dinitrofluorobenzene (DNFB) and quantification of change in ear thickness over baseline (No UV + vehicle) in B6 and Mrl- / - after DNFB challenge with (UV) or without (No UV) UVB exposure (n=10). (G) Flow cytometry contour plots and corresponding quantification of KI67+ Treg frequency in B6 and Mrl-- skin after UV exposure and hapten challenge (n=5). (H) Illustration of expansion of MAIT cells via topical 5-OP-RU (ImM) prior to UVB exposure (lx, 250mJ / cm2) in healthy human skin. (I) Flow cytometry contour plots of human skin MAIT cell (Live CD45+ CD3+ TCR0+ TCRVa7.2+MR1:5’op'RUTetramer+) KI67 expression and corresponding quantification with (UV + 5-OP-RU) and without (UV + Veh) MAIT expansion prior to UVB exposure (n=3). (J) Flow cytometry contour plots of human skin Treg (Live CD45+ CD3+ TCR0+ CD4+ Foxp3+ CD25+) and corresponding quantification with (UV + 5-OP-RU) and without (UV + Veh) MAIT expansion prior to UVB exposure (n=4). Data derived from (B-C, I-J) three, (E) two, or (D) one independent experiment s), represented as means + / - SEM. Data analyzed with (B-C, E-F) two-way ANOVA or (G, I-J) / test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

[0027] FIGS. 13A-13L Local MAIT cell activation promotes Treg expansion in lupus-like skin and protects against UV-induced inflammation (A) Illustration of MAIT cell expansion via topical application of 5-OP-RU (4x, ImM) in B6, Mrl- / -, or MRL / lpr mice (female, 8wks). (B) Flow cytometry contour plots and quantification of skin MAIT cells (live CD45+ ySTCR-CD3+ TCRP+MR1:5'op'RUTetramer+) in B6 and Mrl- / - mice with topical 5-OP-RU or vehicle (n=14). (C) Flow cytometry contour plots and quantification of skin Treg (live CD45+ ySTCR-Atty. Dkt. No. 178981.00043CD3+ TCR0+ CD4+ Foxp3+) in B6 and Mrl- / - mice with topical 5-OP-RU or vehicle (n=14). (D) Feature plots of Foxp3 and Ctla4 expression and quantification of Treg function score for cells captured from skin of 5-OP-RU- or vehicle-exposed 36.Foxp3-GFP mice by scRNAseq. (E) Uniform manifold approximation and projection of skin Treg and corresponding stacked bar plot generated from concatenated flow cytometric data of representative baseline (Veh) and antigen-exposed (5-OP-RU) MRL / lpr mice (n=5). (F) Illustration of expansion of MAIT cells via topical 5-OP-RU (ImM, every 48h x4) prior to UVB exposure (6x, 50mJ / cm2) in MRL / lpr mice (female, 8wks). (G) Flow cytometry contour plots of skin Treg (live CD45+ ySTCR- CD3+ TCR0+ CD4+ Foxp3+) and CD8 T cells (live CD45+ ySTCR- CD3+ TCR0+ CD8+) and quantification of Treg:CD8 ratio in MRL / lpr skin after UVB exposure with (UV + 5-OP-RU) or without (UV + Veh) MAIT cell expansion (n=7-ll). (H) Expression of cytotoxic (Ifng, Gzmb), interferon-stimulated (Irf7, Ifi27l2ra), and disease-associated (Hifla, Tgfbl) genes determined by qPCR relative to Gapdh in MRL / lpr skin with (UV + 5-OP-RU) and without (UV + Veh) MAIT cell expansion prior to UVB exposure (n=5). (I) Representative H&E stain of MRL / lpr skin with (UV + 5-OP-RU) and without (UV + Veh) MAIT cell expansion prior to UVB exposure. Data derived from (B-C) three, (D, E, H) one or (E-F, G) two independent experiment s), represented as means + / - SEM. Data analyzed with (B-C) two-way ANOVA or (E-F, G-H) Welch’s t test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

[0028] FIGS. 14A-14J. Monocyte-derived APCs mediate MAIT cell-driven Treg expansion in the skin via IL-15. (A) Serum IL-15 levels in MRL / lpr (16-20 weeks, female) before (pre) and after (post) systemic treatment with 5-OP-RU or vehicle (n=8). (B) Flow cytometry contour plots and corresponding quantification of skin MAIT cells (live CD45+ ySTCR- CD3+ TCR0+MR1: op-RUTetramer+) in B6 and 1115- - mice treated with UV (UVD6) or MAIT antigen (5-OP-RU) compared to untreated (Ctrl) controls (n=5). (C) Flow cytometry contour plots and corresponding quantification of skin Treg (live CD45+ y§TCR- CD3+ TCR0+ CD4+ Foxp3+) in B6 and 1115- / -mice treated with UV (UVD6) or MAIT antigen (5-OP-RU) compared to untreated (Ctrl) controls (n=5). (D) Flow cytometry contour plots and corresponding quantification of fold change in skin monocyte-derived dendritic cells and other APCs (gating in Supplemental Figure 7) early after UVB (UVD2) in B6 and Mr IF- mice (n=9-10). (E) Flow cytometry contour plots and corresponding quantification of fold change in skin monocyte-derived dendritic cells and other APCs early after MAIT cell antigen (D3 5-OP-RU) in B6 an Mrl- / - mice (n=10-13). (F) FlowAtty. Dkt. No. 178981.00043cytometry histograms and corresponding quantification of skin monocyte-derived dendritic cell IL15Ra expression with (UVD2) and without (No UV) UVB exposure (n=5) or with (D3 5-OP-RU) and without (Veh) MAIT cell stimulation (n=5). (G) Flow cytometry contour plots and corresponding quantification of skin MAIT cells (live CD45+ ySTCR- CD3+ TCRP+MR1: op-RUTetramer+) in B6 and Ccr2- / ~ mice treated with MAIT antigen (5-OP-RU) vehicle (n=5). (H) Flow cytometry contour plots and corresponding quantification of skin Treg (live CD45+ ySTCR-CD3+ TCR0+ CD4+ Foxp3+) in B6 and Ccr2- / ~ mice treated with MAIT antigen (5-OP-RU) compared to vehicle (n=5). Data derived from (A-E) two or (F-G) one independent experiment(s), represented as means + / - SEM. Data analyzed with (B-E) Welch’s / -test or (F-H) two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant. (I) Flow cytometry contour plots and corresponding quantification of skin Flow cytometry gating strategy for identification of skin myeloid cell subsets: moAPCs (live CD45+ CD207- EpCAM- F4 / 80-Ly6Chi CDllc+ MHC11+), Langerhans cells (live CD45+ CD207+ EpCAM+), macrophages (Live CD45+ CD207- EpCAM- F4 / 80+), neutrophils (live CD45+ CD207- EpCAM- F4 / 80-Ly6G+), cDCl (live CD45+ CD207- EpCAM- F4 / 80- Ly6Clo CD1 lb- CD103+), and cDC2 (live CD45+ CD207- EpCAM- F4 / 80- Ly6Clo CDllb+ CD103-) with (5-OP-RU) or without (Veh) MAIT activation (n=5). Data derived from (I) one independent experiment, represented as mean + / - SEM. Data analyzed with (I) two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. ns, not significant. (J) Quantification of skin antigen presenting cells in B6 and 1115- / - mice at baseline (Ctrl) or after antigen (5-OP-RU) or UVB (UVD6; n=10). Data derived from (J) two independent experiments, represented as mean + / - SEM. Data analyzed with two-way ANOVA. *p<0.05. ns, not significant.

[0029] FIGS. 15A-15M. Topical MAIT cell expansion heals active lupus-like skin lesions. (A) Illustration of lesion treatment in MRL / lpr mice (male and female, 12-18wks) via topical MAIT cell activation (5-OP-RU ImM, every 48h x9) in the presence of anti-MRl blocking antibody or isotype control (9mg / kg, every 48h). (B) Representative images of MRL / lpr skin lesions at treatment initiation (DO) and treatment cessation (DI 6). (C) Lesion scores calculated on degree of erythema, scaling, thickness, and alopecia in mice treated with vehicle versus 5-OP-RU with (anti-MRl) or without (Isotype) blockade of antigen presentation to MAIT cells (n=6). (D) Lesion scores calculated on degree of erythema, scaling, thickness, and alopecia in mice treated with 5-OP-RU versus vehicle over time (n=7). (E and F) Flow cytometry contour plots (E) or histogramsAtty. Dkt. No. 178981.00043(F) and corresponding quantification of KI67 on skin Treg in 5-OP-RU treated lesional skin with (anti-MRl) and without (Isotype) MR1 blockade (n=9). (G) Representative H&E stain of MRL / lpr back and ear skin treated with 5-OP-RU with (anti-MRl) or without (Isotype) blockade of MAIT antigen presentation. (H) Illustration of acute induction of inflammation via topical imiquimod (0.06-0.07mg, daily x3) prior to treatment with topical 5-OP-RU (ImM, every 48h x5) or vehicle in B6 and Mrl- / - mice (n=5). (I) Ear thickness of B6 and Mrl- / - mice through course of inflammation induction with imiquimod and 5-OP-RU or vehicle treatment (n=5). (J) Representative H&E stain of control or imiquimod-treated B6 ears with (5-OP-RU) and without (Veh) MAIT cell expansion. (K) Expression of cytotoxic (ffng, Gzmb) genes determined by qPCR relative to Gctpdh in B6 control or imiquimod-treated ears with or without MAIT cell expansion (n=5). (L) Illustration of acute induction of inflammation via topical imiquimod (0.06-0.07mg, daily x3) prior to treatment with topical 5-OP-RU (ImM, every 48h x5) or vehicle + / - IL-15 blockade in B6 mice. (M) Ear thickness of B6 mice through course of inflammation induction with imiquimod and 5-OP-RU or vehicle treatment + / - IL- 15 blockade (n=5). Data derived from (C-F) three or (J, L) one independent experiment(s), represented as means + / - SEM. Data analyzed with (C-F) Welch’s t test, (J) two-way ANOVA, or (L) one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

[0030] FIG. 16. Human skin T cell gating. Representative flow cytometry gating strategy for identification of T cells in human skin.

