COMBINATION THERAPY WITH RAR ALPHA AGONISTS FOR ENHANCING TH1 RESPONSE

DESCRIPTION

[001] This application claims priority to US Provisional Application NO.

62/ 130,240, which was filed on March 9, 2015, and which is incorporated by reference in its entirety.

FIELD

[002] Treatment of cancer and autoimmune diseases using immunotherapy

BACKGROUND

[003] Immunotherapeutic strategies for targeting malignant disease are an active area of translational clinical research, and have been for several decades. While some positive test data has been shown with prior approaches, additional clinically-effective therapeutic strategies should be explored. The art especially desires cancer treatments that will apply to a broader cross-section of patients than presently-available therapies. Likewise, more effective treatments for autoimmune diseases are also desired.

[004] The immune-oncology (I-O) community is seeking approaches and

therapeutics that will enhance the efficacy of PD-1/ CTLA-4/vaccine targeted therapies. These therapeutics are known to drive productive CD4+ and CD8+ T-cell responses to tumor antigens, leading to clinical benefit in cancer patients. The novel discovery described herein is that RARa agonists drive Thl CD4+ T-cell responses, and their use as

monotherapy or in combination with other I-O agents is distinct from the use of RARa agonists as direct tumor cell differentiation agents.

[005] Vitamin A and its derivatives (retinoids) are agonists at retinoic acid receptors, and have activity in cellular growth, differentiation and apoptosis. There are three retinoic acid receptors (RAR-a, β, and γ), and these receptors form heterodimers with members of the complementary retinoid X receptor family (RXR-a, β, and γ). All- trans retinoic acid (ATRA) is an agonist at RAR receptors only. Bexarotene and 13-cis retinoic acid (RA) bind only to RXR receptors. ATRA and bexarotene have been approved for the treatment of human cancers.

[006] ATRA, an RARoc, β, and γ receptor agonist, has been used systemically to treat a subset of acute myeloid leukemia, specifically acute promyelocytic leukemia (APL) patients having an RARoc translocation. In APL, the RARoc gene is aberrantly fused to a fusion partner, typically the APL gene, and the resulting protein binds to DNA and recruits transcriptional co-repressors which impair granulocyte differentiation, key to the

pathogenesis of leukemia. Treatment with ATRA causes the release of co-repressors from the DNA, releases repression of differentiation, and allows the granulocytes to differentiate normally. This treatment, however, is only indicated when the RARoc translocation has occurred and thus has a very limited scope. This narrow indication clearly demonstrates that the utility of ATRA in AML relates to direct effects upon the fusion protein, and not to other effects upon T helper cells, which would not be limited to patients with fusion proteins in their tumor cells. One of the major limitations to the wide scale use of ATRA is its many, severe, toxicities, which may be due to its agonistic effects on RAR or RARy. As such a selective RARoc agonist will have reduced toxicities and have broader utility. The toxicities observed with ATRA include the potentially fatal differentiation syndrome, cardiac toxicity and cutaneous toxicity.

[007] ATRA previously failed to demonstrate activity in a breast cancer study when administered in combination with paclitaxel. Clinical studies of ATRA in lung cancer in combination with cytotoxic chemotherapy are underway, but these aim to exploit direct effects of ATRA upon cell death, most likely via stimulation of RAR (typically measured as a biomarker), hence the use in combination with cytotoxic chemotherapy, which is recognized to generally suppress T-cell responses.

[008] Bexarotene, a synthetic RXR agonist, bexarotene, is approved for the systemic treatment of cutaneous T-cell lymphoma (CTCL). Bexarotene has been tested clinically for activity in other human tumors but failed to show convincing evidence of activity in lung cancer (phase 3 trial in combination with chemotherapy) or breast cancer. 13-cis RA, another RXR agonist, has been tested in treatment of pre-malignant oral leukoplakia, and was shown to induce direct lesion shrinkage, but a meta-analysis suggested evidence was insufficient to support routine usage. 13-cis RA also failed to show compelling activity as monotherapy in breast cancer.

[009] It is well established that Thl CD4+ T-cells are important to the development of productive anti-tumor immunity, with interferon-γ, a critical Thl cytokine, also implicated. In association with tumor-specific CD8+ cytolytic T-cells, promotion of Thl CD4+ T-cell differentiation and stabili2ation has been widely shown to enhance anti-tumor immunity. The role of RAR in Thl cell biology has been hitherto unclear, and implications for the treatment of cancer have been unrecogni2ed. Only with the present work has that pathway been elucidated. Additionally, in the prior art, ATRA has been administered in combination with cytotoxic chemotherapy, which generally suppresses T-cell responses. Only with this discovery, it becomes clear that coadministration of ATRA or other RARa agonists with immunosuppressive cytotoxic agents actually reduces the beneficial impact of the RARa agonist, which generally suppress T-cell responses (i.e., suppresses or entirely prevents the previously unknown immunomodulatory effects from occurring) . The approach of monotherapy with an RARa agonist, or combination use with

immunomodulatory therapeutics, has not been described previously.

[0010] Certain retinoids have been attempted for use in treatment of autoimmune diseases, but have been limited by side effects and potential concerns regarding

teratogenicity. With this study, we are now appreciating that the immune effects of ATRA and other RAR agonist occur through RARa, not RAR or RARy. As such, methods of treatment with agonists specific for RARa can provide benefit and exclude certain side effects associated with RAR or RARy.

[0011] Here we show that RA-RARa is useful for maintenance of the Thl cell lineage. Loss of RA signaling in Thl cells resulted in the emergence of hybrid Thl-Thl7 and Thl 7 effector cells. Global analysis of RARa binding and enhancer mapping revealed that RA-RARoc directly regulated enhancer activity at Thl cell lineage-defining genes while repressing genes that drive Thl 7 cell fate. In the absence of RA signaling, infectious and oral antigen induced inflammation resulted in impaired Thl cell responses with deviation towards a Thl 7 cell phenotype. These findings identify RA-RARoc as a regulatory node that acts to sustain the Thl cell response while repressing Thl 7 cell fate. Thus RARa agonists can used to treat cancer by promoting the Thl cell response and also can be used to treat autoimmune diseases by repressing Thl 7 cells.

SUMMARY

[0012] CD4 ⁺ T-cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4 ⁺ T-cell lineages is useful for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Thl) cells, traditionally

considered the most phenotypically stable Th subset. We found that RA, through its receptor RARa, sustains stable expression of Thl lineage specifying genes as well as repressing genes that instruct Thl7 cell fate. RA signaling is useful for limiting Thl cell conversion into Thl 7 effectors and for preventing pathogenic Thl 7 responses in vivo. Our study identifies RA-RARoc as a component of the regulatory network governing

maintenance and plasticity of Thl cell fate and defines an additional pathway for the development of Thl7 cells.

[0013] In accordance with the description, a method of potentiating anti-tumor immunity comprises administering an RARa agonist to a patient having a tumor, as well as providing at least one other therapy to the patient to treat the tumor. Such at least one other therapy may be chosen from administering a checkpoint inhibitor to the patient having a tumor, administering a vaccine to the patient having a tumor, and treating the patient with T-cell based therapy. [0014] In another embodiment, a method of suppressing a Thl7 response in a patient comprises administering an RARa agonist, as well as at least one other therapy, to the patient.

[0015] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0016] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the

description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1-F. RA Controls the Balance Between Thl and Thl7 Effector Cells. (A) Splenic CD4 ⁺ T-cells from dnR ra and wild-type littermate control mice (WT) mice. Numbers indicate percentage CD62L ^lo CD44 ^hl cells (top left) or CD62L ^hl CD44 ^l0 T-cells (bottom right) gated on CD4 ⁺ cells. (B) Frequency and total number (C) of CD62L ^lo CD44 ^hl in the CD4 ⁺ T-cell population in WT and dnR ra mice (n = 3-4 per group). (D) Intracellular IFN-γ and IL-17A expression in splenic CD4 ⁺ CD44 ^hl T-cells after stimulation with phorbol 12-myristate 13 -acetate (PMA) and ionoymycin. (E) Statistical data from cells as in (D). (F) Quantitative real time PCR analysis of Tbx21, Ron and Gata3 in splenic CD4 ⁺ CD62 ^lo CD44 ^hl cells (as in 1A), sorted by flow cytometry. Data are from two or three independent experiments with similar results. Mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure 9.

[0019] Figures 2A-E. RA Signaling Required for Thl Cell Differentiation and

Repression of Thl7 Cell Fate in Thl Cell Precursors. Sorted naive CD4+ T-cells from dnRara or WT mice were cultured under Thl conditions for 6 days. (A) Intracellular expression of IFN-γ and IL-17A following stimulation with PMA and ionomycin. (B) T-bet and RORyt expression. Grey histograms indicate staining for Tbx21 ^~ / ^~ (left panel) or isotype control antibody (right panel). Numbers show MFI. Numbers in quadrants represent percent T-cells in each. (C) Amount of IL-17A, IL-21, IL-22 and IL-10 in supernatants following restimulation of cells as in (A) with a-CD3 and (X-CD28 for 24 h as measured by multiplex bead array. Triplicate culture wells. (D) Quantitative real time PCR analysis of Thl and Thl 7 cell signature cytokine and TF genes following stimulation with PMA and ionomycin. (E) Naive CD4 ⁺ T-cells from and I/¾ ^eYFP mice were cultured under Thl conditions. IFN-γ (eYFP ⁺ ) cells were sorted on day 7 following stimulation with PMA and ionomycin. Heatmaps displaying the fold changes of genes that were differentially expressed (fold change>1.5, p<0.05) for selected cytokines or cytokine receptors (upper panel) and TFs (lower panel). Samples from three independent experiments. Representative data of at least three (A, B) or two (C-D) independent experiments. Mean ± SEM. See also Figure 10.

[0020] Figures 3A-G. RA Required for Late Phase T-bet Expression. (A) Naive CD4 ⁺ T-cells from dnRara and WT mice were differentiated under Thl conditions with combinations of IFN-γ or IFN-γ antibody. T-bet expression analysed at the indicated timepoints. Histograms gated on CD4 ⁺ T-cells. (B) Flow cytometric analysis of STAT4 phosphorylation in naive CD4 ⁺ T-cells from dnRara and WT mice differentiated under Thl conditions. Cells analysed directly from culture after 3 days (left panel) or on day 6 following treatment with (solid lines) or without (dashed lines) 25ng/ ml IL-12 for 30 min (right panel). Shaded histogram displays pSTAT4 staining in cells cultured under ThO conditions. (C) Cell surface expression of IL-12R 2 on day 6 of culture. (D) Quantitative real-time PCR analysis of IH2rb1 and IH2rb2 on day 6. (E) Quantitative real-time PCR analysis of Stat4 in Thl polarised cells at indicated time points. Expression relative to naive CD4 ⁺ T-cells. (F)

Western-blot analysis of total STAT4 protein on day 6 of Thl culture. (G) Naive CD4 ⁺ T- cells from and control mice were activated under Thl conditions. Frequency of IFN-γ ^-1" (eYFP ⁺ ) cells at indicated timepoints, gated on viable CD4 ⁺ . Data representative of two to three independent experiments. Mean ± SEM. See also Figure 11.

[0021] Figures 4A-B. Loss of RA Signaling in Fully Committed Thl cells Leads to Thl Plasticity and Divergence Towards the Thl 7 Lineage. (A) Naive CD4 ⁺ T-cells from dnRara ^{l l/l l} mice were differentiated under Thl conditions. Thl cells were transduced with TAT-Cre on days 5 and 7 and repolarised under Thl conditions for a further 5 days.

Intracellular expression of T-bet and RORyt. (B) Naive CD4 ⁺ T-cells from Ι/η£ ^Υ¥ν mice were differentiated under Thl conditions. IFN-γ (eYFP ⁺ ) cells were sorted on day 7 and restimulated under Thl conditions for 5 days in the presence of Veh or RAi. Intracellular expression of T-bet and RORyt. Data representative of two independent experiments. See also Figure 12.

[0022] Figures 5A-K. RA-RARoc Regulates Enhancer Activity at Thl Lineage Associated Loci and Represses Thl7 Genes. Naive CD4 ⁺ T-cells from WT and dnRara mice were cultured for 6 days under Thl conditions prior to chromatin precipitation and transcriptional profiling. (A) ChlP-seq binding tracks at Tbx21 locus for RARa in WT Thl cells and p300 binding, H3I 27ac, H3K4mel and H3K4me3 modifications in WT and d R ra Thl cells. (B) Validation of the RARa binding regions in WT Thl cells by ChlP- qPCR. Untr6 region serves as a negative control. Binding events per 1000 cells displayed as 'Enrichment'. (C) The effects of dnRara expression on p300 and H3k27ac abundance at the Tbx21 locus were validated by ChlP-qPCR. (D) Quantitative real-time PCR analysis of Batf, Irf4 and Irfi mRNA in naive CD4 ⁺ T-cells from dnRara or WT-cells differentiated under Thl cell conditions for 0, 24, 48, 72 h. Mean ± SEM, replicate wells. (E) Log2 values of fold changes in gene expression as measured by microarray analyses. Average fold change depicted. (F) ChlP-seq binding tracks at IrfS locus for cells as in (A). (G) Validation of RARa ChlP-seq regions by ChlP-qPCR. (H-J) ChIP analysis of p300 and H3K27ac at selected loci. (K ChIP analysis of H3I 27me3 at the RORc locus. Actb locus serves as a negative control. Data from three independent experiments (E) or representative of two independent experiments (B-D, G-K); Mean ± SD unless noted otherwise. Abbreviation: pro., promoter. See also Figure 13. [0023] Figures 6A-D. RA Signaling Required to Prevent the Generation of Thl7 Cells During Infection with L. monocytogenes. (A) Frequency of LLOp:I-A ^b CD4 ⁺ T-cells isolated from spleen of dnRara and WT mice 7 days after infection with an attenuated strain of L. monocytogenes (Lm-2W). Gated on CD4 ⁺ T-cells. (B) Absolute numbers of LLOp:I-A ^b CD4 ⁺ T-cells as in (A). (C) Intracellular T-bet and RORyt expression gated on LLOp:I-A ^b CD4 ⁺ T-cells. (D) Intracellular staining for IFN-γ and IL-17A following stimulation of splenocytes with LLOp for 6 h, 7 days after infection with Lm-2W. Gated on CD4 ⁺ T-cells. Right panel shows statistical data pooled from 3 independent experiments (3-6 mice per group). Representative data of at least three (A, B), or two independent experiments (C). Mean ± SEM. See also Figure 14.

