WO2024253957A1 - Ciblage de lymphocytes t associés à l'auto-immunité par régulation de facteurs de transcription - Google Patents

Ciblage de lymphocytes t associés à l'auto-immunité par régulation de facteurs de transcription Download PDF

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WO2024253957A1
WO2024253957A1 PCT/US2024/031858 US2024031858W WO2024253957A1 WO 2024253957 A1 WO2024253957 A1 WO 2024253957A1 US 2024031858 W US2024031858 W US 2024031858W WO 2024253957 A1 WO2024253957 A1 WO 2024253957A1
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cells
cell
ahr
vector
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Jaehyuk Choi
Deepak Rao
Calvin LAW
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Brigham and Womens Hospital Inc
Northwestern University
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Brigham and Womens Hospital Inc
Northwestern University
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Definitions

  • SLE Systemic lupus erythematosus
  • B cell activation and autoantibody production in autoimmune diseases generally require help from T cells, which stimulate B cell activation and differentiation via CD40L and IL-21 2,10 .
  • T follicular helper (Tfh) cells are the principal T cell population that helps B cells within follicles of secondary lymphoid organs; however, chronic autoimmune diseases often involve varied B cell-helper T cells with distinct phenotypes, including T peripheral helper (Tph) cells, which couple B cell helper functions to a migratory program targeting inflamed peripheral tissues and accumulate prominently in the joints of patients with seropositive rheumatoid arthritis (RA) and in the circulation of patients with SLE 9,11 .
  • Tph T peripheral helper
  • CXCL13 a B cell chemoattractant that uniquely binds CXCR5.
  • Chemokine (C-X-C motif) ligand 13 (CXCL13) is a chemokine encoded by the CXCL13 gene.
  • CXCL13 production is critical for the accumulation and organization of B cell follicles in secondary lymphoid organs, and overexpression of CXCL13 is sufficient to induce the formation of ectopic lymphoid structures in murine models 12,13 .
  • Circulating levels of CXCL13 have been proposed as a biomarker of global germinal center activity and correlate with disease activity in both SLE and RA 14,15 .
  • CXCL13 is primarily produced by stromal cells and not by T cells; yet, in humans and primates, Tfh and Tph cells appear to be the primary source of this chemokine 14,16,17 .
  • CXCL13 is also highly expressed by T cells in the tumor microenvironment 20,21 .
  • tumor antigen-reactive CD8 + T cells have been found to express CXCL13, raising the possibility that some of these regulatory circuits may be shared with CD8 + T cells 22 ' 24 .
  • T cell functions are strongly influenced by signals derived from the environment, including cytokines, metabolites, and other small molecules, which can push T cells towards specific phenotypes and away from others. These divergent T cell fates establish axes of opposing functional outcomes, which can regulate pathogenic inflammation.
  • the disclosure herein identifies regulators that control the differentiation of CXCL13 + Tph and Tfh cells, which may place this phenotype in the context of other differentiated T cell states.
  • Tfh T follicular helper
  • Tph T peripheral helper
  • SLE systemic lupus erythematosus
  • Human Tfh and Tph cells are marked by high production of the B cell chemoattractant CXCL13, yet regulation of T cell CXCL13 production and the relationship between a CXCL13 + state and other differentiated T cell states remain largely undefined.
  • AHR aryl hydrocarbon receptor
  • Type I interferon IFN
  • SLE Type I interferon
  • compositions and methods for negatively regulating pathological T cell differentiation in autoantibody driven autoimmune disease are provided herein.
  • the negative regulation of pathological T cell differentiation can play a role in treating or mitigating symptoms of an autoimmune disease.
  • compositions and methods for preventing B cell-helper T cell function, thereby inhibiting differentiation of B cells are provided herein.
  • One embodiment is an aryl hydrocarbon receptor (AHR) agonist, wherein the AHR agonist negatively regulates pathological T cell differentiation in autoantibody driven autoimmune disease.
  • the agonist negatively regulates T follicular helper (Tfh) cell and/or T peripheral helper (Tph) cell differentiation.
  • the agonist is conjugated to a T cell targeting moiety, such as anti-T cell antibody.
  • Another embodiment is a vector comprising the AHR agonist.
  • the vector is a lentivirus.
  • Another embodiment is a composition comprising the AHR agonist.