[0031] FIGS. 17A-17K. Inflammatory T cell cytokine production is elevated in lupus-prone skin. (A) Representative flow cytometry gating strategy for identification of T cell subsets in murine skin. (B) Flow cytometry histogram and quantification of Treg AREG expression in B6 and MRL / lpr skin at baseline (n=5). (C) Flow cytometry contour plot and quantification of Treg IL- 10 production in B6 and MRL / lpr skin at baseline (n=5). (D-E) Flow cytometry contour plots and quantification of (D) CD8 and (E) CD4 T cell IFNg production in B6 and MRL / lpr skin at baseline (n=5). (F-G) Flow cytometry contour plots and quantification of (D) CD8 and (E) CD4 T cell TNFa production in B6 and MRL / lpr skin at baseline (n=5). (H-J) Flow cytometry contour plots and quantification of MAIT cell cytokine production in B6 and MRL / lpr skin at baseline (n=5). (K) Flow cytometry contour plots and quantification of skin CD8 T cells in B6 and MRL / lpr mice with (UVD6) and without (No UV) UVB exposure (n=8-l 1). Data derived from (B-J) one or (K) two independent experiment s), represented as means + / - SEM. Data analyzed with (B-I)Atty. Dkt. No. 178981.00043Welch’s / test or (K) two-way ANOVA. **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

[0032] FIGS. 18A-18K. MAIT cells drive broad Treg expansion. (A) Flow cytometry contour plots of skin MAIT cells (live CD45+ ySTCR- CD3+ TCR0+MR1:5'op‘RUTetramer+) in B6 and Mrl- / - mice . (B) Quantification of skin MAIT cells (live CD45+ ySTCR- CD3+ TCR0+MR1:5-°p-RUTetramer+) in B6 and Mrl- / - mice with (UVD6) and without (No UV) UVB exposure (n=17-24). (C) Quantification of skin Treg (live CD45+ ySTCR- CD3+ TCR0+ CD4+ Foxp3+) frequency in B6 and Mrl- / - mice with (UVD6) and without (No UV) UVB exposure (n=17-24). (D) Flow cytometry contour plots and corresponding quantification of Treg (live CD45+ ySTCR- CD3+ TCR0+ CD4+ Foxp3+) in B6 and Mrl- / - skin draining lymph nodes with (UVD6) and without (No UV) UVB exposure. Data concatenated from SDLNs of 5 mice. (E) Flow cytometry contour plots and corresponding quantification of Treg (live CD45+ y5TCR- CD3+ TCR0+ CD4+ Foxp3+) in B6 and Mrl- / - spleens with (UVD6) and without (No UV) UVB exposure (n=5). (F) Feature plots of select Treg markers used to subset Treg from flow cytometric data of B6 and Mrl- / - mice at baseline (No UV) or after UVB exposure (UVD6). (G) Flow cytometry contour plots of skin MAIT cells (live CD45+ ySTCR- CD3+ TCRP+MR1: op'RUTetramer+) in Mrl- / - mice with (5-OP-RU) and without (Veh) antigen exposure. (H) Flow cytometry quantification of MAIT cell (live CD45+ ySTCR- CD3+ TCR0+MR1:5’op'RUTetramer+) frequency in B6 and Mrl- / - skin with (5-OP-RU) and without (Veh) antigen exposure (n=14). (I) Flow cytometry quantification of Treg (live CD45+ ySTCR- CD3+ TCR0+ CD4+ Foxp3+) frequency in B6 and Mrl- / - skin with (5-OP-RU) and without (Veh) antigen exposure (n=14). (J) Uniform manifold approximation and projection of skin Treg and corresponding stacked bar plot generated from concatenated flow cytometric data of representative baseline (Veh) and antigen-exposed (5-OP-RU) B6 mice (n=5). (K) Feature plots of select Treg markers used to subset Treg from flow cytometric data of B6 mice at baseline or after 5-OP-RU exposure. Data derived from (B-C, H-f) three, (D-E, J) one, or (H-I) two independent experiment(s), represented as means + / - SEM. Data analyzed with (B, E, H-I) two-way ANOVA. **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

[0033] FIGS. 19A-19K. Systemic 5-OP-RU expands MAITs and Treg across multiple tissues.(A) Illustration of systemic delivery of MAIT antigen 5-OP-RU or vehicle to MRL / lpr mice (16-20wks, female). (B-K) Flow cytometry contour plots and corresponding quantification of (B, D, F, H, J) MAIT cells or (C, E, G, I, K) Treg in (B-C) skin, (D-E) intestinal epithelial lymphocytes (IEL), (F-G) brain, (H-I) liver, or (J-K) lung with (5-OP-RU) or without (Veh) systemic MAITAtty. Dkt. No. 178981.00043cell activation (n=5). Data derived from (B-K) one independent experiment, represented as means + / - SEM. Data analyzed with Welch’s / -test. *p0.05, **p<0.01. ns, not significant.

[0034] FIGS. 20A-20F. Imiquiniod-iiiduced skin inflammation is heightened in the absence of MAIT cells but can be resolved by antigen-mediated activation of MAIT cells. (A) Illustration of induction of chronic inflammation with TLR7 agonist imiquimod in B6 and B6A / r7- / - mice (8wks, female). (B) Gene expression of Ifng, Ifi2712a, and Tgfbl determined by qPCR relative to Gapdh in B6 and B6.A7r7- / - skin with imiquimod-induced inflammation (n=4-5). (C) Flow cytometry contour plots of skin MAIT cells in B6 and Mrl- / - mice with (IMQ) or without (Ctrl) imiquimod-induced inflammation and with (5-OP-RU) or without (Veh) topical MAIT cell activation. (D) Flow cytometry contour plots of skin Treg in B6 and Mrl-G mice with (IMQ) or without (Ctrl) imiquimod-induced inflammation and with (5-OP-RU) or without (Veh) topical MAIT cell activation. (E) Flow cytometry histograms and corresponding quantification of MAIT cell CD69 (activation) and KI67 (proliferation) expression in B6 skin (n=5). (F) Representative H&E stain of control or imiquimod-treated B6.A7 / 7- - ears with (5-OP-RU) and without (Veh) MAIT cell expansion. Data derived from (B,E) one independent experiment, represented as mean + / - SEM. Data analyzed with (B) Welch’s / -test or (E) one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. ns, not significant.

[0035] FIGS. 21A-21B. (A) Representative fabrication scheme for liposome. (B) Time-course NMR of 5-OP-RU formation reaction in DMSO-de.

[0036] FIGS. 22A-22E. 5-OP-RU activates and expands MAIT cells in healthy and SLE PBMCs in MRl-dependent fashion. (A) Percent MAIT cells in healthy and SLE PBMCs (n=16-21) (B-C) MFIs of activation markers CD69 and ICOS on MAITs and percent proliferating (Ki67+) MAIT cells in unstimulated and PBMCs stimulated with 5-OP-RU (luM) and IL2 for 7 days. (D) Percent MAIT cell expansion with 5-OP-RU + IL2 in healthy and SLE PBMCs after 7 day treatment and fold increase relative to unstimulated cells. (E) Percent MAITs in unstimulated and PBMCs treated with 5-OP-RU+IL2, IL2 alone, or 5-OP-RU+IL2+aMRl IgG. Data analyzed using (A, D) t-test, (B-C) Two-way ANOVA, or (E) one-way ANOVA; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.DETAILED DESCRIPTION

[0037] Described herein are methods for using 5-(2-oxopropylideneamino)-6-D-Atty. Dkt. No. 178981.00043ribitylaminouracil (5-OP-RU) to treat inflammatory skin and systemic disorders that are impacted by reduced mucosal-associated invariant T cells (MAIT cells). MAIT cells are a subset of T cells in the immune system that display innate, effector-like qualities. In humans, MAIT cells are found in the blood, liver, lungs, and mucosa, defending against microbial activity and infection. The MHC class I-like protein, MR1, presents bacterially-produced antigens, inducing MAIT cells to secrete pro-inflammatory cytokines and are capable of lysing bacterially-infected cells. After activation, MAIT cells also produce cytolytic molecules perforin and granzyme B, which form pores in the bacterially-infected cells, leading to apoptosis and the elimination of dangerous microbes from the body. MAIT cells can also be activated through MR1 -independent signaling. For example, in response to extracellular IL-12, which is often secreted by stressed macrophages, MAIT cells produce and secrete interferon-gamma (IFN-y), a cytokine that activates macrophages, assists in maturation of dendritic cells, and promotes expression of MHC class II on antigen presenting cells. In addition to possessing innate-like functions, this T cell subset supports the adaptive immune response and has a memory-like phenotype. MAIT cells have tissue repair capacity at homeostasis.