[0024] Figures 7A-F. Loss of RA signalling Causes dysregulated Thl and Thl7 Response and Increased Pathogenicity in a Model of Gut Inflammation. (A) Schematic illustration of the adoptive transfer experiment. (B) Intracellular expression of IL-17A and IFN-γ among CD4 ⁺ cells from the spleen (Sp), mesenteric lymph nodes (MLN) and lymphocytes from the lamina propria (LPL) of mice as in (A) 7 days after transfer. (C)

Statistical data for frequency of ΓΕΝ-γ ⁺ , IL-17 ⁺ and IFN-Y ⁺ IL-17 ⁺ cells as in (B) in MLN and Sp. (D) Percentile change of original body weight in Rag I recipients treated as in (A) (n = 5-7 per group). Mean ± SD. (E) Frequency of diarrhoea- free mice among R g I recipients as in (A) (OTII recipients n=3, OT-II (d Rard) recipients n=5). (F) Frequencies of IL-17, IFN-γ and Foxp3 in CD4 ⁺ cells isolated from Sp, MLN, LPL and IELs of mice as in (A), 9 days after transfer (n = 5-6 per group). Data from one experiment (B-C), pooled from two independent experiments (D, F), or representative of two independent

experiments (E). Mean ± SEM.

[0025] Figure 8 provides a graphical summary. Retinoic acid (RA) is produced at sites of inflammation. In the presence of Thl instructing cytokines, RA suppress the

differentiation of naive CD4+ T-cells into Thl 7 cells, in part through induction of IRF8 expression and repression of IL-6RA. RA further stabilises the Thl phenotype by maintaining T-bet expression and repressing Runxl. [0026] Figure 9 (related to Figure 1). Expression of Foxp3 in CD4 ⁺ T-cells deficient in RA signalinkate7Eg. (A) Intracellular expression of Foxp3 in CD4 ⁺ T-cells from spleen, thymus and mesenteric lymph nodes (MLN) of wild-type littermate control (WT) and dnRara mice. (B) Total number of CD4 ⁺ Foxp3 ⁺ T-cells in spleen (upper panel) and thymus (lower panel) of WT and dnRara mice. Data are representative of two independent experiments. Mean ± SEM.

[0027] Figure 10 (related to Figure 2). Proliferation and differentiation of CD4 ⁺ T- cells in the absence of RA signalling. (A) Naive CD4+ T-cells from WT and dnRara mice were labeled with CellTrace™ and cultured under Thl conditions for 5 days. Flow cytometry showing dye dilution, gated on viable CD4 ⁺ T-cells. (B) Cell-surface expression of CD44 and CD25 on naive CD4+ T-cells from WT or dnRara mice cultured under Thl conditions for 5 days. (C) Naive CD4+ T-cell from WT and dnRara mice were cultured under ThO or Th2 conditions for 6 days. Cells were analysed by flow cytometry for expression of intracellular RORyt. Gated on CD4 ⁺ T-cells. (D) Sorted naive CD4 ⁺ T- cells from WT and dnRara mice were cultured under Thl7 conditions for 6 days. Intracellular IL- 17A and IFN-γ expression after stimulation with PMA and ionomycin. (E) CD4 ⁺ T-cells from dnRara-Iftig ^YFP and Ιβ£ ^Ύ¥ν mice were cultured under Thl conditions. Quantitative real-time PCR analysis of Cxcr3 and 1112rb2 from IFN-γ (eYFP ⁺ ) cells sorted on day 7.

Samples from three independent experiments. Representative data from two to three independent experiments (A-D). Mean ± SEM.

[0028] Figures 11A-B (related to Figure 3). STAT3 and STAT4 activity in dnRam Thl differentiated cells. (A) Flow cytometric analysis of STAT3 and STAT4

phosphorylation in naive CD4 ⁺ T-cells from dnRara and WT mice differentiated under Thl conditions. Cells analysed after 6 days following treatment with 25ng/ ml IL-12, 20ng/ ml IL- 6 and lOng/ml IL-23 for 30 minutes. Dashed lines represent untreated cells. (B) Bar graph depicts ratio of pSTAT3/pSTAT4 signaling as assessed by MFI.

[0029] Figures 12A-B (related to Figure 4). Cytokine analysis following temporal inhibition of RA signalling in Thl cells. (A) Naive CD4 ⁺ T-cells from dnRara ¹ ^ ^l mice were cultured under Thl conditions. Thl cells were transduced with TAT-Cre on days 5 and 7 and repolarised under Thl conditions for a further 5 days. Intracellular expression of IFN-γ and IL-17A following PMA and ionomycin stimulation. (B) Naive CD4+ T-cells from n ^Y¥V niice were differentiated under Thl conditions. IFN-γ (eYFP ⁺ ) cells were sorted on day 7 and recovered cells underwent secondary re olarisation in Thl conditions for 5 days in the presence of Veh or RAi. Intracellular expression of IFN-γ and IL-17A following PMA and ionomycin stimulation. Data representative of two independent experiments.

[0030] Figures 13A-F (related to Figure 5). RA-RARoc regulates enhancers at Thl genes and represses Thl7 lineage specifying genes. Naive CD4 ⁺ T-cells from dnRara and WT mice were cultured under Thl conditions as in Figure 5. After 6 days, ChIP was performed with the specified antibodies, followed by real-time PCR analysis at selected sites (B-C) or sequencing (A). (A) ChlP-seq binding tracks at Stat4 and Ifng loci for RARoc in WT Thl polarised cells and p300 binding, H3K27ac, H3K4mel and H3K4me3 modifications in WT and dnR^ra Thl cells. (B) Validation of the RARoc ChlP-seq regions in (A) by ChlP- qPCR assays. Untr6 region serves as a negative control. Data presented normalised to input. (C) ChIP analysis of the abundance of p300 at the loci in (B) in WT and dnR ra Thl cells. Data presented normalised to input. (D) ChlP-seq analysis of STAT4 binding at the Tbx21 enhancer and comparison of p300 binding in WT and STAT4 ^_/ - Thl cells. ChlP-Seq data (Vahedi et al. 2012 and Wei et al., 2010) was mapped to the Dec. 2011 (GRCm38/mmlO) mouse genome assembly with the UCSC genome browser along with the ChlP-seq binding track for RARoc at the Tbx21 locus. (E) Quantitative real time PCR analysis of selected genes identified as differentially expressed on genome wide transcriptional profiling analysis of cells as in (A). Mean ± SEM. (F) Cell-surface expression of IL6-Roc by flow cytometry in naive diiR ra and WT CD4 ⁺ T-cells at indicated timepoints. Grey histogram indicates staining for isotype control. Data (B-F) representative of two to three independent experiments. Mean ± SD unless otherwise stated, **p < 0.01; ****p < 0.0001.

[0031] Figure 14A-C (related to Figure 6). Cytokine production by dnRARa T-cells following infection with L. monocytogenes. (A) Splenocytes from diiRara and WT mice infected with Lm-2W were restimulated with LLOp for 24 h. Concentration of IFN-γ, IL- 17 A and IL-4 in supernatants was measured by multiplex bead array (Biorad). Data normalised to total numbers of CD4 ⁺ T-cells. n = 3-4 mice per group. (B) Intracellular staining for IFN-γ and IL-4 following stimulation of splenocytes with LLOp for 6 h, 7 days after infection with L. monocytogenes. Gated on CD3 ⁺ CD4 ⁺ T-cells. (C) Cell surface expression of IL-6R by flow cytometry on LLOp:I-A ^b CD4+ T-cells isolated from spleen of dnR^ra or WT mice 7 days after infection with L. monocytogenes. Data from 4 pooled mice. Numbers indicate MFI. Data representative of two to three independent experiments. Mean ± SEM.

[0032] Figure 15 (related to Figure 7). Gut homing in dnR^ra-OTII CD4+ T-cells. Percentage of OTII or OTII(dnR^ra) CD4 ⁺ cells recovered from LPL, IEL, MLN and Spleen of RAG ^_/ - recipients, 9 days after adoptive transfer (n = 3-4 per group). Data representative of two independent experiments. Mean ± SEM.

DESCRIPTION OF THE SEQUENCES

[0033] Table 1 provides a listing of certain sequences referenced herein.

DESCRIPTION OF THE EMBODIMENTS

I. RARa Agonists

[0034] RARa agonists may include any agent that activates RAR or sustains retinoic acid so that its activity at RAR increases. This includes both substances that initiate a physiological response when combined with a receptor, as well as substances that prevent the catabolism (or breakdown) of retinoids (for example, retinoic acid), allowing the signal from retinoic acid itself to increase. As a nonlimiting list, RARa agonists include, but are not limited to ATRA, AM580, AM80 (tamibarotene), BMS753, BD4, AC-93253, and AR7. Additional RARoc agonists include those provided in US 2012/0149737, which is

incorporated herein by references for its teaching of the chemical structure of additional RARoc agonists. For example, an RAR agonist may include: compound of the following formula, or a pharmaceutically acceptable salt thereof:

[0035] wherein: — R ¹ is independently -X, -R ^x , -0-R ^x -0-R ^A , -0-R ^c , -O-L- R ^C __0-R ^AR , or— 0-L-R ^AR ;— R ² is independently -X, -R ^x , -0-R ^x -0-R ^A , -0-R ^c , -O- L-RC — O— R ^AR , or— 0-L-R ^AR ;— R ³ is mdependentiy -X, -R ^x -0-R ^x -0-R ^A , -0-R ^c , - O-L-RC, — O— R ^AR , or— 0-L-R ^AR ; with the proviso that— R ¹ , -R ² , and— R ³ are not all -O- R ^A ; wherein: each—X is independently— F, --C1,—Br, or—I; each— R ^A is saturated aliphatic Ci-6alkyl; each— R ^x is saturated aliphatic O-ehaloalkyl; each— R ^c is saturated C3-7Cycloalkyl; each— R ^AR is phenyl or Cs-eheteroaryl; each -L- is saturated aliphatic Ci-3alkylene; and wherein: -J- is -C(=0)-NR ^N -; — R ^N is independently -H or -H or -R ^NN ; -R ^NN is saturated aliphatic Ci-4alkyl; =Y— is =CR ^Y — and— = is ~CR ^Z =;— R ^Y is — H;— R ^z is independently— H or — R ^zz ;— R ^zz is independently— F,—CI,—Br,—I,—OH, saturated aliphatic Ci-4alkoxy, saturated aliphatic Ci-4alkyl, or saturated aliphatic Ci-4haloalkyl; =W— is =CR __ _; __R ^W is __H; -RO _ls independently -OH,— OR ^E , -NH _¾ — NHR ^T1 , — NR ^T1 R ^T1 or - NR ^T2 R ^T3 ;— R ^E is saturated aliphatic Ci-6alkyl; each— R ^T1 is saturated aliphatic Ci-6alkyl;— NR ^T2 R ^T3 is independently a2etidino, pyrrolidino, piperidino, piperi2ino, N— (Ci-3alkyl) piperi2ino, or morpholino; with the proviso that the compound is not a compound selected from the following compounds, and salts, hydrates, and solvates thereof: 4-(3,5-dichloro-4- ethoxy-ben2oylamino)-ben2oic acid (PP-02); and 4-(3,5-dichloro-4-methoxy-ben2oylamino)-

[0036] In some embodiments, the RARoc agonist is selective for RARoc and does not produce significant agonistic effects on RAR or RARy. In some instances, about 100% or at least about 99%, 95%, 90%, 85%, 80%, 85%, 80%, 70%, or 60% of the effect of the agonist impacts RARa as compared to combined impact on RAR or RARy.

[0037] In some embodiments, the RARa agonist is at least one substance that prevents the catabolism (or breakdown) of retinoids (for example retinoic acid), allowing the signal from retinoic acid itself to increase. Such agents may include retinioic acid metabolism blocking agents (RAMBAs), which are drugs that inhibit the catabolism of retinoids.

RAMBAs temporarily raise the endogenous levels of al /s/w-retrnoic acid ^' nil inias R .Y in vivo. In doing so, they induce a local retinoid effect and avoid excessive systemic retinoid exposure, thereby avoiding some of the toxicity issues associated with retinoic acid agonists. RAMBAs will act as RARa agonists.

[0038] In some embodiments, RAMBAs include ketocona2ol, liaro2ol, and/ or tararo2ol.

II. Methods of Treating Cancer

[0039] A method of potentiating anti-tumor immunity may be pursued by

administering an RARa agonist to a patient having a tumor. In certain aspects, the method consolidates and/or maintains Thl differentiated state in CD4+ and/or CD8+ T-cells. In some embodiments, a method of potentiating anti-tumor immunity comprises administering an RARa agonist together with an immune enhancer to a patient having a tumor.

[0040] In some embodiments, the patient does not have RARa translocated acute myeloid leukemia. In some embodiments, the patient does not have an RARa translocation. In some embodiments, the RARa agonist is not all-trans retinoic acid.

[0041] In some embodiments, the RARa agonist is administered without

concomitant chemotherapy, such as without traditional small-molecule chemotherapeutic drugs, which would produce a cytotoxic effect that generally suppresses T-cell responses. For some patients, they have had no prior chemotherapy. For other patients, they have had no chemotherapy within at least about 2 weeks, 1, 2, or 3 months. For some patients, they will have no future chemotherapy within at least about 2 weeks, 1, 2, or 3 months, optionally so long as the RARa agonist shows treatment benefit. [0042] Without being bound by theory, we have discovered that RARoc agonists stabilize THO cells that are becoming THl cells, as well as provide for the maintenance of THl cells. Thus, this approach may be used for monotherapy or it may be used in combination with agents that trigger the THO to THl differentiation pathway.