  • Another embodiment is preventing CXCL13+ Tph/Tfh cell generation, comprising contacting a cell population with one or more AHR agonists, or a vector comprising an AHR agonist, or a composition comprising an AHR agonist and a pharmaceutically acceptable carrier, optionally where the contacting is in vivo, in vitro, or ex vivo. In other embodiments the method can be used in situ.
  • Yet another embodiment is a method of inhibiting the function of one or more autoimmunity-associated T cells or T cell populations, comprising administering one or more AHR agonist, or a vector comprising an AHR agonist, or a composition comprising an AHR agonist and a pharmaceutically acceptable carrier, to a subject in need.
  • the subject in need in need has an autoimmune disease.
  • administering is intravenous, subcutaneous, oral, or topical.
  • Yet another embodiment is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering an effective amount of one or more AHR agonists, or a vector comprising an AHR agonist, or a composition comprising an AHR agonist and a pharmaceutically acceptable carrier.
  • administering is intravenous, subcutaneous, oral, or topical.
  • Yet another embodiment is a method of inhibiting the function of one or more autoimmunity-associated T cells or T cell populations, comprising administering an effective amount of one or more AHR agonists, a vector comprising an AHR agonist, or a composition comprising an AHR agonist to a subject in need thereof or to a cell population.
  • the contacting is in vivo, in vitro, or ex vivo.
  • the method may be used in situ.
  • the subject in need has an autoimmune disease.
  • administering is intravenous, subcutaneous, oral, or topical.
  • the present disclosure provides a method of using one or more AHR agonists, or vectors or compositions including an AHR agonist, to prevent B cell differentiation in a cell population or subject in need.
  • the method comprises contacting an effective amount of the AHR agonist with a cell population in vivo, in vitro, or ex vivo, or in situ.
  • the method comprises administering an effective amount of the AHR agonist to a subject in need.
  • AHR agonist may be administered intravenously, subcutaneously, orally, or topically to a subject in need.
  • yet another embodiment is a method of treating an autoimmune disease in a subject in need thereof, comprising obtaining a population of T cells from the subject, treating the population of T cells with an AHR agonist, and administering an effective amount of the treated population of T cells to the subject.
  • the treatment with an AHR agonist reduces the frequency of PD-1+CXCR5- Tph cells in the treated T cell population.
  • administering is intravenous, subcutaneous, oral, or topical.
  • Yet another embodiment comprises treatment with an AHR agonist to stabilize JUN function in a subject in need thereof.
  • transcription factor selected from AHR, JUN, FOS, ATF3, FOSL1, and FOSL2, wherein the transcription factor negatively regulates Tfh and Tph cell function in autoantibody driven autoimmune disease, and methods of use for this purpose.
  • a vector such as a lentivirus, comprising the transcription factor.
  • Another embodiment is a composition comprising the transcription factor.
  • One embodiment is preventing CXCL13+ Tph/Tfh cell generation, comprising contacting a cell population with one or more transcription factors selected from AHR, JUN, FOS, ATF3, FOSL1, and/or FOSL2, or a vector comprising the transcription factor, or a composition comprising the transcription factor and a pharmaceutically acceptable carrier.
  • the contacting is in vivo, in vitro, or ex vivo. In other embodiments the method may be used in situ.
  • Yet another embodiment is a method of inhibiting the function of one or more autoimmunity-associated T cells or T cell populations, comprising administering an effective amount of one or more transcription factors selected from AHR, JUN, FOS, ATF3, FOSL1, and/or FOSL2, or a vector comprising the transcription factor, or a composition comprising the transcription factor and a pharmaceutically acceptable carrier to a subject in need thereof or to a cell population.
  • administering is intravenous, subcutaneous, oral, or topical.
  • Another embodiment is treating an autoimmune disease in a subject in need thereof, comprising administering an effective amount of one or more transcription factors selected from AHR, JUN, FOS, ATF3, FOSL1, and/or FOSL2, or a vector comprising the transcription factor, or a composition comprising the transcription factor and a pharmaceutically acceptable carrier, to the subject in need thereof, wherein the transcription factor causes a target gene in the subject to be overexpressed.
  • administering is intravenous, subcutaneous, oral, or topical.
  • an autoimmune disease in a subject in need thereof comprising administering an effective amount of an AHR agonist and a transcription factor selected from AHR, JUN, FOS, ATF3, FOSL1, and/or FOSL2.