[0038] It was previously found that MAIT cells are reduced in systemic lupus erythematosus (SLE) blood, but activated, suggesting they may be migrating into the tissues . It was thought that, due to the inflammatory activity of MAIT cells, e.g. secretion of IFN-y and production of granzyme B, increasing MAIT cells would facilitate and intensify the harmful effects of SLE and potentially CLE. However, as demonstrated in the Examples, the inventors discovered that increasing MAIT cells using 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) slows the development and progression of CLE lesions and expands regulatory T cells (Tregs) in skin. This unexpected effect suggests a protective role for MAIT cells in inflammatory skin and systemic conditions.

[0039] Accordingly, provided herein is a method for treating or preventing a disorder characterized by reduced mucosal-associated invariant T (MAIT) cells in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU).

[0040] A disorder characterized by reduced MAIT cells is a disorder having reduced MAIT cells in circulation and / or in the skin in comparison to levels of MAIT cells found in circulation or the skin of an individual who does not have that disorder.

[0041] 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) (C12H18N4O7,Atty. Dkt. No. 178981.00043cell antigen. It is a combination of 5-Amino-6-(D-ribitylamino)uracil (5-A-RU) (C9H16N4O6) and methylglyoxal (MGO) (C3H4O2). 5-A-RU is a derivative of uracil and a precursor to riboflavin. It reacts non-enzymatically with dihydroxy acetone, methylglyoxal, or glyoxal to form 5-OP-RU, which activates mucosal-associated invariant T (MAIT) cells. MGO is a reduced derivative of pyruvic acid.

[0042] The disorder may comprise a skin disorder. Skin disorders include, but are not limited to, atopic dermatitis and hi dradenitis suppurativa, as well as autoimmune skin disorders, such as CLE, dermatomyositis, psoriasis, cutaneous vasculitis, scleroderma, pemphigus and rosacea. In exemplary embodiments, the skin disorder comprises CLE. CLE is a group of autoimmune connective tissue disorders localized to the skin that can be associated with SLE.

[0043] As used herein, the terms “treat”, “treatment”, and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). “Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.

[0044] As used herein, “preventing”, “prevent”, and “preventative” refer to partially or completely delaying or precluding the onset or recurrence of a disorder or conditions and / or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0045] As used herein, the term “administering” an agent, such as a therapeutic, to an animal is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a cell or subject by any suitable routeAtty. Dkt. No. 178981.00043for delivery of the therapeutic agent to the desired location.

[0046] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The amount of the pharmaceutical composition that is therapeutically effective may vary depending on the particular condition of the subject. Appropriate dosages may be determined, for example, by extrapolation from cell culture assays, animal studies, or human clinical trials taking into account body weight of the patient, absorption rate, half-life, disease severity and the like. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, the optimum effective amount can be readily determined by one skilled in the art using routine experimentation.

[0047] The terms “subject” and “patient” are used herein interchangeably to refer to a mammal to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. The subject may be a human.

[0048] The 5-OP-RU may be provided in a topical formulation and administered topically. The 5-OP-RU may be provided in the topical formulation at a concentration of between about 0.5 mM and about 5 mM. In exemplary embodiments, the 5-OP-RU is provided at a concentration of about 1 mM. The 5-OP-RU may be applied to at least a portion of skin on the subject. The 5-OP-RU may be applied to most of the skin on the subject. The 5-OP-RU may be applied at least once per day, every seven days. In embodiments, the 5-OP-RU is applied at least once, every two days. The 5-OP-RU may be applied at any effective frequency, such as every day, every other day, every three days, every four days, every five days, every six days, every seven days, etc. Topical formulations disclosed herein may be formulated into a lotion, aerosol foam or spray, gel, cream,Atty. Dkt. No. 178981.00043ointment, paste, powder, transdermal patch, or any other substance appropriate for topical administration.

[0049] In embodiments, the subject is expressing one or more CLE lesions, and the 5-OP-RU may be applied directly to the lesions. CLE skin lesions may be induced or aggravated by exposure to the sun or ultraviolet (UV) light. In some embodiments, the 5-OP-RU is administered at least one time within two days prior to exposure to UV light.

[0050] The method may further comprise administering an additional topical skin therapy to the subject, such as a topical steroid (e.g. a corticosteroid, a glucocorticosteroid, a cortisone) or a UV protectant (e.g. sunscreen, sunblock). The method may further comprise administering a systemic autoimmune disorder therapy.

[0051] In addition to, or instead of, a skin disorder, the subject may have a systemic autoimmune disorder. The systemic autoimmune disorder may comprise SLE. SLE, commonly called lupus, is a chronic (long-lasting) autoimmune disease that can affect many parts of the body. Lupus occurs when the immune system, which normally helps protect the body from infection and disease, attacks its own tissues. This attack causes inflammation, and in some cases permanent tissue damage, which can be widespread - affecting the skin, joints, heart, lung, kidneys, circulating blood cells, and brain. In embodiments, the subject expresses symptoms in organs other than the skin, such as the kidneys.

[0052] For treatment of a systemic autoimmune disorder, the 5-OP-RU may be administered systemically. Any suitable mode of administration may be used. In embodiments, the 5-OP-RU is administered intraperitoneally. The 5-OP-RU may be administered at a concentration of between about 0.5 and about 5 mM. The 5-OP-RU may be administered at least one time every 1 to 7 days. The method may further comprise administering an additional systemic autoimmune disorder therapy, such as an anti-interferon receptor antibody, a corticosteroid, and immunosuppressant, a non-steroidal anti-inflammatory drug (NSAID), or hydroxychloroquine. The method may further comprise administering an additional topical skin therapy.

[0053] As discussed above, the 5-OP-RU may be prepared as a formulation or pharmaceutical composition. Inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofAtty. Dkt. No. 178981.00043Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). The formulations may be designed or intended for systemic, topical, oral, nasal, or transmucosal (including buccal, sublingual, ocular, vaginal and rectal) and parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intraperitoneal, intrathecal, intraocular and epidural) administration. In embodiments, aqueous and non-aqueous liquid or cream formulations are delivered by a parenteral, oral or topical route. In embodiments, the compositions may be present as an aqueous or a non-aqueous liquid formulation or a solid formulation suitable for administration by any route, e.g., oral, topical, buccal, sublingual, parenteral, aerosol, a depot such as a subcutaneous depot or an intraperitoneal or intramuscular depot. The formulations may be lyophilized. The formulations 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. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J., USA) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be formulated for ease of injectability. The composition should be stable under the conditions of manufacture and storage, and must be shielded from contamination by microorganisms such as bacteria and fungi.

[0054] The 5-OP-RU formulation may further comprise a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Suitable pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles and adjuvants.

[0055] In some embodiments the 5-OP-RU is formulated in a liposome. The liposome may comprise a phospholipid, a PEGylated phospholipid lipid, and cholesterol. In exemplary embodiments, the liposome comprises 5-OP-RU, cholesterol, phospholipid 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), and PEGylated phospholipid l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-1000) (DSPE-PEG1000). The liposome comprises about 65 mol% DPPC, about 5 mol% DSPE-PEG1000, and about 30 mol% cholesterol. In exemplary embodiments, the liposome comprises 5-OP-RU, cholesterol, phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), lipid l,2-dioleoyl-3-trimethylammonium-Atty. Dkt. No. 178981.00043propane (DOTAP), and PEGylated lipid DSPE-PEG2000. The liposome comprises about 10 mol% DOPE, 50 mol% DOTAP, 38.5 mol% cholesterol, and 1.5 mol% DSPE-PEG2000. In exemplary embodiments, the liposome comprises 5-OP-RU, cholesterol, phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), lipid l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and PEGylated lipid l,2-dimyristoyl-rac-glycero-3-methoxy(poly(ethylene glycol) (DMG-PEG2000). The liposome comprises about 10 mol% DOPE, 45 mol% DOTAP, 38.5 mol% cholesterol, and 5 mol% DMG-PEG2000.

[0056] “ Cholesterol” is a waxy, fat-like substance made in the liver, and found in the blood and all cells of the body. It is a sterol (i.e., a modified steroid), a type of lipid. Cholesterol composes about 30% of all animal cell membranes. It is required to build and maintain membranes and modulates membrane fluidity over the range of physiological temperatures.

[0057] Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, trehalose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0058] Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01 to 0.1 M and preferably 0.05 M phosphate buffer or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include isotonic solutions, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. A tabulation of ingredients listed by the above categories,Atty. Dkt. No. 178981.00043may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990).

[0059] Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0060] The 5-OP-RU formulation may additionally include a biologically acceptable buffer to maintain a pH close to neutral (7.0-7.3). Such buffers preferably used are typically phosphates, carboxylates, and bicarbonates. More preferred buffering agents are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate. The buffer may comprise about 0.0001-5% (w / v) of the vaccine formulation, more preferably about 0.001-1% (w / v). Other excipients, if desired, may be included as part of the final composition. The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 10%, and preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0061] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The preparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e g., vials of drug, vialsAtty. Dkt. No. 178981.00043of diluent, syringes and needles) for a course of treatment (e.g., 7 days of treatment).

[0062] Sterile injectable solutions can be prepared by incorporating the active chemical compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0063] Capsules are prepared by mixing the chemical compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders. Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants, and disintegrators (in addition to the compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methyl cellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.