A. Types of Cancer

[0043] In some embodiments, the cancer to be treated includes at least one of adrenocortical carcinoma; AIDS-related cancers (Kaposi sarcoma, lymphoma); anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bile duct cancer (e.g., extrahepatic bile duct cancer); bladder cancer; bone cancer; Ewing sarcoma family of tumors; osteosarcoma and malignant fibrous histiocytoma; brain stem glioma; brain cancer; central nervous system embryonal tumors; central nervous system germ cell tumors; craniopharyngioma; ependymoma; breast cancer; bronchial tumors; carcinoid tumor; cardiac (heart) tumors; lymphoma, primary; cervical cancer; chordoma; acute myelogenous leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); chronic myeloproliferative neoplasms; colon cancer; colorectal cancer; ductal carcinoma in situ (DCIS); embryonal tumors, endometrial cancer; esophageal cancer; esthesioneuroblastoma; extracranial germ cell tumor; extragonadal germ cell tumor; eye cancer (e.g., intraocular melanoma, retinoblastoma); fallopian tube cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumors (GIST); germ cell tumor (e.g., ovarian, testicular); gestational trophoblastic disease; glioma; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer;

hypopharyngeal cancer; islet-cell tumors, pancreatic cancer (e.g., pancreatic neuroendocrine tumors); kidney cancer (e.g., renal cell, Wilms tumor); Langerhans cell histiocytosis; laryngeal cancer; lip and oral cavity cancer; lung cancer (e.g., non-small cell, small cell); lymphoma (e.g., B-cell, Burkitt, cutaneous T-cell, Sezary syndrome, Hodgkin, non-Hodgkin); primary central nervous system (CNS); male breast cancer; mesothelioma; metastatic squamous neck cancer with occult primary; midline tract carcinoma involving nut gene; mouth cancer;

multiple endocrine neoplasia syndromes; multiple myeloma/ plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myelodysplastic/myeloproliferative neoplasms; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma; oral cancer;

oropharyngeal cancer; ovarian cancer (e.g., epithelial tumor, low malignant potential tumor); papillomatosis; paraganglioma; parathyroid cancer; penile cancer; pharyngeal cancer;

pheochromocytoma; pituitary tumor; pleuropulmonary blastoma; pregnancy and breast cancer; primary peritoneal cancer; prostate cancer (e.g., castration-resistant prostate cancer); rectal cancer; rhabdomyosarcoma; salivary gland cancer; sarcoma (uterine); skin cancer (e.g., melanoma, Merkel cell carcinoma, nonmelanoma); small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; cancer of unknown primary; urethral cancer; uterine cancer, vaginal cancer; vulvar cancer; or

Waldenstrom macroglobulinemia.

[0044] In some embodiments, the cancer is acute myelogenous leukemia, bile duct cancer; bladder cancer; brain cancer; breast cancer; bronchial tumors; cervical cancer;

chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); colorectal cancer; endometrial cancer; esophageal cancer; fallopian tube cancer; gallbladder cancer; gastric (stomach) cancer; head and neck cancer; hepatocellular (liver) cancer; kidney (e.g., renal cell) cancer; lung cancer (non-small cell, small cell); lymphoma (e.g., B-cell); multiple myeloma/plasma cell neoplasm; ovarian cancer (e.g., epithelial tumor); pancreatic cancer; prostate cancer (including castration-resistant prostate cancer); skin cancer (e.g., melanoma, Merkel cell carcinoma); small intestine cancer; squamous cell carcinoma; testicular cancer; cancer of unknown primary; urethral cancer; uterine cancer.

B. Combination Therapy Approaches for Cancer

[0045] In certain aspects, the RARoc agonist is administered in combination with at least one other therapy, such as an immuno-oncology agent, namely an immune enhancer.

[0046] In some embodiments, at least one other therapy promotes Thl

differentiation. At least one other therapy may be used to maintain Thl immune response. At least one other therapy may be used to reintroduce Thl immune response. In some aspects, the Thl immune response is a Thl immune response to an antigen expressed by the tumor.

[0047] In some embodiments, at least one other therapy is a Thl differentiation therapeutic. A Thl differentiation therapeutic may be chosen from at least one of, but is not limited to, IL-12, STAT-4, T-bet, STAT-1, IFN-γ, Runx3, IL-4 repressor, Gata-3 repressor, Notch agonist, and DLL.

[0048] In some aspects, at least one other therapy is a checkpoint inhibitor. For example the checkpoint inhibitor may be chosen from at least one of anti-PDl, anti-PDLl, anti-CD80, anti-CD86, anti-CD28, anti-ICOS, anti-B7RPl, anti-B7H3, anti-B7H4, anti- BTLA, anti-HVEM, anti-LAG-3, anti-CTLA-4, IDOl inhibitor, CD40 agonist, anti-CD40L, anti-GAL9, anti-TIM3, anti-GITR, anti-CD70, anti-CD27, anti-CD137L, anti-CD137, anti- OX40L, anti-OX40, anti-KIR, anti-B7.1 (also known as anti-CD80), anti-GITR, anti- STAT3, anti CD137 (also known as anti-4-lBB), anti-VIS A, and anti-CSF-lR checkpoint inhibitor. The checkpoint inhibitor may also cause S AT3 depletion. STAT3 depletion may be achieved through antisense technology or small molecule inhibitors, including cell surface receptor inhibitors, kinase inhibitors, and direct STAT3 inhibitors (including STAT3 SH2 domain inhibitors and S AT3 DNA-binding domain inhibitors). S AT3 inhibitors are described in Furtek et al, ACS Chem. Biol. 11:308-318 (2016), which is incorporated herein in its entirety for the disclosure of STAT3 inhibitors.

[0049] Optionally, a checkpoint inhibitor is an antibody. Such an antibody may be chosen from an anti-PDl, anti-PDLl, anti-CD80, anti-CD86, anti-CD28, anti-ICOS, anti- B7RP1, anti-B7H3, anti-B7H4, anti-BTLA, anti-HVEM, anti-LAG-3, anti-CTLA-4, agonistic anti-CD40, anti-CD40L, anti-GAL9, anti-TIM3, anti-GITR, anti-CD70, anti- CD27, anti-CD137L, anti-CD137, anti-OX40L, anti-OX40, anti-KIR, anti-B7.1 (also known as anti-CD80), anti-GITR, anti-STAT3, anti CD137 (also known as anti-4-lBB), anti- VISTA, and anti-CSF-lR antibody.

[0050] In some aspects, the checkpoint inhibitor helps to induce and/ or maintain a therapeutic Thl response.

[0051] In some embodiments, the at least one other therapy is a vaccine, containing one or more antigens expressed or likely to be expressed by a tumor. The vaccine may be based on a variety of delivery methodologies, including, but not limited to, peptides, DNA, RNA, viruses, virus-like particles, or cell-based vectors. Such a vaccine may be administered to stimulate the patient to produce T-cells or antibodies against the antigen, which would then mediate an immune response against the tumor. In such combination therapy the RARa agonist enhances the response to the antigens administered in the vaccine. For example, if the antigen was intended to induce a T-cell response, a co-administered RARa agonist would serve as a Thl -promoting "adjuvant" and would provide further therapeutic utility.

[0052] In some embodiments, the immuno-oncology agent is a bispecific antibody. In some embodiments, the immuno-oncology agent is a BITE (bispecific T-cell engaging antibody). In some embodiments, the bispecific antibody is anti-CD20 and anti-CD3; anti- CD3 and anti-CD19; anti-EpCAM and anti-CD3; or anti-CEA and anti-CD3.

[0053] In some embodiments, the combination therapy is a T-cell based therapy, such as an ex vivo cell based therapy. T-cell receptor technologies allow culturing or engineering of T cells with a T-cell receptor that can recognize a specific major

histocompatibility complex (MHC) and peptide structure on a tumor. For example, a T-cell may be engineered to express an antibody or binding fragment thereof, where the antibody or fragment is specific for an antigen expressed by the tumor cell. This allows the T cells to target the patient's cancer cells. This culturing or engineering can be done ex vivo and the cells transplanted back into the patient to combine in the present methods. See Kim et al., Arch. Pharm. Res., DOI 10.1007/sl2272-016-0719-7 (published online Feb. 19, 2016), which is incorporated herein in its entirety for the disclosure of T-cell receptor therapy.

C. Methods of Treating Autoimmune Diseases

[0054] In certain embodiments, a method of suppressing a Thl7 response in a patient comprises administering an RARa agonist. Such a treatment may occur in a patient that has an autoimmune disease. In some embodiments, Thl 7 cells with an IFNg+ and/ or IL17+ signature are suppressed.

[0055] Without being bound by theory, we have found that RARa agonist drive away from production of TH17 cells and towards TH1 cells.

D. Types of Autoimmune Diseases

[0056] In some aspects, the autoimmune disease is chosen from autoimmune diseases with an IFNg+IL17+ T-cell signature. In some embodiments, the autoimmune disease may be Juvenile Idiopathic Arthritis, Rheumatoid Arthritis, Crohn's disease, or Multiple Sclerosis.

[0057] In certain modes, the autoimmune disease is chosen from alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/ systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's).

[0058] In one embodiment, the autoimmune disease is not psoriasis and/ or lupus.

E. Combination Therapy for Autoimmune Diseases

[0059] In certain embodiments, a combination therapy approach may be utilized by also administering one or more compounds that function to suppress T-cells, such as known treatments for autoimmune diseases.

[0060] Potential combination therapy agents include abatacept, adalimumab, anakinra, azathioprine, certolizumab, certolizumab pegoltacrolimus, corticosteroids (such as prednisone), dimethyl fumarate, etanercept, fingolimod, glatiramer acetate, golimumab, hydroxychloroquine, infliximab, leflunomide, mercaptopurine, methotrexate, mitoxantrone, natalizumab, rituximab, sulfasalazine, teriflunomide, tocilizumab, tofacitinib, vedolizumab.

[0061] Further aspects are provided through the following nonlimiting examples.

EXAMPLES

Example 1. RA-RARcc Regulates the Balance Between Thl and Thl7 Cells

[0062] To directly assess the role of RA in Th cell differentiation in vivo we used mice carrying a sequence encoding a dominant negative form of the RA receptor RARoc (RARoc403) targeted to ROSA26 downstream of a /oxP-flanked 'stop' (lsl) cassette. [0063] C57B1/ 6 diiR ra mice have been described previously (Pino-Lagos et al., 2011). Mice were bred and maintained at Charles River Laboratory, UK in pathogen- free conditions. All animal experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.

[0064] As shown previously (Pino-Lagos et al., 2011), interbreeding with mice expressing Cre recombinase from the Cd4 promoter generates Cd4 ^cre dnRara ^U/ ' ^ω progeny (diiRara mice) in which RA signaling is abrogated within the T-cell compartment. In contrast to arer/- mice, expression of this dnRARoc disrupts the RA dependent activity of RARoc while retaining the ligand independent effects, allowing the specific analysis of RA dependent functions.

[0065] To investigate the role of RA in the generation of Th cell subsets under steady-state conditions, the expression of cytokines within CD4 ⁺ T-cells with an activated, CD44 ^hl , phenotype was determined. Sort purified, naive CD4 ⁺ CD25-CD44 ^lo CD62L ^hl T-cells were cultured with T-cell depleted splenocytes (APCs) and anti-CD3 under polarisation conditions for ThO, Thl, Th2 and Thl7 cell-associated subsets.

[0066] Experimental conditions were as follows. Naive

CD4 ⁺ CD25 ^neg CD44 ¹ °CD62L ^hl T-cells were isolated by cell sorting by F ACS Aria (BD) after enrichment with a CD4 ⁺ T-cell negative selection kit (Miltenyi Biotec). T-cell depleted splenocytes were prepared using a CD3 ⁺ microbead selection kit (Miltenyi Biotec) followed by irradiation at 3000 rad. Naive CD4+ T-cells were cultured for 3 days with irradiated T- cell-depleted splenocytes at a ratio of 1:5 in the presence of 5 g/ml of anti-CD3 (145-2C11) under ThO cell conditions (IL-2 100 IU/ml, anti-IL-4 (11B11) and anti-IFN-γ (XMG1.2), 10 g/ml each); Thl cell conditions (100 IU/ml of IL-2, 10 ng/ml of IL-12, and anti-IL-4); Th2 cell conditions (100 IU/ml of IL-2, 10 ng/ml of IL-4, anti-IL-12 (C17.8), and anti-IFN- γ (XMG1.2); or Thl7 cell conditions, 5 ng/ml TGFp, 20 ng/ml IL-6, 10 ng/ml IL-Ιβ, anti- IL-4, and anti-IFN -γ). Cells were expanded for an additional 3-4 days. Where indicated, 10 ng/ml IFN-γ or 10 μg/ml anti-IFN-γ was added. In secondary repolarisation assays, where specified, LE540 (1 μΜ) or DMSO (vehicle control) was added to the media.

Cytokines were from R&D. Anti-CD3 was from BioXcell and other antibodies were from BD Biosciences. All cell cultures were performed in complete RPMI containing 10% fetal bovine serum (FBS), 55 μΜ β-mercaptoefhanol, HEPES, non-essential amino acids, glutamine, penicillin and streptomycin.

[0067] For analysis of cytokine production, cells were restimulated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml ionomycin in the presence of monensin for 4-5 h at 37°C in a tissue culture incubator. Cell surface staining was carried out in PBS with 2% FBS. For live cell analysis or cell sorting, dead cells were excluded by staining with SYTOX blue (Invitrogen). For intracellular staining, cells were first stained with LIVE/DEAD Fixable Violet or near IR Dead Cell Stain (Invitrogen), followed by staining for cell-surface markers and then resuspended in fixation/ permeabilisation solution (Cytofix/ Cytoperm kit or Transcription Factor Buffer kit; BD Bioscences). Intracellular staining carried out in accordance with the manufacturer's instructions. Intracellular phosphorylated STA proteins were stained with Phosflow Lyse/Fix Buffer, and Phosflow Perm Buffer III (BD Biosciences) according to the manufacturer's protocol. Data were collected with a LSR Fortessa (BD) and results were analy2ed with Flowjo software (Tree Star). All the antibodies for staining cell surface markers, cytokines or transcription factors were purchased from either BD Biosciences or eBiosciences.

[0068] Cytokine levels in supernatants were measured using a multiplex bead-based assay (Bio-Rad Laboratories) in a Luminex FlexMap3D System (Luminex Corporation).