  • the AHR agonist and transcription factor are administered consecutively or sequentially.
  • the AHR agonist may be in a vector or composition.
  • the transcription factor may be in a vector or composition.
  • administering is intravenous, subcutaneous, oral, or topical.
  • Yet another embodiment is a gRNA targeting a gene selected from CD3D, LCP2, ATF4, IFNAR2, or STAT2, wherein the gRNA has a sequence according to one of the sequences in Table 5.
  • These genes were selected from a larger selection of genes upregulated in Tph/Tfh cells in the current RNA-Seq dataset or in previously published RNA-Seq datasets of RA synovial T cells 16 , as well as from genes previously correlated with CXCL13 expression, or genes in the AP-1 family. 19,36 When CD3D, LCP2, ATF4, IFNAR2, or STAT2, were knocked out, CXCL13 in a cell or cell population decreased.
  • Another embodiment is a CRISPR-Cas9-gRNA ribonucleoprotein (crRNP) complex comprising at least one gRNA as described herein.
  • Another aspect is a vector or a composition comprising the crRNP complex or vector, and a pharmaceutically acceptable carrier.
  • Yet another embodiment is a method of decreasing CXCL13 production in a cell, comprising contacting the cell with the crRNP complex, vector, or composition as described herein, optionally where the contacting is in vivo, in vitro, or ex vivo.
  • the crRNP complex knocks out a target gene. In other embodiments the method may be used in situ.
  • Yet another embodiment is a method of treating an autoimmune disease in a subject in need thereof, comprising administering the crRNP complex described herein to a subject in need thereof.
  • the crRNP complex knocks down a target gene.
  • administering is intravenous, subcutaneous, oral, or topical.
  • an additional therapeutic agent is administered to the subject in need, optionally wherein the additional therapeutic agent is anifrolumab.
  • methods of treatment described herein are used to treat an autoimmune disease selected from rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, and/or systemic sclerosis.
  • RA rheumatoid Arthritis
  • SLE Systemic Lupus Erythematosus
  • cutaneous lupus cutaneous lupus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, and/or systemic sclerosis.
  • ANCA anti-neutrophil cytoplasmic antibody
  • the autoimmune disease is RA or SLE.
  • Figures la-j Imbalanced CXCL13 + Tph and Tfh cells versus IL-22 + CD96hi cells in SLE patients.
  • Fig la Differentially expressed proteins on gated memory CD4 + T cells from systemic lupus erythematosus patients compared to controls, p-values from t-test with Bonferroni correction.
  • Fig. lb UMAP feature plots showing expression of indicated proteins on memory CD4 + T cells.
  • Fig. 1c UMAP showing Cell Neighborhood Analysis (CNA) results (red, enriched in SLE; blue, enriched in controls).
  • Fig. Id UMAP showing CD4 + T cell assigned to clusters.
  • Th22 score compared to CD96 hl T cells, p-values from left to right: 6.24e-8, 9.53e-3 , 1.1 le-7, 4.75e-3.
  • IL22 relative expression p-values from left to right: 0.0063, 0.0075.
  • CXCL13 relative expression p-values from left to right: 0.0128, 0.0012, 0.0283.
  • Data for Figs, le, Ih, and li are shown as mean ⁇ DS, and min/max/median for Fig. Ij.
  • p-values (NS ⁇ 0.05, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001) were obtained by Mann-Whitney test in e, by Friedman test with Dunn multiple comparisons test in Fig. Ih, by ratio paired T-test in Fig- li, or by Wilcoxon test in j.
  • FIGs 2a-2n AHR controls a CXCL13-IL-22 differentiation axis in human T cells.
  • Fig. 2a Schematic of arrayed CRISPR-deletion screen to detect regulators of CXCL13 production.
  • Fig. 2b CXCL13 levels by ELISA in supernatants of cells from CRISPR screen, with screen results of 2 independent experiments using different donors on x- and y-axes.
  • DMSO normalized to control condition
  • Fig. 2f UMAPs of scRNA- seq data of memory CD4 + T cells stimulated under indicated conditions for 2 weeks.
  • Fig. 2g UMAP of scRNA-seq data with cells coloured by enrichment of Tph gene signature.
  • Fig. 2h UMAP of rheumatoid arthritis (RA) synovial CD4 + T cell clusters (left) and feature plot of expression of CXCL13 (right).