[0064] Tablets can be coated with sugar, e.g., as a flavor enhancer and sealant. The chemical compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasti ng substances, such as mannitol, in the formulation. Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty that some patients experience in swallowing solid objects. A lubricant can be used in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils. Tablets can also contain disintegrators. Disintegrators are substances that swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins, and gums. As further illustration, com and potato starches,Atty. Dkt. No. 178981.00043methyl cellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.

[0065] Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments and soluble in basic environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0066] As one skilled in the art will also appreciate, the formulation can be prepared with materials (e.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and / or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.

[0067] The preferred route may vary with, for example, the subject’s pathological condition or age or the subject's response to therapy or that is appropriate to the circumstances. The formulations can also be administered by two or more routes, where the delivery methods are essentially simultaneous, or they may be essentially sequential with little or no temporal overlap in the times at which the composition is administered to the subject.

[0068] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations, but nonetheless, may be ascertained by the skilled artisan from this disclosure, the documents cited herein, and the knowledge in the art.

[0069] Miscellaneous

[0070] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.”

[0071] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minusAtty. Dkt. No. 178981.00043>10% of the particular term.

[0072] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0073] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0074] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternativeAtty. Dkt. No. 178981.00043terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0075] “ Substantial identity” of amino acid sequences means that a polynucleotide or polypeptide comprises a sequence that has at least 85% sequence identity to a reference sequence (SEQ ID NO) using a sequence alignment program; preferably BLAST using standard parameters. A preferred percent identity of polynucleotides and polypeptides can be any integer from 85% to 100%. A preferred percent identity may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a reference sequence.

[0076] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0077] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0078] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of theAtty. Dkt. No. 178981.00043above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0079] EXAMPLES

[0080] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0081] Example 1

[0082] We have uncovered that expanding mucosal associated invariant T (MAIT) cells in the skin of mice that have lupus skin disease by painting MAIT specific antigen onto the skin resolves severe skin lesions.

[0083] Methods

[0084] Frequency and number of MAIT cells in CD3+TCR0+ T cells in B6 and CLE (8-weeks female MRL-lpr) ears were quantified by flow cytometry using 5-OP-RU loaded MR1 -tetramer (FIG. 1). To test if expanding MAIT cells resolves active lupus skin lesions, female MRL-lpr mice with severe skin lesions (lesion score>10) were treated topically with the MAIT cell antigen 5-OP-RU (ImM) every 48hr for 18 days (FIG. 2A). Lesion scores were quantified over time by the extent of skin erythema, scaling, thickness, and alopecia. To test if expanding MAIT cells slows CLE development, female MRL-lpr mice were treated topically with the MAIT cell antigen 5-OP-RU (ImM) weekly starting at 8 weeks of age, and skin lesions were photographically documented and quantified by extent of erythema, scaling, thickness, and alopecia (FIG. 3A). To assess how MAIT cell expansion affects skin immune and inflammatory environment, 8-week-old female MRL-lpr mice were treated with 5-OP-RU topically every 48 hours for 4 total applications (FIG.4). Skin immune and inflammatory environment was assessed by flow cytometry and qPCR. To test if MAIT cells were required for the expansion of Treg after painting the skin with MAIT cell antigen, MAIT cell-deficient (B6.A / 7- / -J mice were treated topically with 5-OP-RU (ImM) every 48 hours for 4 total applications and 8 days later MAIT cells and Treg were quantified by flow cytometry (FIG. 6). To test if MAIT cells were required for Treg expansion in other injury models, B6 and B6.Mrl- / - mice were treated with Imiquimod (IMQ, 5%, every 3 days for 4 weeks) and MAIT cells and Treg were quantified by flow cytometry at the end of the treatment (FIG. 6). To define the profile of MAIT cells and Treg stimulated by topical application of 5-OP-RU, B6.FoxP3-GFP mice were treated with 5-OP-RU topically every 48 hours for 4 total applications and 8 days later MAIT cells and Treg were enriched and FACS sorted for scRNAseq (FIG. 7). ToAtty. Dkt. No. 178981.00043test if systemic administration of 5-OP-RU also stimulates MAIT and Treg expansion in the skin and suppresses inflammation in lupus mice, 17-week old female MRL-lpr mice were injected i.p. with ImM 5-OP-RU or vehicle (water) 6 times over nine days and skin and blood were analyzed on day 22 after the first treatment (FIG. 8). MAIT and Treg in the skin were quantified by flow cytometry. Serum cytokine levels were quantified by Luminex assay.

[0085] Results

[0086] MRL-lpr mice have fewer MAIT cells in nonlesional skin, compared to the healthy B6 mouse skin (FIG. 1). Topical application of MAIT cell antigen 5-OP-RU onto active lupus skin lesions (lesion score > 10) leads to complete and persistent healing of skin lesions, compared to vehicle-treated mice (FIG. 2A-C). Weekly topical application of the MAIT cell antigen 5-OP-RU, beginning prior to lesion development (FIG. 3A), reduced the prevalence of severe skin lesions compared to vehicle treated controls (FIG. 3B). Of the mice that did develop lesions, those that received 5-OP-RU displayed accelerated lesion healing (FIG. 3C). 5-OP-RU topical application (FIG. 4A) effectively expanded MAIT cells in MRL-lpr skin (FIG. 4B). Surprisingly, MAIT expansion was accompanied by a concurrent increase in regulatory T cells (Treg) in the skin (FIG.4C). Moreover, MAIT cell expansion with 5-OP-RU reduced the high baseline expression of inflammatory (11-6, Inf) and cytotoxic (Ifng, Gzmb) genes in MRL-lpr skin, to the low levels of healthy B6 skin (FIG. 4E). The Treg expanded following 5-OP-RU-mediated MAIT cell expansion, are not recruited but are expanded locally in the skin (i.e. treatment of mice with FTY720 throughout topical application of 5-OP-RU did not suppress 5-OP-RU mediated Treg expansion in the skin, FIG. 5). Treg do not expand when MAIT antigen 5-OP-RU is applied to the skin of MAIT-defi cient mice (FIG. 6). Treg do no expand when MAIT-deficient mice are treated with another inflammatory stimulus (Imiquimod, TLR7 / 8 agonist, FIG. 6). Treg that expand following MAIT cell activation with 5-OP-RU are tissue resident effector Treg with immunosuppressive and tissue-healing properties (Foxp3+Ill0+Il2ra+Icos+Ccr7+, FIG. 7). Systemically administered MAIT cell antigen 5-OP-RU increased the levels of skin MAIT cells and Treg (FIG. 8A-8C) and decreased the blood concentration of inflammatory cytokines (Thl and Th2: IFNg, IL13, IL2) and chemokines (CXCL1, MIPla, GCSF) (FIG. 8D).

[0087] Conclusions

[0088] MAIT cells are reduced in non-lesional CLE skin. Topical application of the MAIT cell antigen 5-OP-RU heals active CLE lesions and slows the development and progression of CLE-Atty. Dkt. No. 178981.00043like lesions, suggesting a protective role for these cells in the skin. The concurrent expansion of Treg supports a potential immunosuppressive mechanism by which MAIT cells downregulate the expression of inflammatory and cytotoxic mediators in MRL-lpr skin associated with lesion development. MAIT cells are required for local skin Treg expansion in other models of skin injury (e g. TLR7 / 8 stimulation, FIG. 6 and UV light, see below). Systemic administration of MAIT cell antigen 5-OP-RU in lupus mice expands MAIT cells and Treg in the skin and suppresses systemic inflammation.

[0089] Example 2

[0090] We have uncovered that expanding MAIT cells in the skin of mice that have lupus skin disease by painting MAIT specific antigen onto the skin prior to skin exposure to ultraviolet (UV) light suppresses UV-induced immune activation in these mice.

[0091] Methods

[0092] To test if MAIT cells expand after UV in healthy (B6) and lupus (MRL-lpr) skin, 8 week old female mice were exposed to low-dose chronic UVB (50mJ / cm2, lx per day for 6 days) and MAIT and Treg were quantified in the skin one day after the last dose by flow cytometry (FIG.9). To test if MAIT cells are required for UV light-induced expansion of Treg, B6 and MAIT-deficient (B6.AYr7- / -) mice were exposed to UV as in FIG. 9 and Treg were quantified by flow cytometry (FIG. 12). To test if expanding MAIT cells in lupus skin, which is photosensitive, suppresses UV-induced immune activation, 8-week-old female MRL-lpr mice were treated with 5-OP-RU topically every 48 hours for 4 total applications, prior to exposure to low-dose chronic UVB (50mJ / cm2, lx per day for 6 days). MAIT cells (MR1 :5-OP-RU-tetramer+) and CD8 T cells were quantified by flow cytometry as was CD8 T cell activation (CD69 MFI) (FIG. 12).

[0093] Results

[0094] Skin exposure to UV light stimulated MAIT cell and Treg expansion in healthy (B6) but not non-lesional lupus skin (MRL.lpr) (FIG. 9). Treg did not expand in the skin of MAIT celldeficient mice (B6JV / r7- / -) after skin exposure to UV light (FIG. 12). Pre-treating lupus mouse skin with MAIT cell antigen (FIG. 10A) restored MAIT cell expansion after UV in these mice (FIG. 10B) and decreased the number of CD8 T cells and their activation (CD69 levels, FIG. 10C).