[0069] Expression analysis was performed as follows. Total RNA was extracted from cells with RNeasy Mini kit (Qiagen) and cDNA was synthesized with Qscript RT kit (Quanta). Quantitative gene expression analysis was performed using Taqman primer probe sets (Applied Biosystems), listed in Table 2. Expression of target genes was normalized to β- actin.

[0070] Sorted naive CD4 ⁺ T-cells from dnRara or WT mice were polarised under Thl conditions. On day 6 of culture cells were harvested and total RNA was extracted for microarray study or ChlP. RNA isolation, microarray and data processing performed by Miltenyi Biotec. For gene-expression analysis for the dnRara Thl dataset Agilent microarray chips were used. Total RNA was extracted from cells lysed in Trizol LS reagent (Life Technologies). RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified with the Nanodrop ND-1000 UV-spectrophotometer (NanoDrop Technologies).

[0071] Transcriptome analysis was performed using Agilent Whole Mouse Genome Oligo Microarrays 8X60K in accordance with manufacturer's protocol. Data analysis was performed using R/bioconductor and software packages therein (www.R-project.org;

www.bioconductor.org) or MS-Office Excel (Microsoft Inc.). Background corrected intensity values were normalized between arrays using quantile normalization. Quality controls include comparison of intensity profiles and a global correlation analysis. Differentially expressed genes were identified by statistical group comparisons on

normalized (background corrected and quantile normalized) log2 transformed fluorescence intensities using Student's t-test (two-tailed, equal variance). Reporters showing a p-value≤ 0.05 and a median fold-change in expression > 1.5 or≤ -1.5 were considered as reliable candidates for altered gene expression. In addition, at least two of the replicate samples in the group with higher expression were required to have detection p-values≤ 0.01.

[0072] Statistical significance was calculated by unpaired two-tailed Student's t test with Graphpad Prism software, p values <0.05 were considered significant, p values are denoted in figures by: *, p < 0.05; **, p < 0.01; p < 0.001; p < 0.0001.

[0073] Examination of the peripheral CD4 ⁺ T-cell compartment revealed equivalent frequencies and absolute numbers of CD44 ^hl CD62 ^lo CD4 ⁺ memory cells in 8-week old dnRara mice and in Cre ^~ , wild-type, littermate controls (WT) (Figure 1A-C). dnRara effector cells displayed reduced production of IFN-γ compared to their WT counterparts with a >5- fold increase in the frequency of IL-17 ⁺ cells (Figure ID— E). Examination of transcripts for the signature lineage-determining TFs showed reduced mRNA expression of Tbx21 and significantly higher expression of Ron in dnRara effector CD4 ⁺ T-cells (Figure IF). Loss of RA signaling had no impact on Th2 effectors with equivalent levels of Gata3 expression between dnRara and WT mice (Figure IF) and similar frequencies of IL-4 producing CD4 ⁺ T-cells (data not shown).

[0074] The frequency and numbers of Foxp3 ⁺ T-cells in the periphery and thymus of dnRara mice were similar to control mice (Figure 9 A— B), indicating that the increase in Thl 7 cells was not a consequence of reciprocal regulation by RA of Foxp3 ⁺ CD4 ⁺ T-cells and Thl7 cells (Mucida et al, 2007). Therefore, it is likely that under steady-state conditions RA is involved in differentiation of Thl cells, while also limiting the differentiation of Thl 7 cells.

Example 2. RA Promotes Thl Cell Differentiation and Inhibits Development of Thl7 Cells from Thl Cell Precursors

[0075] We considered two alternative explanations why dnRara mice exhibit reduced memory effector Thl cells, in parallel with enhanced Thl7 cells. The first possibility was that RA is required for the development of Thl cells while independently suppressing the primary differentiation of Thl 7 cells. The alternative possibility was that RA is involved in restraining conversion of Thl cells to Thl7 cells. In order to resolve these two possibilities, naive CD4 ⁺ T-cells were differentiated in the presence of Thl or Thl7 polarising cytokines. dnRara expressing CD4 ⁺ T-cells differentiated under Thl cell conditions showed a markedly reduced capacity for IFN-γ production (Figure 2A). Diminished cytokine production was not a consequence of impaired proliferative responses as naive CD4 ⁺ T-cells differentiated under Thl cell conditions showed robust proliferation, equivalent to WT-cells (Figure 10A). In addition, up-regulation of the activation markers CD25 and CD44 indicated that dnRara T-cells were not impaired in their ability to differentiate into effector cells (Figure 10B). Analysis of TF expression showed that ablating RA signaling resulted in a dramatic reduction in the expression of T-bet in CD4 ⁺ T-cells differentiated under Thl cell conditions (Figure 2B). Strikingly, a substantial proportion of dnRara Thl cells expressed RORyt and co-expression of T-bet and RORyt was observed at the single cell level.

Although we did not observe intracellular IL-17A in cells following brief stimulation with phorbol myristate (PMA) and ionomycin, analysis of supernatants from Thl polarised cells, reactivated on day 6 of culture on anti-CD3 and anti-CD28 coated plates for 24 h in non- polarising media, showed increased expression of IL-17A alongside other Thl7 cell- associated cytokines (IL-21 and IL-22) (Figure 2C). Furthermore, mRNA analysis of dnRara Thl polarised cells revealed dramatic increases in expression of certain signature Thl 7 cell genes (Figure 2D). Notably, these Thl cells displayed the hallmarks of pathogenic Thl 7 cells with high amounts of 1123 r expression but reduced amounts of IL10 mRNA and protein (Figure 2C and 2D) (Basu et al., 2013).

[0076] In order to assess whether enhanced Thl7 responses were a general feature of CD4 ⁺ T-cells in which RA signaling is disrupted, naive CD4 ⁺ T-cells from dnRara mice were differentiated under Thl 7 polarising conditions. In contrast to our observations above, we did not observe an increase in the frequency of IL-17 ⁺ cells in dnRara mice during primary differentiation into Thl 7 cells (Figure IOC), suggesting that RA restrains Thl 7 cell differentiation only in the context of a Thl polarising cytokine milieu. In support of this, RORyt expression was not observed in dnRara expressing naive CD4 ⁺ T-cells differentiated under ThO or Th2 conditions (Figure 10D).

[0077] The simultaneous expression of RORyt and T-bet in dnRara Thl cells suggested that RA-RARa might act to constrain the deviation of Thl committed cells towards the Thl 7 cell lineage. To determine whether the RORyt ⁺ cells represented a distinct T-cell population that arose directly from naive CD4 ⁺ T-cells or from previously committed Thl cells, Ifng ^eYFF (Great) reporter mice were interbred with the dnRara mice to allow the tracking of IFN-y ⁺ cells.

[0078] Naive CD4 ⁺ T-cells from or littermate control mice were activated under Thl polarising conditions. Ι £ ^Ύ¥ν (GREAT) mice w ^T ere purchased from the Jackson Laboratory. On day 7 of culture, following restimulation with PMA and ionomycin, _e YPP ⁺ _ce vj _{s were} sorted and total RNA was extracted for transcriptional profiling using Affymetrix Mouse Gene 2.0 ST arrays. Pre-processing and statistical analysis of gene expression data were done using Partek Genomics Suite 6.6. CEL files were imported and expression intensities were summarised, normalised and transformed using Robust

Multiarray Average algorithm. Two additional samples from eYFP ⁺ dnRara or wild-type cells sorted without prior restimulation were included in the normalisation. These samples were not included in the analysis of differentially expressed genes. Differentially expressed genes were detected using fold-change and t-test analysis. P values <0.05 and fold change in expression > 1.5 or≤ -1.5 were considered significant.

[0079] Certain signature Thl7 cell genes, including Thl7 cell cytokines and receptors for cytokines that promote Thl 7 cell differentiation (1117f, 1121, 111 r1 , Item, and 1123 r), were highly expressed in dnRara IFN-γ expressing cells relative to WT mice, confirming a hybrid Thl -Thl 7 cell phenotype (Figure 2E). Of note, these Thl -Thl 7 cells retained high expression of I/12rb2 and Cxcr3 mRNA, equivalent to WT Thl cells, while also expressing Il23r (Figure 10E). Genes associated with the Th2 cell subset such as Gata3 and 114 were also dysregulated in dnRara Thl cells consistent with a role for T-bet in repression of GAT A3 (Zhu et al., 2012). These findings show that, in the absence of RA signaling, committed Thl cell precursors can give rise to cells with a Thl7 cell expression signature providing a new perspective on the origins of Thl-Thl7 cells. Collectively these data demonstrate that RA is not only required for Thl cell differentiation, but is also involved in suppressing Thl7 cell development in Thl polarised cells.

Example 3. RA-RARa is Required for Late Phase, STAT4 Dependent T-bet

Expression in Thl Cells

[0080] Early expression of T-bet following TCR activation is dependent on IFN-γ, whereas late expression of T-bet (post-termination of TCR signaling) has been shown to be dependent on IL-12 (Schuk et al., 2009). To distinguish a requirement for RA signaling in Thl cell commitment from maintenance of Thl cell fate, we examined the kinetics of T-bet expression in naive CD4 ⁺ T-cells cultured under Thl polarising conditions.

[0081] Western blot analysis of differentiated Thl cells was as follows. Differentiated Thl cells were lysed in RIPA buffer supplemented with protease inhibitors. Lysates were electrophoresed on 10% gels (Biorad), transferred to nitrocellulose and blotted with anti- STAT4 or anti-actin followed by anti-rabbit-horseradish peroxidase conjugated antibody. All antibodies were from Cell Signaling Technology.

[0082] Induction of T-bet was observed with comparable amounts of T-bet expression between WT and diiRara T-cells at day 3 of culture, indicating that RA-RARa signaling is not required for early Thl lineage commitment (Figure 3A). However, T-bet expression was not sustained in diiR ra Thl cells, with substantially diminished expression of T-bet by day 5 of culture. Given that IFN-γ promotes T-bet expression, the expression of T-bet was examined in the presence of recombinant IFN-γ, in order to avoid potential indirect effects caused by reduced IFN-γ production in diiRara Thl cells. Exogenous IFN-γ enhanced early T-bet expression in both diiRara and WT Thl cells but did not rescue the late (>72 h) impairment in T-bet expression (Figure 3A). IFN-γ signaling, as measured by ST ATI phosphorylation, was not impaired at either timepoint (data not shown).

[0083] The late IL-12-dependent peak of T-bet expression observed in the presence of blocking IFN-γ antibodies was abrogated in diiRara Thl cell polarised cells (Figure 3A) suggesting impaired STAT4 activity. At day 3 of culture, comparable amounts of phosphorylated STAT4 (pSTAT4) were observed between dnRara and WT mice. By contrast, at day 6 of culture, IL-12 induced pSTAT4 was markedly impaired in dnRara T- cells (Figure 3B) despite comparable expression of ΙΣ-12Ι β2 mRNA and protein expression and increased expression of IH2rb1 mRNA (Figure 4C and 4D). Analysis of Stat4

expression, demonstrated impaired induction of Stat4 in the absence of RA signaling (Figure 4E) with reduced amounts of total S AT4 protein (Figure 4F). These findings suggest that the observed reduction in pSTAT4 in dnRara Thl cells is a consequence of diminished STAT4 expression. Consistent with deviation towards the Thl 7 cell lineage, we observed enhanced pSTAT3 activity in Thl cell polarised dnRara cells with an increased ratio of pSTAT3/pSTAT4 (Figure 11 A).

[0084] To evaluate whether the impairment in T-bet and STAT4 expression correlated with changes in IFN-γ, the time-course of IFN-γ expression following initiation of Thl cell polarisation was analysed in naive expressing CD4 ⁺ T-cells. The kinetics of IFN-γ induction, as measured by frequency of eYFP ⁺ cells, closely mirrored WT- cells during the first 72 hours of culture but expression was not sustained in the absence of RA signaling (Figure 3G). Collectively these data show that RA plays a temporal role in Thl differentiation, maintaining Thl cell commitment through regulation of T-bet and STAT4.

Example 4. RA-RARa Regulates Thl Cell Plasticity

[0085] Alterations in the stable expression of lineage-determining TFs are thought to underlie Th cell stability or plasticity. The emergence of Thl -Thl 7 cells together with the loss of T-bet expression, suggested a role for RA in the regulation of Thl cell plasticity. However, diminished T-bet and STAT4 activity from day 3 of primary Thl cell

differentiation prevented assessment of lineage stability in fully differentiated Thl cells. To determine whether RA-RARa was required for long-term Thl cell fate, we differentiated naive CD4+ T-cells from dnRara ^hl ^ ^hl mice under Thl cell conditions, treated them with TAT-Cre (Wadia et al, 2004) on days 5 and 7 and restimulated them under Thl cell conditions for a further 5 days.

[0086] The treatment conditions with TAT-Cre were as follows. Sort purified naive CD4+ T-cells were differentiated under Thl conditions. After 5 days, cells were washed twice in serum free medium prior to treatment with 50 μg/ ml TAT-Cre (Millipore) or medium alone (mock treatment). Cells were incubated at 37°C for 45 minutes. The reaction was quenched with medium containing 20% FBS followed by further washing. Cells were expanded for 2 days followed by retreatment with TAT-Cre or media as before. Cells were then restimulated under Thl cell conditions for 3 days and expanded for a further 2 days prior to analysis.

[0087] The temporal loss of RA signaling in Thl cells resulted in decreased T-bet expression with a reciprocal increase in RORyt expression (Figure 4A).—50% of cells expressed RORyt, which suggests that ongoing RA-RARoc activity is involved in sustaining T-bet and suppressing Thl 7 cell fate. Alterations in the lineage determining TFs did not impact on the cytokine phenotype (Figure 12A). This may in part reflect T-bet independent regulation of the Ifng locus at late stages in Thl cell development.

[0088] To further examine the role of RA in Thl cell stability, naive CD4 ⁺ T-cells from I/¾ ^YFP mice were differentiated under Thl cell polarising conditions. eYFP ⁺ (IFN-y ⁺ ) cells were FACS-sorted on day 7 of culture and restimulated under Thl cell conditions in the presence of the RAR inhibitor LE540 (RAi) or vehicle control (Veh). Inhibition of RA signaling in fully committed Thl cells propagated for a further 5 days under Thl conditions resulted in down-regulation of T-bet and the emergence of cells co-expressing RORyt (Figure 4B). Diminished T-bet expression was associated with modest reductions in IFN-γ expression (Fig 12B). Taken together these data establish that loss of RA signaling in fully committed Thl cells leads to transdifferentiation to progeny with features of the Thl 7 lineage and support a model where RA constrains late stage plasticity of Thl cells.