  • Fig. 2i UMAP of in vitro cultured memory CD4 + T cells from Fig.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor CH-223191.
  • FIG. 3a-31 JUN coordinates with AHR to divert T cells away from CXCL13 and towards IL-22.
  • Fig. 3a Time course RNA-Seq analysis with maSigPro cluster 6 identified as AHR response gene set by pathway enrichment.
  • Fig. 3b Transcription factor motif and common co-occurrence enrichment analysis.
  • Fig. 3c AHR CUT&RUN peak heatmap, signal profile and motif analysis.
  • Fig. 3d Representative AHR binding regions.
  • Fig. 3e Comparison of AHR binding in cells treated with AHR agonist or AHR antagonist.
  • Fig. 3f Pathway enrichment analysis of AHR-bound peak associated genes.
  • Figs. 3g, 3h CXCL13(Fig.
  • Fig. 3g Illustration of JUN-targeting sgRNAs used in CRISPR screens and validation experiments (top), and Western blot detection of JUN protein expression in T cells nucleofected with control (sgCTRL) or JUN-targeting guide (JUN-sg5, bottom).
  • Fig. 3j Representative flow cytometry plots of CXCL13 detection by intracellular flow cytometry in memory CD4+ T cells nucleofected with control (upper left) or JUN-sg5 (lower left).
  • TCDD AHR agonist
  • AHRinh AHR inhibitor CH-223191.
  • FIG. 4a JUN shares binding regions with AHR as coregulator of AHR transcription program.
  • Fig. 4a Top 10 HOMER motifs (left) from called JUN CUT&RUN peaks in TCDD-treated memory CD4 + T cells with pathway enrichment analysis (middle and right).
  • Fig. 4b Venn diagram of CUT&RUN peaks bound by AHR (left) and JUN (right).
  • Fig. 4c Venn diagram of JUN and AHR co-bound CUT&RUN peak-associated genes that overlap with AHR-induced genes identified by Time-course RNA-Seq in Fig. 4a.
  • Fig. 4d Representative JUN binding peaks in relation to AHR peaks in memory CD4 + T cells treated with TCDD.
  • FIG. 4e Overall JUN binding signal at AHR peaks in cells treated with AHR agonist (TCDD, green) or inhibitor (AHRinh, purple) and representative JUN binding peaks at CXCL13 (middle) and 1L22 (right) in memory CD4 + T cells treated with AHRinh.
  • Fig. 4f Analysis of JUN binding peaks lost with AHR inhibitor. Venn diagram of JUN bound CUT&RUN peaks in memory CD4 + T cells treated with AHRinh and TCDD (top) and heatmap of total JUN-bound regions in the same conditions (bottom).
  • Fig. 4g Example Western blot of JUN and phospho- JUN (JUN-pS73) in memory CD4 + T cells stimulated as indicated.
  • FIG. 4j Representative JUN binding peaks from CUT&RUN of AHRinh treated memory CD4+ T cells either expressing the control vector (Empty) or JUN overexpression vector (JUN OE) at the CXCL13 and IL22 gene loci.
  • Fig. 4k KEGG pathway (top) and Elsevier Pathway (bottom) enrichment analysis of genes associated with upregulated peaks (by Diffbind) in JUN OE versus empty vector control.
  • Fig. 41 GSEA analysis of Th22 (red) and Tph (teal) gene signature enrichment in JUN-overexpressing T cells and empty vector control when treated with TCDD (top) or AHRinh (bottom). Data in Fig.4h are shown as mean ⁇ S.D.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor, CH-223191.
  • Figure 5a-5m Increased IFN in systemic lupus erythematosus patients promotes Tph cell differentiation and inhibits AHR.
  • Fig. 5b Serum CXCL13 levels in systemic lupus erythematosus patients treated longitudinally with anti-IFNAR antibody (anifrolumab) or placebo, stratified by IFN signature level, IFN High (left) and IFN Low (right). Sample size for each cohort indicated in figure.
  • FIG. 5c Schematic of longitudinal Lupus patient derived scRNA-seq data.
  • Fig. 5d UMAP clustering of memory CD4 + T cells from Lupus patients pre-anifrolumab treatment (left), and feature plot highlighting clusters enriched in indicated gene signatures (right).