[0095] Conclusions

[0096] MAIT cells are required for Treg expansion in response to UV light. Restoring MAIT cells in lupus skin attenuates T cell activation in lupus skin, thus modulating the photosensitive skinAtty. Dkt. No. 178981.00043response in these mice.

[0097] Example 3

[0098] We have uncovered that type I interferon, which is elevated in lupus skin and blood, suppresses MAIT cell proliferation.

[0099] Methods

[0100] Peripheral blood mononuclear cells (PBMCs) from healthy blood were treated with 5-OP-RU (luM) and / or IL-2 (50 U / ml) and / or IFNa (100 U / ml, does not induce cell death) for 7 days and MAIT expansion was quantified by flow cytometry using 5-OP-RU loaded MR1 -tetramer (FIG. 11).

[0101] Results

[0102] MAIT antigen 5-OP-RU in combination with IL-2 expanded MAIT cells in the in vitro PBMC cultures. IFNa led to -50-80% reduction in MAIT cell expansion.

[0103] Conclusion

[0104] Type I interferon suppresses MAIT cell expansion, which suggests that MAIT cells may be better recovered in lupus with concurrent antigen (5-OP-RU) and anti-IFNAR blocking antibody treatment.

[0105] Example 4

[0106] Barrier injury by UVB light elicits coordinated Treg and MAIT cell expansion in healthy skin that is impaired in lupus-prone skin

[0107] In healthy skin, multiple regulatory T cell programs cooperate to maintain barrier homeostasis and promote repair after injury1 8. UVB-induced barrier disruption is known to drive local expansion of immunosuppressive Tregs in mouse models9 14Using ex vivo human skin explants, we showed that a single UVB exposure is likewise sufficient to stimulate local Treg expansion and proliferation in human skin (FIGS. 9, 16). However, barrier injury also generates rapid “alarm” cues that preferentially activate innate-like T cells, including MAIT cells15 16, prompting us to ask if skin exposure to UVB also activates MAIT cells in human skin explants. Skin explant studies reveal MAIT cell upregulation of proliferation marker KI67 24 hours after UVB exposure, suggesting their UV response may be analogous to Treg.

[0108] Barrier repair programs are compromised in inflamed tissues, such as the skin of lupus patients, where low and / or dysfunctional Treg have been implicated in disease pathogenesis, including the photosensitive response to UVB, which affects up to 80% of SLE patients17 20. ToAtty. Dkt. No. 178981.00043test if UVB-induced Treg and MAIT cell responses are altered in inflamed skin in vivo, we quantified their levels before and after exposure to a chronic course of low-dose UVB in healthy (C57BL / 6, B6) and non-lesional lupus-like skin (MRL / lpr model of spontaneous CLE, 8 weeks, FIG. 9). Of note, despite the absence of skin lesions at this early age, the MRL / lpr skin showed heightened immune activation and Treg dysfunction, evidenced by increased frequency of IFNg-producing CD8+ and CD4+ T cells, as well as reduced Treg production of IL- 10 and amphiregulin (FIG. 17B-G). In B6 skin, UVB elicited ~3-fold increase in both Treg and MAIT cell levels (FIG.9). In contrast, Treg levels were significantly lower in MRL / lpr compared to B6 skin after UVB, in both absolute number and frequency (FIGS9. 9). The dysfunction in the MAIT cell response in lupus-like skin was even more pronounced as MAIT cells in MRL / lpr skin failed to expand at all following exposure to UVB (FIGS9. 9). Consistent with the murine findings, bulk RNA-seq of human skin biopsies collected prior to and 24 hours after UVB exposure of healthy and lupus (SLE) volunteers showed that both Treg and MAIT cell gene signature scores significantly increased after UVB in healthy, but not lupus skin (FIG. 9). Together, these data reveal a parallel Treg and MAIT cell response to skin injury, that is impaired in lupus-prone skin.

[0109] MAIT cells are required for UVB-induced Treg activation and immune suppression

[0110] Given their coordinated response to UVB-induced injury in healthy skin and the complete absence of MAIT expansion in lupus-like skin, we hypothesized that MAIT cells may be required for UVB-triggered immunoregulatory responses in the skin, including local Treg expansion. To test this, we exposed B6 and MAIT cell-deficient (Mrl- -) mice to chronic, low-dose UVB and quantified cutaneous Treg levels by flow cytometry (FIGS. 12A, 18A-C). Strikingly, in the absence of MAIT cells, UVB failed to elicit robust Treg expansion or proliferation as seen in MAIT cell-replete B6 skin (FIGS. 12B-C, 18C). Treg did not preferentially accumulate in the skindraining lymph nodes or spleen of Mrl-- mice (FIG. 18D-E), suggesting the lack of expansion in the skin is not due to failed trafficking. To understand whether the absence of MAIT cells altered Treg heterogeneity and function-related phenotypes in response to UVB, we profiled the Treg in B6 and Mr 1-1- skin by spectral cytometry using a Treg-focused antibody panel. Uniform manifold approximation and projection (UMAP) revealed six distinct subsets defined based on published flow cytometry data of skin Treg21(FIGS. 12D, 18F). While UVB induced a broadly proportional increase across all Treg subsets in B6 skin, the Treg composition shifted slightly in favor of CD103- clusters 5 and 6 mMrl-l- skin, despite no increase in the overall Treg abundance (FIG.Atty. Dkt. No. 178981.0004312D). Additionally, after UVB exposure, Treg in B6 skin increased surface expression of proteins associated with activation (ICOS) and immunosuppression (CTLA4), whereas Treg Mrl- / - skin failed to upregulate these proteins (FIG. 12E). Given the altered phenotype of Treg in MATT celldeficient skin after UVB, we hypothesized that Treg in Mr 1-1- skin may lack immunosuppressive function. To test this, we compared UVB-mediated suppression of contact hypersensitivity (CHS) in B6 and Mr 1-1- skin using the established DNFB sensitization-challenge model, in which UVB-induced immunosuppression is Treg dependent (FIG. 12F)9-22 24Mice received a chronic low-dose UVB course before DNFB sensitization on the back skin and subsequent distal ear challenge; CHS severity was quantified as increase in ear thickness measured in a blinded fashion (FIG. 12F). Vehicle controls showed no ear swelling in either genotype, whereas DNFB sensitization and challenge elicited CHS, i.e. increase in ear thickness, in both B6 and Mrl- / - mice (FIG. 12F). Consistent with prior studies, UVB markedly reduced CHS reaction in B6 skin, as evidenced by significantly diminished increase in ear thickness in UV + DNFB compared to the DNFB only group9'22 24(FIG. 12F). However, the UV-mediated suppression was lost mMrl- / - mice, which developed ear swelling despite UVB exposure (FIG. 12F). Notably, Treg proliferation was significantly reduced in the skin of UV + DNFB treated Mrl-I- mice (FIG. 12G).

[0111] Having established in mice that MAIT cells support cutaneous Treg expansion in response to UVB injury, we next asked whether activating MAIT cells would amplify the UVB-induced Treg response in intact human skin. We pre-treated human skin explants with either MAIT cell antigen 5-OP-RU or vehicle, exposed them to an acute dose of UVB, and profiled Treg and MAIT cells by flow cytometry 48 hours later (FIG. 12H). 5-OP-RU stimulated MAIT cell proliferation, which was paralleled by enhanced expansion of Treg beyond that observed with UVB alone (FIG.12I-J), indicating that MAIT cells also potentiate UVB-associated Treg response in human skin. Taken together, these findings reveal a conserved MAIT-Treg axis in cutaneous injury responses.

[0112] TCR-driven MAIT cell activation supports skin Treg and suppresses inflammation in lupus-like skin

[0113] To understand if the MAIT-Treg relationship is specific to UVB-induced MAIT activation, we tested whether TCR-mediated activation of MAIT cells also supports cutaneous Treg proliferation. Topical administration of MAIT cell cognate antigen 5-OP-RU led to ~3-fold expansion in MAIT cells, which was paralleled by ~4-fold increase in cutaneous Treg levels (FIGS.13A-C, 18G-I). Dimensionality reduction of Treg spectral flow cytometry data showed that 5-OP-Atty. Dkt. No. 178981.00043RU stimulated expansion across the 6 Treg subsets identified following UVB exposure (FIG. 18J-K). Importantly, Treg did not expand in 5-OP-RU treated Mr 1-1- skin, establishing a requirement for MAIT cells in this response (FIG. 13B-C). Using Foxp3 reporter mice (B6Foxp3-GFP), we FACS-sorted Treg from vehicle- or 5-OP-RU-treated skin for single cell RNA-seq, which confirmed that antigen-mediated MAIT cell activation induced a marked expansion of Treg across subsets (FIG. 13D). Moreover, Treg in 5-OP-RU-treated skin exhibited high expression of Foxp3 as well as genes involved in immune tolerance (e.g. Ctla4, 1110, Entpdl), resulting in a higher transcriptional immunoregulatory score (FIG. 13D).