Example 5. RA-RARa regulates Enhancer Activity at Lineage Determining Thl Cell Genes

[0089] To better understand the molecular mechanism by which RARoc regulates Th cell fate, we performed genome wide analysis of RARoc binding in WT Thl cells by ChlP- Seq, combined with transcriptional profiling of dnRara expressing Thl cells in order to identify functional targets of RARoc. [0090] Immunoprecipitation and DNA sequencing was performed by Active Motif (Carlsbad, CA). The following antibodies were used: anti-H3K27me3 (Millipore 07^4-49), anti-p300 (Santa Cru2 sc-551X), anti-H3K4mel (Active Motif 39287), anti-H3K4me3 (Active Motive 39159), anti-H3K27ac (active Motif 39133), anti-RAR (Diagenode

C15310155). Illumina sequencing libraries were prepared from the ChIP and Input DNAs. For ChIP q-PCR, enrichment calculated as binding events per 1000 Cells using Active Motif s normalisation scheme.

[0091] The experimental procedures were as follows. 20-60 million Thl polarised cells from WT and dnRara mice were fixed, washed and snap-fro2en according to the Cell Fixation protocol from Active Motif (www.activemotif.com/ documents/ 1848.pdf).

Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslmking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop

spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin was precleared with protein A agarose beads (Invitrogen). Following immunoprecipitation with specified antibodies, complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation and used for the preparation of Illumina sequencing libraries and for ChIP qPCR analysis.

A. ChlP-qPCR

[0092] Quantitative PCR (qPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad). See Table 3 for Primer details. The resulting signals were normalized for primer efficiency by carrying out qPCR for each primer pair using Input DNA. By using standards of known quantities of DNA it was possible to calculate the number of genome copies pulled down for each of the sites tested, and thus to calculate the copies pulled down per starting cell number, presented as 'Enrichment'. For RARa ChIP qPCR a gene desert on chromosome 6 (Untr6) was used for a negative control site (Active Motif Catalog No: 71011).

B. ChIP Sequencing (Illumina)

[0093] Illumina sequencing libraries were prepared from the ChIP and Input DNAs using standard procedures and libraries were sequenced on HiSeq 2500. ChlP-seq and microarray data are available under GEO accession number GSE60356. C. ChipSeq Analysis

[0094] For each sample the 50bp SE reads in FastQ format from the sequencer were aligned to the mouse reference genome (mmlO) using Novoalign v2.07.11

(http://www.novocraft.com). The resulting alignment file was converted to BAM format using samtools (http:/ / samtools.sourceforge.net/) and the PCR duplicates were removed using picard tools (http://picard.sourceforge.net). Only uniquely mapped reads from each sample were selected for further analysis. Significantly enriched regions from each sample were identified with MACS v2.0.10_20131216 (Zhang et al. 2008, Feng J et al. 2011) (with q=0.10) using the input sample for background correction. In some instances, peaks were identified by visual inspection and confirmed by ChIP qPCR. In case of H3K4mel and H3I 27me3 samples, "—broad" setting was used to merge nearby enriched regions. For visuali2ation purposes, the input signal was subtracted from each ChIP sample and was converted into bigWig format using "bedGraphToBigWig" utility from UCSC tools (http://genome.ucsc.edu/util.html). The identified significantly enriched regions were annotated to find the associated genes using "FindNeighbouringGenes" utility from USeq package (useq.sourceforge.net/). Associated genes represent the closest transcriptional start site from the centre of the peak.

D. ChipSeq Results

[0095] Selected loci were validated by ChlP-qPCR. RARoc binding was identified at 1766 sites in 1567 genes. RARoc binding was detected at 10.3% (76 of 740 genes) of genes down-regulated in the absence of RA signaling (Table 4) (hereafter referred to as positively regulated) and 4.8% (56 of 1169) of the up-regulated genes (Table 5). In keeping with its classical role as a positive regulator of transcriptional activation there was significant enrichment of RARoc binding at genes positively regulated by RA (Fisher exact test, p<0.0001). However, the presence of RARoc at a subset of the negatively regulated genes indicates that RA-RARa also plays a role in transcriptional repression within Thl cells.

[0096] RA-RARoc dependent loci included Thl cell lineage-defining genes (Tbx21 and Stat4-Stat1). In addition to targeting the Tbx21 promoter (Figure 5A and Figure 5C), modest RARa binding was observed at the conserved T-bet enhancer element, 12kb upstream of the transcriptional start site (TSS) (Yang et al., 2007). This was confirmed by ChlP-qPCR (Figure 5C). Intergenic RARa was also detected at the Stat4-Stat1 locus and an Ifng enhancer element (Figure 13 A-B) .

[0097] RA binding to nuclear RARa results in recruitment of co-activator complexes containing the histone acetyl-transferases p300 and CBP (Kamei et al., 1996). p300 is highly enriched at enhancer regions where it acetylates H3I 27, a marker of active enhancers (Rada-Iglesias et al., 2010), suggesting a possible role for RA-RARa in regulating enhancer activity. To test this, we mapped genome wide binding of p300, H3K4mel, H3K4me3 and H3I 27ac histone modifications in dnRara and WT Thl cells, validating selected regions by ChIP q-PCR. Active enhancers were operationally defined as regions with increased intensity of H3K4mel, p300 and H3K27ac with low or absent H3K4me3 (Rada-Iglesias et al, 2010).

[0098] RARa binding at the Tbx21, Stat and Ifng \oci co-localised with p300 binding at enhancer regions (Figure 5 A and 13 A). dnRARoc lacks the activation function 2 (AF2) domain which is required for RA-dependent recruitment of coactivators. Consistent with this, dnRara expressing T-cells exhibited a significant reduction in p300 occupancy and H3K27ac deposition at the Tbx21 enhancer, supporting the direct regulation of enhancer activity by RA-RARa (Figure 5 A and 5C). p300 binding at the Ifng and putative Stat4 intergenic enhancers was also dependent on RA-RARa (Figure 13A and 13C). Loss of p300 binding at the Stat4-Stat1 intergenic enhancer in dnK ra Thl cells correlated with reduced Stat4 transcripts whereas Statl expression was actually increased, suggesting that this enhancer element regulated Stat4 transcription. A recent study identified a role for STAT4 in the regulation of Thl enhancers (Vahedi et al., 2012). Given that STAT4 expression was reduced in dnRara Thl cells, it was possible that the loss of p300 was in part due to reduced expression of STAT4. To address this issue we assessed the binding of STAT4 in WT Thl cells and compared p300 occupancy in WT and Stat4 ^~ l- Thl cells using publically available ChlP-seq data (Table 6) (Vahedi et al., 2012; Wei et al., 2010). Although STAT4 binding was observed at the Tbx21 enhancer, loss of STAT4 was not associated with obvious differences in p300 binding (Figure 13D) arguing for a direct contribution of RARa to p300

recruitment and enhancer activity. Collectively these data show that RA regulates expression of certain Thl cell lineage genes through remodeling of enhancer regions.

Example 6. RA-RARa Represses Thl7 Cell Fate in Thl cells Through Direct Regulation of Thl7 Cell Genes

[0099] The earlier finding that Thl cells acquired features of Thl7 cells in the absence of RA signaling led us to evaluate direct regulation of Thl7 cell instructing genes by RA-RARa. We first investigated effects of RA on the Thl7 cell pioneer factors BATF and IRF4. As previously reported (Basu et al., 2013), these genes were expressed in WT Thl cells. Strikingly, kinetic analysis of Batf and Irf4 expression in naive cells stimulated under Thl cell conditions revealed dramatic up-regulation of IRF4 (40- to 60-fold) during the initial phase of Thl cell polarisation with comparable expression between diiR ra and WT- cells (Figure 5D). Loss of RA signaling resulted in derepression of BATF-IRF4 target genes, Rorc, 1123 r, 1122, 1121 and 1112rb1 (Figure 5E). This suggested that 'balancing' factors must be induced in an RA dependent manner to restrict the actions of BATF-IRF4 complexes at Thl7 cell genes. IRF8, an alternative binding partner for IRF4, previously shown to suppress Thl7 differentiation (Ouyang et al., 2011), was one of the RARa target genes most suppressed in dnRara Thl cells. In WT Thl cells, induction of Irfi expression paralleled Irf4 expression. However, in dnRara cells Irf8 expression was not sustained past 24 h (Figure 5D). RARoc bound at a putative upstream enhancer (Figure 5F-G) and in the absence of RA signaling, reduced p300 and H3I 27ac were observed at this locus (Figure 5H-I). Together these data show that RA directly regulates expression of IRF8 in Thl differentiating cells and suggests a potential mechanism by which BATF-IRF4 activity is constrained within early Thl cells.

[00100] Transcriptional activation of BATF-IRF4 target genes is dependent on

STAT3 and RORyt (Ciofani et al., 2012). Various genes for cytokines and cytokine receptors associated with STAT3 activation (1121 , 111 r1 , Il6ra andll23r) were derepressed in dnRara Thl cells (Figure 5E). RARoc targeted the promoter and an upstream enhancer in the Il6ra locus (Figure 5G) with increased H3k27ac observed at the enhancer element in dnRara Thl cells (Figure 5J). Consistent with this, dnRara Thl cells failed to down regulate mRNA and cell surface IL6-Roc expression during Thl polarisation (Figure 13E and 13F). These findings suggest that RA regulates Thl cell plasticity in part by inhibiting responsiveness to IL-6.

[00101] RORyt was not a direct target of RARoc. However, disruption of RA signaling resulted in increased expression of Runxl , a TF associated with transactivation of Ron (Figure 13E) (Zhang et al., 2008). ChIP analysis confirmed direct regulation of short and long Runxl isoform promoters by RA-RARoc (Figure 5G). In Thl cells, the Ron locus is epigenetically silenced by T-bet (Mukasa et al., 2010). However, in dnRara cells, the repressive H3I 27me3 mark was reduced at RORyt isoform specific exon (Figure 5J), consistent with loss of T-bet. These findings suggest that increased RORyt expression in the absence of RARoc signaling is in part due to increased accessibility of the Ron locus, with unrestrained activation by Runxl. Collectively these data indicate that RA-RARoc antagonises the activity of the core Thl 7 cell instructing TFs (IRF4, BATF, STAT3 and RORyt), both directly and indirectly, to suppress the Thl7 cell gene program. Notably, Th2 cell-associated genes were not identified as targets of RARoc (Table 5 and 4) suggesting that direct repression of alternative cell fates by RA-RARoc is specific to the Thl 7 cell program. Example 7. Thl-like Thl7 Cells Emerge During Infection with L. monocytogenes in the Absence of RA Signaling

[00102] To assess the significance of these findings for immune responses in vivo, WT and dnRara mice were infected intravenously with an attenuated strain of L.

monocytogenes (AActA), Lm-2W, which allows tracking of CD4 ⁺ T-cells specific for listeriolysin O peptide LLO 190-201 (LLOp).

[00103] LLO190-201 was synthesised by PiProteomics and was >95% pure, as determined by HPLC. LLO:I-A ^b monomers were provided by NIH Core Tetramer Facility. PE labeled LLO:I-A ^b dextramers were synthesised by Immudex. Recombinant Lm-2W strain was provided by Marc Jenkin's Laboratory. LE540 was purchased from Alpha Laboratories.

[00104] Mice were infected i.v. with 1 x 10 ⁶ cfu L. monocytogenes and spleens were harvested 7 days later. For FACS analysis, single cell suspensions were enriched for CD4 ⁺ T-cells with a CD4 ⁺ T-cell negative selection microbead kit (Miltenyi Biotec) and stained with PE labeled, LLO:I-A ^b dextramer (Immudex) and cell surface antibodies. For analysis of cytokine production, supernatants were collected from splenocytes restimulated with LLO peptide (PiProteomics) at 10 μg/ ml for 24 h or intracellular cytokine staining was performed following stimulation with LLO peptide for 6 h in the presence of monensin.

[00105] At the peak of the response, CD4 ⁺ T-cells were isolated from the spleen and LLOp antigen specific T-cells were assayed for expression of cytokines and the TFs, T-bet and RORyt. dnRara mice mounted an effector T-cell response of similar magnitude to WT mice with comparable frequencies and total numbers of CD44 ^hl LLOp:I- A ^b -specific CD4 ⁺ T-cells (Figure 6 A— B). In WT mice, Lm-2W induced a Thl cell restricted response, as evidenced by high T-bet expression within the LLOp specific T-cell fraction (Figure 6C). LLOp:I-A ^b+ CD4+ T-cells from dnRara mice expressed lower amounts of T- bet and a substantial proportion expressed RORyt, with co-expression of these TFs observed in a subset of cells (Figure 6C). At day 7 post-infection, a significant proportion of CD4 ⁺ T-cells isolated from the spleen of dnRara mice were IL-17 ⁺ or dual IL-17A ⁺ IFN-y ⁺ with a trend towards reduced frequency of IFN-y ⁺ cells (Figure 6D). Measurement of cytokine protein concentrations from splenocytes restimulated with LLOp confirmed reduced amounts of IFN-γ and concomitant increase in IL-17A (Figure 14A). We did not detect IL-4 production by intracellular staining or protein secretion (Figure 14A-B).

Consistent with our in vitro data showing down-regulation of IL6-Roc on WT Thl cells, cell surface IL-6Roc was not detectable on WT LLOp:I-A ^b+ CD4 ⁺ T-cells. However, dnR ra LLOp:I-A ^b+ CD4 ⁺ T-cells retained expression of IL-6Roc (Figure 14C), supporting a potential role for IL-6 signaling in the regulation of Thl cell plasticity. These findings establish that RA-RARa signaling in T-cells constrains the emergence of Thl 7 cells in a Thl cell instructing microenvironment in vivo.

Example 8. RA Regulates the Thl-Thl7 Cell Axis in the Gut and Prevents the Development of Intestinal Inflammation

[00106] RA is constitutively synthesised by a subset of DCs in the gut. To address the physiological importance of RA signaling in the regulation of pathogenic intestinal CD4 ⁺ T-cells, we interbred dnRara mice with OTII mice that transgenically express an ovalbumin (OVA) specific TCR and transferred naive CD4 ⁺ T-cells from

OTII(dnR«ra) or WT OTII mice into RagH- hosts. C57B1/6 OTII(dnR«ra), OTII andR ^~ g1 ^~ / ^~ mice were bred and maintained at the Rockefeller University specific pathogen free animal facility. Recipients were maintained on an OVA-containing diet for 7 days to induce differentiation within the transferred cells and migration to the intestinal tissue. Consistent with the infection experiments, feeding OTII(dnR<2ra) recipient mice OVA resulted in a shift in the Thl -Thl 7 cell balance with a deficiency in IFN-γ producing cells and increased frequency of IL-17 ⁺ and dual IFN-Y ⁺ IL-17 ⁺ cells in the mesenteric lymph node (MLN), lamina propria lymphocytes (LPL) and spleen (Sp), 7 days after transfer (Figure 7B and 7C). To address the functional significance of the dysregulated cytokine response in dnRara T- cells, mice were orally challenged with OVA and evaluated for development of intestinal inflammation and diarrhoea (Figure 7A).

[00107] Rag1-/ ^~ mice were kept on a sulfatrim-containing diet and only exposed to autoclaved supplies. Naive OTII CD4 cells (defined as CD4 ⁺ CD25 ^_

Vb5 ⁺ Va2 ⁺ CD44 ) were sorted from 8-12 weeks old female C57B16 OTII(dnR«ra) or C57B16 OTII mice using a FACS Aria cell sorter (Becton Dickinson), and 2 x 10 ⁶ cells in ΙΟΟμΙ PBS were retro-orbitally transferred to 12 weeks old Ragl ^{_/ "} females. 12h after the adoptive transfer, the drinking water was replaced by a 1% Grade II ovalbumin (OVA, Sigma) and 0.5% Splenda (McNeil Nutritionals) solution for 7 days. Body weight was measured at 5pm every day. For monitoring diarrhea development, the faeces texture after 7 days of OVA, 2h after a gavage challenge with 50mg Grade III OVA (Sigma) in 200 μΐ PBS on days 9 and 10 and without further challenge on day 12 was analysed. A mouse was diagnosed with diarrhoea if the faeces had the characteristic soft and light appearance at two consecutive occasions. For the single gavage challenge experiment, mice were subjected to the challenge on day 9 only and the faeces were analysed after 2h. To determine T-cell frequencies, lymphocytes were isolated as previously described (Mucida et al., 2007) on day 7 (from mesenteric lymph node (MLN) and spleen only) or day 9 (from the intestinal epithelium, lamina propria, MLN and spleen) after the start of oral OVA exposure of the recipient mice. For cytokine staining, isolated lymphocytes were stimulated for 3h in RPMI medium supplemented with 10% FBS, 55 μΜ β-mercaptoethanol, lOOng/ml PMA (Sigma),

500ng/ml Ionomycin (Sigma) and 10μg/ml brefeldin A (Sigma) prior to the incubation with antibodies. Cells were first stained with antibodies against T-cell surface markers, followed by permeabilization using either Fix/Perm buffer (BD Pharmingen) for cytokine stainings, or using the Foxp3 Mouse Regulatory T-cell Staining Kit (eBioscience) for Foxp3 staining. The fluorescent-dye- conjugated antibodies used were obtained from BD-Pharmingen (anti- CD4, 550954; anti-CD25, 553866; anti-IL-17a, 559502; anti-Vb5, 553190) or eBioscience (anti-CD44, 56-0441; anti-CD45.2, 47-0454; anti-TCR-β, 47-5961; anti-IFN-γ, 25-7311; anti- Foxp3, 17-5773; anti-Va2, 48-5812). Stained cells were analysed using a LSR-II flow cytometer (Becton Dickinson) and population frequencies were determined using the Flowjo software (Tree Star).

[00108] Recipients of OTII(dnRara cells developed accelerated wasting disease relative to mice that received WT OTII cells (Figure 7D). Whereas all of the recipients of OTII(dnRi?ra) cells developed severe diarrhoea by day 12 (Figure 7E), recipients of WT-cells remained diarrhoea free. Cytokine production was also assessed after the first gavage and confirmed an increased frequency of IL-17 ⁺ cells with concomitant reduction in IFN-y ⁺ cells. Notably, enhanced IL-17 responses were not a consequence of impaired Foxp3+ conversion (Figure 7E). Homing of transferred cells to the gut was not affected in this model with similar frequencies of CD4 ⁺ T-cells detected in the gut tissues (Figure 15A). We conclude that loss of RA signaling leads to deviation from Thl to Thl7 phenotype both in the periphery and the gut where these Thl7 cells are associated with significant intestinal inflammation.

Example 9. Discussion

[00109] Dysregulated Th cell responses underlie the pathogenesis of autoimmune and allergic disease. In contrast to T regulatory (Treg) cells and Thl 7 cells, the Thl cell lineage is thought to be relatively stable. However, the factors that control maintenance of the Thl cell lineage were not previously known. This study identifies RA- RARoc as a central regulatory node in the transcriptional network governing Thl cell stability. We found that RA-RARoc directly sustained the expression of lineage determining Thl cell-associated genes during naive T-cell differentiation whilst also repressing signature Thl7 cell-associated genes. Ablation of RA signaling in Thl committed cells resulted in enhanced Thl cell plasticity with deviation towards a Thl 7 cell phenotype. Using ChlP-seq to identify regulatory elements, we found that RARoc bound at enhancers and recruitment of p300 to these regions was dependent on RA signaling. In vivo, both Thl 7 and Thl -Thl 7 cells emerged during infection with L. monocytogenes and in a model of oral tolerance. In the latter, their presence was associated with significant pathology.

[00110] Enhancers play a role in directing cell fate through the regulation of lineage specifying genes. Enhancer profiling in WT and dnRara T-cells revealed RA dependent activation of enhancers at genes involved in Thl identity (Tbx21 , Stat4, Ifng and Irj ). RA dependent changes in p300 and H3I 27ac were reflected at the transcriptional level suggesting that, in addition to its classical role as a transcriptional regulator, RA regulates gene expression in an enhancer dependent manner. Although the ability of RA-RARoc to target p300-CBP complexes to nucleosomes is well established, regulation of enhancers by RA has not been widely studied. We propose that unliganded RARoc at enhancer elements acts as a gatekeeper, enabling initiation of enhancer activation once T-cells sense RA in the microenvironment. A similar role has been demonstrated for STAT proteins (Vahedi et al., 2012), suggesting that environmental cues act as checkpoints for initiation of enhancer activation and T-cell fate. Although H3K4mel modifications are present at early timepoints during T-cell differentiation, conversion to 'active' status requires acquisition of H3K27ac, which is often not evident until later stages of differentiation (Larjo et al., 2013). Consistent with a temporal role for enhancers in maintenance of gene expression, RA signaling was not required for initiation of transcription of target genes but rather acted to maintain their expression. These data highlight the importance of enhancers in maintenance of cell identity and plasticity. It is possible that RA-RARa regulation of enhancers represent the major mechanism by which RA regulates cell fate. A recent study identified enrichment of RARoc at enhancers in embryonic stem cells (Chen et al., 2012). Given that the RA-RARa axis is a highly conserved signaling pathway, which plays a role in regulating cell fate specification during embryogenesis and cell differentiation, it will be important to evaluate a broader role for RA-RARa in regulation of enhancer functionality, both in alternative Th cell subsets and outside of the immune system.

[00111] In addition to sustaining expression of Thl cell-associated genes, we found that RA actively silences genes implicated in Thl 7 cell differentiation. Among genes known to regulate the Thl7 cell program, Runx and Il6ra were directly repressed by RA- RARa. In addition, BATF-IRF4 target genes were derepressed in the absence of RA signaling. In Thl 7 cells, BATF-IRF4 complexes act co-operatively as pioneer factors at certain Thl7 genes (Ciofani et al., 2012), modulating chromatin accessibility to facilitate binding of STAT3 and RORyt. Based on their expression in alternative Th cell subsets, it has been suggested that BATF-IRF4 complexes play a universal role in establishing binding of lineage specific TFs (Ciofani et al., 2012). However, BATF deficiency does not impact on Thl cell differentiation (Schraml et al., 2009). An alternative model is that up-regulation of BATF and IRF4 confers plasticity in early Thl cells, poising chromatin specifically at Thl 7 cell-associated genes. IRF8, an alternative binding partner for BATF, negatively regulates Thl7 cell differentiation (Ouyang et al., 2011). Our results identified IRF8 as a member of the Thl cell transcriptional network whose expression was dependent on RA signaling. Induction of IRF8 would be expected to limit plasticity of Thl cells by repressing Thl7 differentiation, potentially by competing for binding to BATF. In support of a role for IRF8 in regulation of Thl -Thl 7 axis, patients with mutations in IRF8 have impaired Thl responses (Hambleton et al., 2011) and single nucleotide polymorphisms (SNPs) in Irf8 are associated with several autoimmune diseases in which IFN-y ⁺ Thl7 cells play a pathogenic role (Franke et al., 2010; Graham et al., 2011). It will be of interest to identify transcriptional targets of BATF, IRF4 and IRF8 in Thl cells.

[00112] RA signaling was able to maintain appropriate Thl cell responses and suppress the development of IL-17 ⁺ and IFN-Y ⁺ IL17 ⁺ cells. Hybrid Thl-Thl7 cells are implicated in the pathogenesis of several autoimmune diseases. Their development has been attributed to the plasticity of Thl7 cells. Our findings suggest that these cells might alternatively reflect Thl plasticity and suggest a novel developmental pathway for Thl 7 cells. Thl derived 'Thl 7' cells expressed high levels of the receptor for IL-23, a determinant of Thl7 pathogenicity (Basu et al., 2013), and were associated with significant gut inflammation and pathology in a model of oral tolerance. Further experiments are required to test the prediction that pathogenic Thl7 and IFN-y ⁺ IL-17 ⁺ cells which arise in autoimmunity emerge from Thl cells when RA is deficient or its signaling perturbed.

[00113] A range of inflammatory stimuli can induce RA synthesis and signaling during the course of an immune response. Our results suggest that in a Thl cell instructing microenvironment the dominant action of RA is to repress Thl7 cell fate and promote Thl cell responses. We did not observe enhanced Thl 7 cell responses during primary Thl 7 cell differentiation suggesting that the impact of RA on T-cell stability may vary both temporally and among tissues. Previously we have shown in a model of skin allograft rejection that impaired Thl responses in dnRara mice were accompanied by increased Th2 cell cytokines (Pino-Lagos et al., 2011). We did not identify direct repression of Th2 cell-associated genes by RARoc. However, T-bet suppresses GATA3 (Zhu et al., 2012) and in the presence of a Th2 skewing micro-environment, such as the skin, impaired expression of T-bet in the absence of RA signaling renders cells susceptible to Th2 deviation. Thus, the effects of RA on T-cell fate are likely dependent on external and intrinsic factors which shape T-cell polarity. In summary, we show that RA signaling plays a role in regulating stability and functional plasticity of Thl cells. Regulation of enhancer activity at lineage determining genes by RA-RARoc provides mechanistic evidence for reciprocal regulation of Thl and Thl7 cell programs. In the absence of RA signaling, down modulation of T-bet, STAT4 and IFN-γ, and loss of repression of Thl7 cell genes, creates a permissive environment for trans differentiation of Thl cells to Thl 7 cells. This study identifies the RA-RARa axis as a potential node for intervention in diseases in which dysregulation of the Thl -Thl 7 cell axis is observed.

Example 10. Embodiments Described Herein

[00114] The following embodiments, outline some of the aspects of the technology and approaches described herein:

[00115] Embodiment 1. A method of potentiating anti-tumor immunity in a patient having a tumor comprising

[00116] (a) administering an RARoc agonist to the patient having a tumor and

[00117] (b) providing at least one other therapy to the patient to treat the tumor.

[00118] Embodiment 2. The method of embodiment 1, wherein the at least one other therapy is chosen from:

[00119] (a) administering a checkpoint inhibitor to the patient having a tumor;

[00120] (b) administering a vaccine to the patient having a tumor; and

[00121] (c) treating the patient with T-cell based therapy.

[00122] Embodiment 3. The method of any one of embodiments 1-2, wherein the RARoc agonist is chosen from

[00123] (a) ATRA

[00124] (b) AM580

[00125] (c) AM80 (tamibarotene)

[00126] (d) BMS753

[00127] (e) BD4

[00128] (f) AC-93253 [00129] (g) AR7

[00130] (h) compound of the following formula, or a pharmaceutically acceptable salt thereof:

[00131] wherein: — R ¹ is mdependentiy -X, -R ^x , -0-R ^x -0-R ^A , -0-R ^c , -

O-L-RC, — O— R ^AR , or— 0-L-R ^AR ;— R ² is independently -X, -R ^x , -0-R ^x -0-R ^A , -0-R ^c , __0-L-R ^c ,— O— R ^AR , or— 0-L-R ^AR ;— R ³ is independently -X, -R ^x -0-R ^x -0-R ^A , -O- R ^c , -O-L-RC, — O— R ^AR , or— 0-L-R ^AR ; with the proviso that— R ¹ ,— R ² , and— R ³ are not all - 0~R ^A ; wherein: each—X is independently— F, --C1,—Br, or—I; each— R ^A is saturated aliphatic 0-6alkyl; each— R ^x is saturated aliphatic O-ehaloalkyl; each— R ^c is saturated C ₃ - 7cycloalkyl; each— R ^AR is phenyl or Cs-eheteroaryl; each -L- is saturated aliphatic 0- ₃ alkylene; and wherein: -J- is -C(=0)-NR ^N -; — R ^N is independently -H or -H or -R ^NN ; -R ^NN is saturated aliphatic G-4alkyl; =Y— is =CR ^Y ~ and— = is — CR ^Z =; ~R ^Y is — H;— R ^z is independently— H or — R ^zz ;— R ^zz is independently— F,—CI,—Br,—I,—OH, saturated aliphatic G-4alkoxy, saturated aliphatic G-4alkyl, or saturated aliphatic G-4haloalkyl; =W— is =CR __ _; __R ^W is __H; -RO s mdependentiy -OH,— OR ^E , -NH _¾ — NHR ^T1 , — NR ^T1 R ^T1 or - NR ^T2 R ^T3 ;— R ^E is saturated aliphatic Ci-6alkyl; each— R ^T1 is saturated aliphatic Ci-6alkyl;— NR ^T2 R ^T3 is independently a2etidino, pyrrolidino, piperidino, piperi2ino, N— (Ci- ₃ alkyl) piperi2ino, or morpholino; with the proviso that the compound is not a compound selected from the following compounds, and salts, hydrates, and solvates thereof: 4-(3,5-dichloro-4- ethoxy-ben2oylamino)-ben2oic acid (PP-02); and 4-(3,5-dichloro-4-methoxy-ben2oylamino)-

[00132] Embodiment 4. The method of any one of embodiments 1-3, wherein the RAR agonist is a RAMBA. [00133] Embodiment 5. The method of embodiment 4, wherein the RAMBA is at least one chosen from ketocona2ol, liaro2ol, and tararo2ol.