[0114] Since MAIT cell responses are impaired in non-lesional MRL / lpr compared to healthy skin and reside in an inflammatory tissue environment (FIG. 9), we next tested whether TCR-dependent activation of MAIT cells could restore their levels and support immunoregulatory functions observed in healthy B6 skin. Despite low basal MAIT cell levels, topical 5-OP-RU robustly expanded MAIT cells and stimulated ~4-fold increase in Treg levels (FIG. 4B-4C). As in B6 skin, Treg expanded across subsets in MRL / lpr non-lesional skin (FIG. 13E). Consistent with an immunoregulatory effect of MAIT and Treg expansion, 5-OP-RU treatment reduced the frequency of IFNg- and TNFa-producing CD8+ and CD4+ T cells in MRL / lpr skin (FIG. 4D). Additionally, topical 5-OP-RU reduced the expression of cytotoxic (Gzmb, Ifng) and inflammatory ( Tufa, 1 / 6) genes in MRL / lpr skin to levels approaching those in healthy B6 skin (FIG. 4E).

[0115] Because UV light is a potent disease trigger in lupus and MRL / lpr mice model SLE-like photosensitivity23, we next tested whether topical antigen-mediated MAIT cell expansion prior to UVB exposure could attenuate the inflammatory response to barrier injury by the disease-relevant stimulus (FIG. 13F). In contrast to vehicle-treated MRL / lpr skin, in which UVB failed to elicit Treg expansion and instead drove accumulation of CD8+ T cells (FIGS. 9, 18), pre-expansion of MAIT cells significantly increased the Treg:CD8 ratio after UVB exposure (FIG. 13G). Consistent with reduced inflammatory activation, 5-OP-RU-treated skin exhibited lower expression of cytotoxic (Ifng, Gzmb), interferon-stimulated (Irf7, Ifi27 / 2ci), and disease-associated (Hifla, Tgfhl) transcripts compared to vehicle controls following UVB (FIG. 13H). These immunologic changes were accompanied by improved skin histopathology with 5-OP-RU application (FIG. 131), further supporting an immunoregulatory effect of MAIT cell expansion in the context of UVB-induced cutaneous inflammation. Altogether, these findings demonstrate that antigen-driven MAIT cell expansion can promote Treg accumulation even in the inflammatory skin tissueAtty. Dkt. No. 178981.00043environment, dampen baseline inflammation, and suppress additional pathogenic responses normally triggered by UVB exposure.

[0116] Using healthy and patient-derived PBMCs, we also demonstrate that MR1 presentation of the antigen is able to activate MAIT cells from lupus patient blood, despite their lower levels compared to healthy controls. Engagement with 5-OP-RU stimulated MAIT cell activation (i.e. increased CD69 and ICOS), proliferation (increased %Ki67), and expansion of SLE MAIT cells to a similar degree as healthy MAIT cells, in an MR1 -dependent fashion (FIG. 22).

[0117] Monocyte-derived APCs couple MAIT activation to Treg expansion through IL-15-dependent signaling

[0118] Given that the MAIT cell-dependent effects on Treg have not been previously described, we first aimed to identify soluble mediators that could mechanistically link MAIT activation to Treg expansion. Systemic administration of 5-OP-RU induced MAIT cell expansion across multiple tissues including the skin; in other tissues MAIT cell levels remained stable or were reduced, likely due to the kinetics of 5-OP-RU exposure (FIG. 19A-K). Notably, Treg mirrored changes in MAIT cells and expanded in the skin, recapitulating our findings with topical 5-OP-RU (FIGS. 13B-13D, 4B-4C, 19A-K). To identify candidate soluble mediators, we performed Luminex multi-analyte profiling of serum and found that IL- 15 was the only cytokine selectively increased in the serum of 5-OP-RU- treated mice, compared to vehicle controls (FIG. 14A). IL-15 is also induced in human skin following UVB exposure26, a context in which we observed MAIT cell and Treg expansion (FIG. 9). Although IL-15 is more commonly appreciated as a T cell growth factor supporting CD8 T cells, multiple studies have established roles for IL- 15 in promoting Treg proliferation, stability, and suppressive function27 31.

[0119] To test this hypothesis, we treated B6 and IL-15-deficient (1115- / -) mice with topical 5-OP-RU or vehicle (as in FIG. 13 A), or exposed them to chronic, low-dose UVB (as in FIG. 9), and quantified skin MAIT cells and Treg by flow cytometry. While MAIT cell expansion was preserved in 1115-,'- skin under both stimuli, subsequent Treg accumulation was lost, indicating that IL-15 is required for Treg expansion downstream of MAIT cell activation (FIGS. 14B-C). To ensure the lack of Treg expansion in the absence of IL-15 was not due to deficiency in a myeloid cell subset that may present antigen to Treg, we assessed myeloid cell numbers in B6 skin compared to B6. / / 75- / - and found no significant differences (FIG. 24J).

[0120] Because MAIT cells are not known to produce IL-15, we next sought to identify the cellularAtty. Dkt. No. 178981.00043source of IL-15 that links MAIT activation to Treg expansion in skin. Keratinocytes, fibroblasts, and several dendritic cell subsets (Langerhans cells (LCs), monocyte-derived dendritic cells (moDCs)) all reside in the skin and are reported producers of IL-15, with fibroblasts and moDCs having been specifically linked to IL-15-driven Treg support29,31’32. Single cell RNA-seq analysis of skin tissues following a single dose of UVB revealed that 1115 is upregulated primarily in the myeloid cell cluster, rather than keratinocytes or fibroblasts, which have previously been reported to express IL-15. Thus, we hypothesized that an IL-15 producing myeloid cell population mediates MAIT-driven Treg expansion.

[0121] To identify the relevant subset, we asked which myeloid cells preferentially expanded after UVB in B6 compared to Mr 1-1- mice and whether this same subset expanded with 5-OP-RU treatment, during the early window of MAIT activation prior to Treg accumulation. After two doses of either 5-OP-RU or UVB, monocyte-derived antigen presenting cells (Ly6Chi CDllc+ MHCII+; moAPCs), were the only myeloid population that preferentially expanded in MAIT-dependent manner across both stimuli (FIGS. 14D-E). Neither LCs, conventional dendritic cells, nor neutrophils expanded with antigen-mediated MAIT cell activation (FIG. 14E). Consistent with the role for IL- 15 delivery, after 2 doses of UVB or 5-OP-RU moAPCs upregulated IL- 15Roc (FIG.14F), which is critical fortheir ability to trans present IL-15 to other cells33. Importantly, IL-15Ra induction on moAPCs was not observed in MAIT cell-deficient skin.

[0122] Having identified moAPCs as the candidate source of IL- 15 driving Treg expansion downstream of MAIT cells in the skin, we next tested whether they are required for MAIT-driven Treg expansion in the skin. Given that MAIT cell and Treg expansion in response to UVB and 5-OP-RU are sustained in the presence of SIP receptor blocking molecule, FTY720 (FIG. 5A-D), we inferred that preventing moAPC migration into skin would be sufficient to assess their requirement for MAIT cell-driven Treg expansion. We therefore used CCR2-deficient mice, in which recruitment of monocyte-derived APCs, but not other myeloid cell subsets, to inflamed tissues is impaired (FIG. 5E)34, and stimulated MAIT cells with topical 5-OP-RU. CCR2 deficiency did not affect MAIT cell expansion, but it abrogated Treg accumulation (FIG. 14G-H), indicating that moAPCs are necessary for MAIT-driven Treg expansion. Collectively, these data support the model in which MAIT activation in skin promotes the expansion and / or recruitment of moAPCs that, in turn, drive Treg expansion via IL- 15 trans-presentation through IL-15Ra.

[0123] Engaging the MAIT — IL-15 — Treg axis promotes resolution of establishedAtty. Dkt. No. 178981.00043inflammatory skin lesions

[0124] Beyond heightened sensitivity to UV, lupus patients develop skin lesions independent of UVB exposure, so we next asked whether topical MAIT cell expansion could treat established severe skin lesions. MRL / lpr mice were allowed to develop spontaneous lesions that were monitored and scored for erythema, scaling, thickness, and alopecia, as well as extent of skin area involved. When lesions reached a score >10, we initiated therapeutic intervention. In Cohort 1, mice received topical 5-OP-RU or vehicle every 48 hours (alternating days) together with systemic isotype control IgG or anti-MRl blocking antibody (FIG. 15 A) to test efficacy and MR1 dependence. In Cohort 2, mice received topical 5-OP-RU or vehicle every 48 hours for nine total doses and were followed thereafter to assess durability. In Cohort 1, 5-OP-RU-treated mice receiving isotype control IgG exhibited near-complete lesion resolution after nine doses, whereas vehicle-treated mice did not improve (FIG. 15B-C). Anti-MRl blockade blunted the therapeutic effect of topical 5-OP-RU, indicating that lesion resolution requires MRl-dependent MAIT engagement. In Cohort 2, lesion resolution in 5-OP-RU-treated mice was sustained for up to 40 days after treatment cessation, whereas vehicle-treated lesions failed to fully resolve and continued to flare over the monitoring period (FIG. 15D). To determine whether lesion resolution was associated with MAIT-driven Treg responses, we assessed Treg phenotype in Cohort 1 skin 24 hours after the final dose. Anti-MRl treatment significantly reduced Treg proliferation, as measured by KI67 (FIG. 15E-F). At this same time point, histopathology of both back and ear skin was improved in 5-OP-RU + isotype-treated mice relative to mice receiving anti-MRl IgG (FIG. 15G).