[00134] Embodiment 6. The method of any one of embodiments 1-5, wherein the method consolidates and/ or maintains Thl differentiated state in CD4+ and/ or CD8+ T-cells.

[00135] Embodiment 7. The method of any one of embodiments 1-6, wherein the RARoc agonist is administered without concomitant chemotherapy.

[00136] Embodiment 8. The method of embodiment 7, wherein the patient has had no prior chemotherapy.

[00137] Embodiment 9. The method of embodiment 7, wherein the patient has had no chemotherapy within at least about 2 weeks, 1, 2, or 3 months.

[00138] Embodiment 10. The method of any one of embodiments 7-9, wherein the patient will have no future chemotherapy within at least about 2 weeks, 1, 2, or 3 months.

[00139] Embodiment 11. The method of any one of embodiments 1-10, wherein the at least one other therapy is an immune enhancer.

[00140] Embodiment 12. The method of any one of embodiments 1-11, wherein at least one other therapy promotes Thl differentiation.

[00141] Embodiment 13. The method of any one of embodiments 1-12, wherein at least one other therapy is used to maintain Thl immune response.

[00142] Embodiment 14. The method of any one of embodiments 1-13, wherein at least one other therapy is used to reintroduce Thl immune response.

[00143] Embodiment 15. The method of any one of embodiments 1-14, wherein the Thl immune response is a Thl immune response to an antigen expressed by the tumor.

[00144] Embodiment 16. The method of any one of embodiments 1-15, wherein at least one other therapy is a Thl differentiation therapeutic. [00145] Embodiment 17. The method of embodiment 16, wherein the Thl differentiation therapeutic is chosen from IL-12, STAT-4, T-bet, STAT-1, IFN-γ, Runx3, IL-4 repressor, Gata-3 repressor, Notch agonist, and DLL.

[00146] Embodiment 18. The method of any one of embodiments 1-17, wherein at least one other therapy is a checkpoint inhibitor.

[00147] Embodiment 19. The method of embodiment 18, wherein the checkpoint inhibitor is chosen from anti-PDl, anti-PDLl, anti-CD80, anti-CD86, anti- CD28, anti-ICOS, anti-B7RPl, anti-B7H3, anti-B7H4, anti-BTLA, anti-HVEM, anti-LAG- 3, anti-CTLA-4, IDOl inhibitor, CD40 agonist, anti-CD40L, anti-GAL9, anti-TIM3, anti- GITR, anti-CD70, anti-CD27, anti-CD137L, anti-CD137, anti-OX40L, anti-OX40, anti- KIR, anti-B7.1 (also known as anti-CD80), anti-GITR, anti-STAT3, anti CD137 (also known as anti-4-lBB), anti- VISTA, and anti-CSF-lR checkpoint inhibitor.

[00148] Embodiment 20. The method of embodiment 18, wherein the checkpoint inhibitor causes STAT3 depletion.

[00149] Embodiment 21. The method of embodiment 18, wherein the checkpoint inhibitor is an antibody.

[00150] Embodiment 22. The method of embodiment 19, wherein the antibody checkpoint inhibitor is chosen from an anti-PDl, anti-PDLl, anti-CD80, anti- CD86, anti-CD28, anti-ICOS, anti-B7RPl, anti-B7H3, anti-B7H4, anti-BTLA, anti-HVEM, anti-LAG-3, anti-CTLA-4, IDOl inhibitor, agonistic anti-CD40, anti-CD40L, anti-GAL9, anti-TIM3, anti-GITR, anti-CD70, anti-CD27, anti-CD137L, anti-CD137, anti-OX40L, anti- OX40, anti-KIR, anti-B7.1 (also known as anti-CD80), anti-GITR, anti-STAT3, anti CD137 (also known as anti-4-lBB), anti- VISTA, and anti-CSF-lR antibody.

[00151] Embodiment 23. The method of any one of embodiments 18-22, wherein the checkpoint inhibitor helps to induce and/ or maintain a therapeutic Thl response.

[00152] Embodiment 24. The method of any one of embodiments 1-23, wherein at least one other therapy is an antigen, a tumor antigen, and/ or a cancer vaccine. [00153] Embodiment 25. The method of any one of embodiments 1-24, wherein at least one other therapy is a bispecific antibody.

[00154] Embodiment 26. The method of embodiment 25, wherein the bispecific antibody is a bispecific T-cell engaging antibody.

[00155] Embodiment 27. The method of embodiment 26, wherein the bispecific antibody is chosen from anti-CD20 and anti-CD3; anti-CD3 and anti-CD 19; anti- EpCAM and anti-CD3; and anti-CEA and anti-CD3.

[00156] Embodiment 28. The method of any one of embodiments 1-27, where at least one other therapy is a T-cell based therapy.

[00157] Embodiment 29. The method of embodiment 28, wherein the T-cell based therapy is ex vivo cell based therapy.

[00158] Embodiment 30. The method of any one of embodiments 1-29, wherein the patient has at least one of melanoma, renal cell cancer, non-small cell lung cancer (including squamous cell cancer and/ or adenocarcinoma), bladder cancer, non- Hodgkins lymphoma, Hodgkin's lymphoma, and head and neck cancer.

[00159] Embodiment 31. The method of any one of embodiments 1-29, wherein the patient has adrenocortical carcinoma; AIDS-related cancers (Kaposi sarcoma, lymphoma); anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bile duct cancer (e.g., extrahepatic bile duct cancer); bladder cancer; bone cancer; Ewing sarcoma family of tumors; osteosarcoma and malignant fibrous histiocytoma; brain stem glioma; brain cancer; central nervous system embryonal tumors; central nervous system germ cell tumors; craniopharyngioma; ependymoma; breast cancer; bronchial tumors; carcinoid tumor; cardiac (heart) tumors; lymphoma, primary; cervical cancer; chordoma; acute myelogenous leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); chronic myeloproliferative neoplasms; colon cancer; colorectal cancer; ductal carcinoma in situ (DCIS); embryonal tumors, endometrial cancer; esophageal cancer; esthesioneuroblastoma; extracranial germ cell tumor;

extragonadal germ cell tumor; eye cancer (e.g., intraocular melanoma, retinoblastoma); fallopian tube cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumors (GIST); germ cell tumor (e.g., ovarian, testicular); gestational trophoblastic disease; glioma; hairy cell leukemia; head and neck cancer;

hepatocellular (liver) cancer; hypopharyngeal cancer; islet-cell tumors, pancreatic cancer (e.g., pancreatic neuroendocrine tumors); kidney cancer (e.g., renal cell, Wilms tumor);

Langerhans cell histiocytosis; laryngeal cancer; lip and oral cavity cancer; lung cancer (e.g., non-small cell, small cell); lymphoma (e.g., B-cell, Burkitt, cutaneous T-cell, Se2ary syndrome, Hodgkin, non-Hodgkin); primary central nervous system (CNS); male breast cancer; mesothelioma; metastatic squamous neck cancer with occult primary; midline tract carcinoma involving nut gene; mouth cancer; multiple endocrine neoplasia syndromes;

multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myelodysplastic/myeloproliferative neoplasms; nasal cavity and paranasal sinus cancer;

nasopharyngeal cancer; neuroblastoma; oral cancer; oropharyngeal cancer; ovarian cancer (e.g., epithelial tumor, low malignant potential tumor); papillomatosis; paraganglioma;

parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pituitary tumor; pleuropulmonary blastoma; pregnancy and breast cancer; primary peritoneal cancer; prostate cancer (e.g., castration-resistant prostate cancer); rectal cancer; rhabdomyosarcoma; salivary gland cancer; sarcoma (uterine); skin cancer (e.g., melanoma, Merkel cell carcinoma, nonmelanoma); small intestine cancer; soft tissue sarcoma; squamous cell carcinoma;

testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; cancer of unknown primary; urethral cancer;

uterine cancer, vaginal cancer; vulvar cancer; or Waldenstrom macroglobulinemia.

[00160] Embodiment 32. The method of embodiment 31, wherein the cancer is chosen from acute myelogenous leukemia, bile duct cancer; bladder cancer; brain cancer; breast cancer; bronchial tumors; cervical cancer; chronic lymphocytic leukemia (CLL);

chronic myelogenous leukemia (CML); colorectal cancer; endometrial cancer; esophageal cancer; fallopian tube cancer; gallbladder cancer; gastric (stomach) cancer; head and neck cancer; hepatocellular (liver) cancer; kidney (e.g., renal cell) cancer; lung cancer (non-small cell, small cell); lymphoma (e.g., B-cell); multiple myeloma/plasma cell neoplasm; ovarian cancer (e.g., epithelial tumor); pancreatic cancer; prostate cancer (including castration- resistant prostate cancer); skin cancer (e.g., melanoma, Merkel cell carcinoma); small intestine cancer; squamous cell carcinoma; testicular cancer; cancer of unknown primary; urethral cancer; uterine cancer.

[00161] Embodiment 33. The method of any one of embodiments 1-32, wherein the patient does not have RARa translocated acute myeloid leukemia.

[00162] Embodiment 34. The method of any one of embodiments 1-33, wherein the RARa agonist is not all-trans retinoic acid.

[00163] Embodiment 35. A method of suppressing a Thl7 response in a patient comprising administering an RARa agonist and at least one other therapy to the patient.

[00164] Embodiment 36. The method of embodiment 35, wherein the patient has an autoimmune disease and the method treats the autoimmune disease.

[00165] Embodiment 37. The method of any one of embodiments 35-36, wherein the Thl7 cells with an IFNg+ and/ or IL17+ signature are suppressed.

[00166] Embodiment 38. The method of any one of embodiments 35-37, wherein the RARa agonist is chosen from

[00167] (a) ATRA

[00168] (b) AM580

[00169] (c) AM80 (tamibarotene)

[00170] (d) BMS753

[00171] (e) BD4

[00172] (f) AC-93253

[00173] (g) AR7

[00174] (h) compound of the following formula, or a pharmaceutically acceptable salt thereof:

[00175] wherein: — R ¹ is independently -X, -R ^x , -0-R ^x , -0-R ^A , -0-R ^c , -

O-L-RC, — O— R ^AR , or — 0-L-R ^AR ;— R ² is independentiy -X, -R ^x , -0-R ^x , -0-R ^A , -0-R ^c , __0-L-R ^c ,— O— R ^AR , or— 0-L-R ^AR ;— R ³ is independentiy -X, -R ^x -0-R ^x , -0-R ^A , -O- R ^c , -O-L-R ^C ,— O— R ^AR , or— 0-L-R ^AR ; with the proviso that— R ¹ ,— R ² , and — R ³ are not all - 0~R ^A ; wherein: each—X is independentiy— F, --C1,—Br, or—I; each — R ^A is saturated aliphatic Ci-6alkyl; each — R ^x is saturated aliphatic O-ehaloalkyl; each — R ^c is saturated C3- 7cycloalkyl; each — R ^AR is phenyl or Cs-eheteroaryl; each -L- is saturated aliphatic Ci-3alkylene; and wherein: -J- is -C(=0)-NR ^N -;— R ^N is independentiy -H or -H or -R ^NN ; -R ^NN is saturated aliphatic G-4alkyl; =Y— is =CR ^Y — and— = is ~CR ^Z =;— R ^Y is — H;— R ^z is independentiy— H or — R ^zz ;— R ^zz is independently— F,—CI,—Br,—I,—OH, saturated aliphatic G-4alkoxy, saturated aliphatic G-4alkyl, or saturated aliphatic G-4haloalkyl; =W— is =CR __ _; __R ^W is __H; -RO _ls independently -OH,— OR ^E , -NH _¾ — NHR ^T1 ,— NR ^T1 R ^T1 or - NR ^T2 R ^T3 ;— R ^E is saturated aliphatic Ci-6alkyl; each — R ^T1 is saturated aliphatic Ci-6alkyl;— NR ^T2 R ^T3 is independentiy a2etidino, pyrrolidino, piperidino, piperi2ino, N— (Ci-3alkyl) piperi2ino, or morpholino; with the proviso that the compound is not a compound selected from the following compounds, and salts, hydrates, and solvates thereof: 4-(3,5-dichloro-4- ethoxy-ben2oylamino)-ben2oic acid (PP-02); and 4-(3,5-dichloro-4-methoxy-ben2oylamino)-

[00176] Embodiment 39. The method of any one of embodiments 35-38, wherein the RARa agonist is coadministered together with a T-cell suppressive agent.

[00177] Embodiment 40. The method of any one of embodiments 35-39, wherein the RARa agonist is coadministered together with abatacept, adalimumab, anakinra, a2athioprine, certoli2umab, certoli2umab pegoltacrolimus, corticosteroids (such as prednisone), dimethyl fumarate, etanercept, fingolimod, glatiramer acetate, golimumab, hydroxychloroquine, infliximab, leflunomide, mercaptopurine, methotrexate, mitoxantrone, natalizumab, rituximab, sulfasala2ine, teriflunomide, tocili2umab, tofacitinib, or

vedolizumab.