[0125] Because spontaneous MRL / lpr lesions resemble open wounds and MAIT cells and Treg have known beneficial roles in wound healing1 3-56, we next asked whether MAIT cell engagement could also suppress lupus-like inflammation in the absence of overt skin wound. We therefore employed the TLR7 agonist, imiquimod. In a chronic imiquimod model (4 weeks)35, MAIT celldeficient (Mrl-I-) skin exhibited higher expression oilfrig, Ifi27l2a, and Tgfbl compared with WT B6 skin (FIG. 20A-B), consistent with an endogenous role for MAIT cells in restraining lupus-associated inflammatory programs. To test the effects of MAIT activation on lesion resolution in a setting that better models a lupus flare, imiquimod was applied for three consecutive days to induce acute ear inflammation (ear thickness), after which topical 5-OP-RU or vehicle was initiated and continued every 48 hours for four doses (FIG. 15H). By the end of treatment, earAtty. Dkt. No. 178981.00043thickness was significantly reduced in 5-OP-RU-treated WT B6 mice relative to vehicle controls, whereas Mr 1-1- ears remained inflamed irrespective of treatment, demonstrating an MR1-MAIT requirement for resolution (FIG. 151). Consistent with this mechanism, 5-OP-RU induced expansion of both MAIT cells and Tregs in WT skin, whereas Tregs failed to expand in MAIT-deficient skin (FIG. 20C-D). MAIT cells from 5-OP-RU-treated WT skin also showed increased CD69 and KI67 compared to vehicle-treated controls, indicating local activation and proliferation (FIG. 20E). Histologic improvement in WT mice treated with 5-OP-RU was accompanied by reduced expression of cytotoxic mediators, including Ifrig and Gzrnb (FIG. 15J-K, FIG. 20F).

[0126] Finally, to directly test whether immunoregulatory effects of MAIT cells and the resolution of imiquimod-induced skin inflammation require IL- 15, we blocked IL- 15 signaling with an IL-15 neutralizing antibody during 5-OP-RU treatment (FIG. 15L). As predicted by our mechanistic studies, IL-15 blockade abrogated the protective effects of MAIT cell activation, with ears remaining thick and inflamed, whereas isotype-treated controls showed resolution of inflammation (FIG. 15M). Together, these findings demonstrate that activation of the MAIT-IL-15-Treg axis is sufficient to promote resolution of established lupus-like skin inflammation and lesions.

[0127] Example 5

[0128] Background

[0129] Skin disease affects up to 80% of patients with systemic lupus erythematosus (SLE), a chronic autoimmune and inflammatory disease36. Cutaneous LE (CLE) causes painful, scarring, and disfiguring chronic skin lesions and significantly reduces quality of life36’37. Moreover, exposure to ultraviolet B (UVB) sunlight rays exacerbates local disease and can provoke systemic flares in SLE38 40. There are currently no FDA-approved targeted topical therapies to treat CLE or photosensitive reactions. Treatment is limited to topical steroids and systemic immunosuppression, which require chronic use, have broad side effects and poor efficacy41. Therefore, there is a critical need for a novel targeted topical therapy in SLE. Here, we develop a new approach to topically deliver a small molecule that will promote the local and targeted expansion of mucosal-associated invariant T (MAIT) cells, thereby promoting tissue repair and suppressing inflammation in SLE skin.

[0130] Therapeutic efforts in CLE have largely focused on blocking inflammatory pathways or eliminating pathogenic immune cell populations36’42. Although it has long been recognized that anti-inflammatory regulatory T cells (Treg) are low in number and dysfunctional in SLE skin43,44,Atty. Dkt. No. 178981.00043therapeutic efforts to directly activate or restore this immune cell subset have been challenging. This challenge largely reflects our limited understanding of the cellular and molecular circuits that sustain Treg abundance and function within the skin. In recent studies, we have uncovered that MAIT cells are important for Treg immune-suppressive functions in both spontaneous skin lesions and UV light-induced skin disease flares (Examples 1-4). MAIT cells are innate like T cells that express a conserved T cell receptor (TCR), through which they recognize microbial vitamin B2 metabolites presented on an MHC-I like molecules MR145,46. While MAIT cells are functionally pleiotropic, depending on the tissue or injury context (e.g., promoting wound closure via amphiregulin production or eliminating bacterial pathogens via IFNg production47 49), their role in CLE skin has not been studied. We have uncovered a protective role for MAIT cells in lupus skin (Examples 1-4).

[0131] As shown in Examples 1-4, in addition to their anti-inflammatory effects in non-lesional CLE-prone skin, topically treating active CLE skin lesions (score >10) with 5-OP-RU led to their complete and sustained resolution. Concurrent administration of the anti-MRl antibody to block the presentation of 5-OP-RU to MAIT cells prevented lesion resolution in MRL / lpr skin, establishing that the therapeutic effect of 5-OP-RU is MAIT cell-specific. In addition to healing active CLE-like lesions, topical 5-OP-RU suppressed UV-induced skin inflammation, as evidenced by decreased skin thickness and increased Treg:CD8 ratio in 5-OP-RU, compared to vehicle-treated skin. To demonstrate that the therapeutic potential of targeted MAIT expansion is not limited to one CLE model, we asked if topical application of 5-OP-RU resolves inflammation in the TLR7 agonist inducible model of CLE50. Applying 5-OP-RU to already inflamed ears of B6 mice (after 3 doses of TLR-7 agonist) decreased ear thickness back to basal levels. 5-OP-RU had no therapeutic effects in MAIT-deficient. These findings support MAIT cells as a novel cellular target in CLE that can be activated in the skin locally in an antigen-specific fashion.

[0132] The lead small-molecule antigen that stimulates MAIT cells, 5-OP-RU, is chemically unstable and must be generated in situ, posing a critical barrier to translation31,52. To expand MAIT cells locally, we combine the inactive metabolite 5-A-RU with 2-methylglyoxal (2-MGO) in a test tube to generate 5-OP-RU and immediately apply it to the skin. Even though studies estimate low conversion efficiency, one dose is sufficient to trigger local MAIT proliferation in 24 hr (not shown). While these observations support the potency of the antigen, they highlight the need to develop novel strategies for antigen delivery that have therapeutic potential for CLE patients. WeAtty. Dkt. No. 178981.00043address this barrier by developing a novel delivery platform for localized MAIT antigen delivery to CLE lesions: a liposomal formulation to shield the active antigen from the aqueous phase.

[0133] Relevance to translational science and rural health: This project tackles a fundamental translational barrier: the chemical instability of the lead MAIT antigen (5-OP-RU), which prevents its clinical development. By engineering two delivery platforms, we propose tractable and scalable approaches to stabilize MAIT activation in the skin. These innovative approaches could accelerate the translational pipeline for MAIT-directed therapies, first in CLE and potentially in other inflammatory and photosensitive skin diseases. Positive results will establish the feasibility of a targeted, steroid-sparing topical therapy that can be manufactured, stored, and applied under real-world conditions. This is particularly important for patients in rural regions where long travel distances, harsh winter weather, and limited public transportation restrict access to dermatology specialists and make frequent laboratory monitoring for systemic immunosuppression difficult to sustain. This problem is amplified by the scarcity of dermatologists in rural areas (4.11 per 100,000 in urban vs 0.085 per 100,000 in rural counties53). A stable, easy-to-apply topical formulation that can be safely managed in primary care or at home would reduce dependence on systemic agents, lower barriers to treatment adherence, and expand access to disease-modifying therapy for rural patients with lupus skin disease.

[0134] Approach: 5-OP-RU is a potent MAIT-specific activator54-55. 5-OP-RU is the product of 5-amino-6-D-ribitylaminouracil (5-A-RU), a key intermediate in vitamin B2 synthesis, and 2-MGO, an adduct of mammalian glycolysis or bacterial metabolism54. This non-peptide antigen forms a complex with MR1 and is presented to the MAIT TCR, stimulating their proliferation and cytokine production47. Nonetheless, its instability in aqueous solution (ti / 2 < 2h at 37°C), due to a chemically unstable a-iminocarbonyl Schiff base motif, has hampered 5-OP-RU’ s clinical relevance46-55Here, we propose innovative drug-delivery approaches to stabilize 5-OP-RU and enable topical dosing that results in robust, therapeutically effective MAIT expansion in CLE skin.

[0135] Develop a novel liposome formulation for the MAIT cell antigen 5-OP-RU to enable in vivo MAIT expansion and lesion resolution in CLE-like skin

[0136] Liposomes and related constructs (e.g., lipid nanoparticles) have revolutionized modem medicine56. Their capabilities for delivering therapeutic payloads, from macromolecular constructs (e.g., mRNA, proteins) to small molecules (e.g., doxorubicin, cytarabine), are well-established, as are their well -tolerated safety profdes in clinical contexts36 60. For example, clinical-stageAtty. Dkt. No. 178981.00043liposomal formulations such as Doxil (doxorubicin) or Vyxeos (daunorubicin and cytarabine) remain pivotal in the management of several cancer indications. These widespread adoptions stem from liposomes’ capabilities to 1) reduce toxicity, 2) enhance pharmacokinetics (PK) and biodistribution (BD), and 3) enable extended and controlled exposure via efficient endosomal escape61 63. We posit that the core-shell structure of a liposome serves as the ideal platform for the encapsulation of 1) 5-A-RU (into the lipid bilayer shell) and 2-MGO (into the core), or 2) preformed 5-OP-RU (FIG. 21A). The PEGylated liposomes enhance 5-OP-RU's bioavailability via 1) an in-situ soft-matter “nanoreactor” approach, where 5-A-RU and 2-MGO react post-delivery upon rupture of the liposome or 2) shielding of the preformed 5-OP-RU from premature degradation. Moreover, the resulting liposomes modulate partitioning into the stratum corneum and enable controlled release, thereby improving MAIT engagement and therapeutic benefit.