[00178] Embodiment 41. The method of any one of embodiments 35-40, wherein the autoimmune disease is chosen from an autoimmune disease with an

IFNg+IL17+ T-cell signature.

[00179] Embodiment 42. The method of any one of embodiments 35-41, wherein the autoimmune disease is chosen from Juvenile Idiopathic Arthritis, Rheumatoid Arthritis, Crohn's disease, and Multiple Sclerosis.

[00180] Embodiment 43. The method of any one of embodiments 35-42, wherein the autoimmune disease is chosen from alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/ systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or granulomatosis with polyangiitis ( egener's).

Example 11. Items Described Herein

[00181] While not limiting, certain items are described through the application and in the listing of the following items:

[00182] Item 1. A method of potentiating anti- tumor immunity comprising administering an RARoc agonist to a patient having a tumor.

[00183] Item 2. The method of item 1, wherein the RARoc agonist is chosen from a. ATRA

b. AM580

c. AM80 (tamibarotene)

d. BMS753 e. BD4

f. AC-93253

g. AR7

h. compound of the following formula, or a pharmaceutically acceptable

thereof:

wherein: — R ¹ is independently -X, -R ^x , -0-R ^x -0-R ^A , -0-R ^c , -0-L-R ^c , -O- R ^AR , or— 0-L-R ^AR ;— R ² is independentiy -X, -R ^x , -0-R ^x -0-R ^A , -0-R ^c , -O- L-RC — O— R ^AR , or— 0-L-R ^AR ;— R ³ is independentiy -X, -R ^x -0-R ^x -0-R ^A , - O-RC __0-L-R ^c ,— O— R ^AR , or— 0-L-R ^AR ; with the proviso that — R ¹ ,— R ² , and— R ³ are not all — O— R ^A ; wherein: each—X is independentiy— F,—CI,—Br, or—I; each— R ^A is saturated aliphatic Ci-6alkyl; each — R ^x is saturated aliphatic O-ehaloalkyl; each— R ^c is saturated C3- ₇ cycloalkyl; each — R ^AR is phenyl or Cs-eheteroaryl; each -L- is saturated aliphatic G-3alkylene; and wherein: -J- is — C(=0)~NR ^N — ;— R ^N is independentiy— H or — H or — R ^NN ;— R ^NN is saturated aliphatic Ci-4alkyl; =Y— is =CR ^Y - and -Z= is -CR ^Z =;— R ^Y is -H; -R ^z is independentiy -H or -R ^zz ; -R ^zz is independentiy— F,—CI,—Br,—I,—OH, saturated aliphatic G-4alkoxy, saturated aliphatic Ci-4alkyl, or saturated aliphatic Ci-4haloalkyl; =W— is =CR ^W — ;— R ^w is — H;— R° is independentiy -OH,— OR ^E , -NH _¾ — NHR ^T1 ,— NR ^T1 R ^T1 or— NR ^T2 R ^T3 ;— R ^E is saturated aliphatic G-6alkyl; each — R ^T1 is saturated aliphatic G-6alkyl; — NR ^T2 R ^T3 is independentiy a2etidino, pyrrolidino, piperidino, piperizino, N— (Ci-3alkyl) piperizino, or morpholino; with the proviso that the compound is not a compound selected from the following compounds, and salts, hydrates, and solvates thereof: 4-(3,5- dichloro-4-ethoxy-benzoylamino)-benzoic acid (PP-02); and 4-(3,5-dichloro-4- methoxy-benzoylamino) -benzoic acid (PP-03). [00184] Item 3. The method of any one of items 1-2, wherein the method consolidates and/ or maintains Thl differentiated state in CD4+ and/ or CD8+ T- cells.

[00185] Item 4. The method of any one of items 1-3, wherein the RARoc agonist is administered without concomitant chemotherapy.

[00186] Item 5. The method of item 4, wherein the patient has had no prior chemotherapy.

[00187] Item 6. The method of item 4, wherein the patient has had no chemotherapy within at least about 2 weeks, 1, 2, or 3 months.

[00188] Item 7. The method of any one of items 4-6, wherein the patient will have no future chemotherapy within at least about 2 weeks, 1, 2, or 3 months.

[00189] Item 8. The method of any one of items 1-7, wherein the RARoc agonist is administered in combination with at least one other therapy.

[00190] Item 9. The method of item 8, wherein the at least one other therapy is an immune enhancer.

[00191] Item 10. The method of any one of items 8-9, wherein at least one other therapy promotes Thl differentiation.

[00192] Item 11. The method of item 10, wherein at least one other therapy is used to maintain Thl immune response.

[00193] Item 12. The method of any one of items 9-11, wherein at least one other therapy is used to reintroduce Thl immune response.

[00194] Item 13. The method of any one of items 11-12, wherein the Thl immune response is a Thl immune response to an antigen expressed by the tumor.

[00195] Item 14. The method of any one of items 8-13, wherein at least one other therapy is a Thl differentiation therapeutic. [00196] Item 15. The method of item 14, wherein the Thl differentiation therapeutic is chosen from IL-12, STAT-4, T-bet, STAT-1, IFN-γ, Runx3, IL-4 repressor, Gata-3 repressor, Notch agonist, and DLL.

[00197] Item 16. The method of any one of items 8-15, wherein at least one other therapy is a checkpoint inhibitor.

[00198] Item 17. The method of item 16, wherein the checkpoint inhibitor is chosen from anti-PDl, anti-PDLl, anti-CD80, anti-CD86, anti-CD28, anti- ICOS, anti-B7RPl, anti-B7H3, anti-B7H4, anti-BTLA, anti-HVEM, anti-LAG-3, anti- CTLA-4, IDOl inhibitor, anti-CD40, anti-CD40L, anti-GAL9, anti-TIM3, anti-GITR, anti- CD70, anti-CD27, anti-CD137L, anti-CD137, anti-OX40L and anti-OX40 checkpoint inhibitor.

[00199] Item 18. The method of item 17, wherein the checkpoint inhibitor is an antibody.

[00200] Item 19. The method of any one of items 16-18, wherein the checkpoint inhibitor helps to induce and/ or maintain a therapeutic Thl response.

[00201] Item 20. The method of any one of items 8-19, wherein at least one other therapy is an antigen, a tumor antigen, and/ or a cancer vaccine.

[00202] Item 21. The method of any one of items 1-20, wherein the patient has at least one of melanoma, renal cell cancer, non-small cell lung cancer (including squamous cell cancer and/ or adenocarcinoma), bladder cancer, non-Hodgkins lymphoma, Hodgkin's lymphoma, and head and neck cancer.

[00203] Item 22. The method of any one of items 1-20, wherein the patient has Adrenocortical Carcinoma; AIDS-Related Cancers (Kaposi Sarcoma,

Lymphoma); Anal Cancer; Appendix Cancer; Astrocytomas; Atypical Teratoid/Rhabdoid Tumor; Basal Cell Carcinoma; Bile Duct Cancer; Bladder Cancer; Bone Cancer; Ewing Sarcoma Family of Tumors; Osteosarcoma and Malignant Fibrous Histiocytoma; Brain Stem Glioma; Brain Tumor; Central Nervous System Embryonal Tumors; Central Nervous System Germ Cell Tumors; Craniopharyngioma; Ependymoma; Breast Cancer; Bronchial Tumors; Carcinoid Tumor; Cardiac (Heart) Tumors; Lymphoma, Primary; Cervical Cancer; Chordoma; Chronic Lymphocytic Leukemia (CLL); Chronic Myelogenous Leukemia (CML); Chronic Myeloproliferative Neoplasms; Colon Cancer; Colorectal Cancer; Duct, Bile, Extrahepatic; Ductal Carcinoma In Situ (DOS); Embryonal Tumors, Endometrial Cancer; Esophageal Cancer; Esthesioneuroblastoma; Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor; Eye Cancer (Intraocular Melanoma, Retinoblastoma); Fallopian Tube Cancer; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumors (GIST); Germ Cell Tumor (Ovarian, Testicular);

Gestational Trophoblastic Disease; Glioma; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer; Hypopharyngeal Cancer; Islet Cell Tumors, Pancreatic Neuroendocrine Tumors; Kidney (Renal Cell, Wilms Tumor); Langerhans Cell Histiocytosis; Laryngeal Cancer; Lip and Oral Cavity Cancer; Lung Cancer (Non-Small Cell, Small Cell); Lymphoma (Burkitt, Cutaneous T-Cell, Se2ary Syndrome, Hodgkin, Non-Hodgkin); Primary Central Nervous System (CNS); Male Breast Cancer; Mesothelioma; Metastatic Squamous Neck Cancer with Occult Primary; Midline Tract Carcinoma Involving NUT Gene; Mouth Cancer; Multiple Endocrine Neoplasia Syndromes; Multiple Myeloma/Plasma Cell

Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes;

Myelodysplastic/Myeloproliferative Neoplasms; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Oral Cancer; Oropharyngeal Cancer; Ovarian Cancer (Epithelial Tumor, Low Malignant Potential Tumor); Papillomatosis; Paraganglioma; Parathyroid Cancer; Penile Cancer; Pharyngeal Cancer; Pheochromocytoma; Pituitary Tumor; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Primary Peritoneal Cancer; Prostate Cancer; Rectal Cancer; Rhabdomyosarcoma; Salivary Gland Cancer;

Sarcoma (Uterine); Skin Cancer (Melanoma, Merkel Cell Carcinoma, Nonmelanoma); Small Intestine Cancer; Soft Tissue Sarcoma; Squamous Cell Carcinoma; Testicular Cancer; Throat Cancer; Thymoma and Thymic Carcinoma; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Unknown Primary; Urethral Cancer; Uterine Cancer, Vaginal Cancer; Vulvar Cancer; or Waldenstrom Macroglobulinemia.

[00204] Item 23. The method of any one of items 1-12, wherein the patient does not have RARoc translocated acute myeloid leukemia. [00205] Item 24. The method of any one of items 1-23, wherein the

RARoc agonist is not all-trans retinoic acid.

[00206] Item 25. A method of suppressing a Thl 7 response in a patient comprising administering an RARoc agonist.

[00207] Item 26. The method of item 25, wherein the patient has an autoimmune disease.

[00208] Item 27. The method of any one of items 25-26, wherein the

Thl 7 cells with an IFNg+ and/ or IL17+ signature are suppressed.

[00209] Item 28. The method of any one of items 25-27, wherein the

RARoc agonist is chosen from a. ATRA

b. AM580

c. AM80 (tamibarotene)

d. BMS753

e. BD4

f. AC-93253

g. AR7

h. compound of the following formula, or a pharmaceutically acceptable salt thereof:

wherein:— R ¹ is independentiy -X, -R ^x , -0-R ^x , -0-R ^A , -0-R ^c , -0-L-R ^c , -O- R ^AR , or— 0-L-R ^AR ;— R ² is independently -X, -R ^x , -0-R ^x , -0-R ^A , -0-R ^c , -O- L-R ^C — O— R ^AR , or— 0-L-R ^AR ;— R ³ is independently -X, -R ^x , -0-R ^x , -0-R ^A , - O-R ^C __0-L-R ^c ,— O— R ^AR , or— 0-L-R ^AR ; with the proviso that— R ¹ ,— R ² , and— R ³ are not all— O— R ^A ; wherein: each—X is independently— F, --C1,—Br, or—I; each— R ^A is saturated aliphatic 0-6alkyl; each— R ^x is saturated aliphatic O-ehaloalkyl; each— R ^c is saturated C3- ₇ cycloalkyl; each— R ^AR is phenyl or Cs-eheteroaryl; each -L- is saturated aliphatic G-3alkylene; and wherein: -J- is— C(=0)~NR ^N — ;— R ^N is independently— H or— H or— R ^NN ;— R ^NN is saturated aliphatic Ci-4alkyl; =Y— is =CR ^Y - and -Z = is -CR ^Z =;— R ^Y is -H; -R ^z is independentiy -H or -R ^zz ; -R ^zz is independently— F,—CI,—Br,—I,—OH, saturated aliphatic G-4alkoxy, saturated aliphatic Ci-4alkyl, or saturated aliphatic Ci-4haloalkyl; =W— is =CR ^W — ;— R ^w is— H;— R° is independentiy -OH,— OR ^E , -NH _¾ — NHR ^T1 ,— NR ^T1 R ^T1 or— NR ^T2 R ^T3 ;— R ^E is saturated aliphatic Ci-6alkyl; each— R ^T1 is saturated aliphatic G-6alkyl;— NR ^T2 R ^T3 is independentiy azetidino, pyrrolidino, piperidino, piperizino, N— (Ci-3alkyl) piperizino, or morpholino; with the proviso that the compound is not a compound selected from the following compounds, and salts, hydrates, and solvates thereof: 4-(3,5- dichloro-4-ethoxy-benzoylamino)-benzoic acid (PP-02); and 4-(3,5-dichloro-4- methoxy-benzoylamino) -benzoic acid (PP-03).

[00210] Item 29. The method of any one of items 25-28, wherein the

RARoc agonist is coadministered together with a T-cell suppressive agent.

[00211] Item 30. The method of any one of items 25-29, wherein the

RARoc agonist is coadministered together with abatacept, adalimumab, anakinra,

azathioprine, certolizumab, certolizumab pegoltacrolimus, corticosteroids (such as prednisone), dimethyl fumarate, etanercept, fingolimod, glatiramer acetate, golimumab, hydroxychloroquine, infliximab, leflunomide, mercaptopurine, methotrexate, mitoxantrone, natalizumab, rituximab, sulfasalazine, teriflunomide, tocilizumab, tofacitinib, vedolizumab.

[00212] Item 31. The method of any one of items 25-30, wherein the autoimmune disease is chosen from an autoimmune disease with an IFNg+IL17+ T-cell signature.

[00213] Item 32. The method of any one of items 25-31, wherein the autoimmune disease is chosen from Juvenile Idiopathic Arthritis, Rheumatoid Arthritis, Crohn's disease, and Multiple Sclerosis.

[00214] Item 33. The method of any one of items 25-32, wherein the autoimmune disease is chosen from alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/ systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, granulomatosis with polyangiitis (W egener's)

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EQUIVALENTS

[00254] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[00255] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/ -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.