[0137] Liposome Formulation

[0138] Components of the formulation include cholesterol (starting from 30% mol), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine as the main lipid (DPPC, starting at 65% mol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-1000) as the PEGylated lipid (DSPE-PEG1000, starting at 5% mol64). Thin-film hydration is used as the fabrication route (FIG. 21A). This process comprises the formation of a lipid film, which is acquired via rotary evaporation of the organic phase (i.e., cholesterol, DPPC, and DSPE-PEG1000 in chloroform); the aqueous phase (e.g., PBS) is then added, and the mixture undergoes subsequent extrusion to acquire the liposomes of interests at the appropriate size (lOOnm65). For route 1), 5-A-RU is incorporated into the organic phase, and 2-MGO is added to the aqueous phase. For route 2) 5-OP-RU is pre-formed in DMSO following the reported method that we have preliminarily reproduced in our lab (FIG. 2 IB)53. We note that the formation of 5-OP-RU via this route is nearly quantitative, whereas aqueous mixing of 5-A-RU and 2-MGO yields <1% 5-OP-RU (~100-fold higher)55. Formulated constructs exhibited an average hydrodynamic diameter of -130 nm, and a zeta potential of -25 mV.

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[0140] EMBODIMENTS

[0141] Embodiment 1. A method for treating or preventing a disorder characterized by reduced mucosal-associated invariant T (MAIT) cells in a subject in need thereof, the method comprising:Atty. Dkt. No. 178981.00043administering to the subject a therapeutically effective amount of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU).

[0142] Embodiment 2. The method of embodiment 1, wherein the disorder comprises a skin disorder.

[0143] Embodiment 3. The method of embodiment 2, wherein the skin disorder is an autoimmune skin disorder.

[0144] Embodiment 4. The method of embodiment 2 or 3, wherein the skin disorder comprises cutaneous lupus erythematosus (CLE).

[0145] Embodiment 5. The method of any one of embodiments 2-4, wherein the 5-OP-RU is administered topically.

[0146] Embodiment 6. The method of embodiment 5, wherein the 5-OP-RU is provided in a topical formulation at a concentration of between about 0.5 mM and about 5 mM.

[0147] Embodiment 7. The method of embodiment 6, wherein the 5-OP-RU is provided at a concentration of about 1 mM.

[0148] Embodiment 8. The method of any one of embodiments 2-7, wherein the 5-OP-RU is applied to at least a portion of skin on the subject.

[0149] Embodiment 9. The method of embodiment 8, wherein the 5-OP-RU is applied at least one time every seven days.

[0150] Embodiment 10. The method of embodiment 9, wherein the 5-OP-RU is applied at least one time every two days.

[0151] Embodiment 11. The method of any one of embodiments 8-9, wherein the subject is expressing CLE lesions.

[0152] Embodiment 12. The method of embodiment 11, wherein 5-OP-RU is applied to the CLE lesions.

[0153] Embodiment 13. The method of any one of embodiments 2-12, wherein the 5-OP-RU is administered within two days prior to exposure to UV light.

[0154] Embodiment 14. The method of any one of embodiments 2-13, further comprising administering an additional topical skin therapy to the subject.

[0155] Embodiment 15. The method of embodiment 14, wherein the additional topical skin therapy comprises a topical steroid.Atty. Dkt. No. 178981.00043

[0156] Embodiment 16. The method of embodiment 14 or 15, wherein the additional topical skin therapy comprises aUV protectant.

[0157] Embodiment 17. The method of any one of embodiments 1-16, wherein the disorder comprises a systemic autoimmune disorder.

[0158] Embodiment 18. The method of embodiment 17, wherein the systemic autoimmune disorder comprises systemic lupus erythematosus (SLE).

[0159] Embodiment 19. The method of embodiment 17 or 18, wherein the 5-OP-RU is administered systemically.

[0160] Embodiment 20. The method of embodiment 19, wherein the 5-OP-RU is administered by intraperitoneal injection.

[0161] Embodiment 21. The method of embodiment 20, wherein the 5-OP-RU is administered at a concentration of between about 0.5 mM and about 5 mM.

[0162] Embodiment 22. The method of embodiment 21, wherein the 5-OP-RU is administered at a concentration of between about 1 mM.

[0163] Embodiment 23. The method of any one of embodiments 19-20, wherein the 5-OP-RU is administered at least one time every 1 to 7 days.

[0164] Embodiment 24. The method of any one of embodiments 17-23, further comprising administering an additional systemic autoimmune disorder therapy.

[0165] Embodiment 25. The method of embodiment 24, wherein the additional systemic autoimmune disorder therapy comprises an anti -interferon receptor antibody.

[0166] Embodiment 26. The method of any one of embodiments 1-25, wherein the 5-OP-RU is in a liposome comprising l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-1000) (DSPE-PEG1000).

[0167] Embodiment 27. The method of embodiment 26, wherein the liposome comprises;a) l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(methoxy(polyethyleneglycol)-1000) (DSPE-PEG1000), and cholesterol;b) l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(methoxy(polyethyleneglycol)-2000) (DSPE-PEG2000), and cholesterol; orAtty. Dkt. No. 178981.00043c) l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), and l,2-dimyristoyl-rac-glycero-3- methoxy(poly(ethylene glycol) (DMG-PEG2000), and cholesterol.

[0158] Embodiment 28. A liposome comprising 5-OP-RU, cholesterol, a phospholipid, and a PEGylated lipid.

[0159] Embodiment 29. The liposome of embodiment 28, wherein the phospholipid is selected from DOPE and DPPC.

[0160] Embodiment 30. The liposome of embodiment 28 or 29, wherein the PEGylated lipid is selected from DSPE-PEG1000, DSPE-PEG2000, and DMG-PEG2000.

[0161] Embodiment 31. The liposome of any one of embodiments 28-30, further comprising DOTAP.

Claims

Atty. Dkt. No. 178981.00043CLAIMSWe claim:

1. A method for treating or preventing a disorder characterized by reduced mucosal-associated invariant T (MAIT) cells in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU).

2. The method of claim 1, wherein the disorder comprises a skin disorder.

3. The method of claim 2, wherein the skin disorder is an autoimmune skin disorder.

4. The method of claim 2, wherein the skin disorder comprises cutaneous lupus erythematosus (CLE).

5. The method of claim 2, wherein the 5-OP-RU is administered topically.

6. The method of claim 5, wherein the 5-OP-RU is provided in a topical formulation at a concentration of between about 0.5 mM and about 5 mM.

7. The method of claim 6, wherein the 5-OP-RU is provided at a concentration of about 1 mM.

8. The method of claim 2, wherein the 5-OP-RU is applied to at least a portion of skin on the subject.

9. The method of claim 8, wherein the 5-OP-RU is applied at least one time every seven days.

10. The method of claim 9, wherein the 5-OP-RU is applied at least one time every two days.

11. The method of claim 8, wherein the subject is expressing CLE lesions.

12. The method of claim 11, wherein 5-OP-RU is applied to the CLE lesions.

13. The method of claim 2, wherein the 5-OP-RU is administered within two days prior to exposure to UV light.

14. The method of claim 2, further comprising administering an additional topical skin therapy to the subject.

15. The method of claim 14, wherein the additional topical skin therapy comprises a topical steroid.

16. The method of claim 14, wherein the additional topical skin therapy comprises a UV protectant.

17. The method of claim 1, wherein the disorder comprises a systemic autoimmune disorder.Atty. Dkt. No. 178981.0004318. The method of claim 17, wherein the systemic autoimmune disorder comprises systemic lupus erythematosus (SLE).

19. The method of claim 17, wherein the 5-OP-RU is administered systemically.

20. The method of claim 19, wherein the 5-OP-RU is administered by intraperitoneal injection.

21. The method of claim 20, wherein the 5-OP-RU is administered at a concentration of between about 0.5 mM and about 5 mM.

22. The method of claim 21, wherein the 5-OP-RU is administered at a concentration of between about 1 mM.

23. The method of claim 19, wherein the 5-OP-RU is administered at least one time every 1 to 7 days.

24. The method of claim 17, further comprising administering an additional systemic autoimmune disorder therapy.

25. The method of claim 24, wherein the additional systemic autoimmune disorder therapy comprises an anti-interferon receptor antibody.

26. The method of claim 1, wherein the 5-OP-RU is in a liposome.

27. The method of claim 26, wherein the liposome comprises;d) l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(methoxy(polyethyleneglycol)-1000) (DSPE-PEG1000), and cholesterol;e) l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(methoxy(polyethyleneglycol)-2000) (DSPE-PEG2000), and cholesterol; orf) l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), and l,2-dimyristoyl-rac-glycero-3- methoxy (poly (ethylene glycol) (DMG-PEG2000), and cholesterol.

28. A liposome comprising 5-OP-RU, cholesterol, a phospholipid, and a PEGylated lipid.

29. The liposome of claim 28, wherein the phospholipid is selected from DOPE and DPPC.

30. The liposome of claim 28, wherein the PEGylated lipid is selected from DSPE-PEG1000, DSPE-PEG2000, and DMG-PEG2000.Atty. Dkt. No. 178981.0004331. The liposome of claim 28, further comprising DOTAP.

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