  • Fig. 5e Violin plots comparing Tph (left) and CD96 hl (right) signature enrichments in respective clusters found in d, gene signatures were taken from gene sets used in Fig. 1g. p ⁇ 2.2e-16 for Tph and p ⁇ 2.2e- 16 for CD96 hl .
  • ELISA for CXCL13 (left) and IL-22 (right) from memory CD4+ T cells stimulated with or without IFN- a for 24 hours prior to addition of AHR agonist/inhibitor or DMSO control as indicated (n 6). From left to right, p-value for CXCL13: 8.5e-5, 4.05e-4, 2.9e-5, and for IL-22: 2.35e- 3, 8.76e-4, 0.0338.
  • Fig. 5k Venn diagram of overlapped DAR regions in IFN- P treated CD4+ T cells (left) or Control (right) with SF PD-l hl and AHRinh treated CD4+ T cells.
  • Fig. 51 Accessible regions near the CXCL13 gene loci in CD4+ T cells treated with IFN-P or control for 72h.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor, CH-223191.
  • FIG. 6a-6k IFN opposes IL-2 and JUN to promote CXCL13+ Tph cells.
  • Fig. 6a CXCL13 (left) and IL-22 (right) expression levels measured by ELISA in supernatants of cells from regulators of IFN induced CXCL13 CRISPR arrayed screen, with screen results of 2 independent experiments using different donors on x- and y-axes.
  • Fig. 6k Graphical representation of proposed model in type I IFN regulation of IL-2, AHR and JUN to promote Tph cells in systemic lupus erythematosus. Data in Figs. 6c, 6d, 6e and 6g shown was mean ⁇ S.E.M. p- value (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001) was obtained by linear mixed model with random effect for patients applied in Fig. 6b, by 2-way ANOVA in Fig. 6c, and by ratio paired T-test in Figs. 6d, 6e, 6f, 6g and 6j.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor, CH-223191.
  • Figures 7a-7e Clinical associations of PD-1 + /ICOS + and CD96 111 cell clusters.
  • Fig. 7a Heatmap of marker expression on mass cytometry cell clusters (left) and MASC association statistics for each cluster comparing systemic lupus erythematosus vs controls (right).
  • SLE OR odds ratio of representation in systemic lupus erythematosus vs control.
  • CI confidence interval.
  • Fig. 7d Association of indicated cluster proportions with prednisone dose or equivalent at time of sample collection.
  • Fig. 7e Cluster proportions of PD-1 + /ICOS + (left) and CD96 hl (right) clusters in SLE patients stratified by immunosuppressant drug use at time of sample collection. Spearman correlation statistics shown in Figs. 7b-7d. Data in Fig. 7e shown as median ⁇ interquartile, and statistically tested with Mann-Whitney test.
  • FIG. 8a Example of flow cytometry sorting of CD4 + T cell subsets for bulk RNA-seq analysis.
  • Fig. 8c Multiset Venn diagram of the number of differentially expressed genes between CD96 hl cells and indicated CD4 + T cell subsets. Fig.
  • FIG. 9a-91 AHR controls T cell production of CXCL13.
  • Fig. 9b Representative Western blot for AHR protein expression in cells nucleofected with sgAHR and sgCD8a control.
  • 9d, 9e, 9f, 9g, 9j, and 91 are shown as mean ⁇ S.D.
  • p-values (NS>0.05, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001) are obtained by ratio paired t-test for Figs. 9a, 9d, 9e, 9f, 9g, 9h, 9j, 91 or Wilcoxon test in Figs. 9c and 9k.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor, CH-223191.
  • Figures lOa-lOd Effects of chronic AHR modulation in CD4+ T cells.
  • Fig. 10a Figures lOa-lOd: Effects of chronic AHR modulation in CD4+ T cells.
  • ELISA data for indicated cytokines in supernatants of memory (top) and naive (bottom) CD4 + T cells re-stimulated each week for 3 weeks, normalized to DMSO 1 week result for each donor (n 3-4 donors).
  • Fig. 10b CXCL13 expression (by fragments per kilobase of transcript per million mapped reads, FPKM) in bulk RNA-seq samples of cell stimulated under indicated conditions.
  • Fig. 10c GSEA enrichment plots of Tph gene signature in naive or memory CD4 + T cells stimulated with TGF-P plus either AHR agonist TCDD or inhibitor (AHRinh) CH-223191.
  • Fig. lOd GSEA enrichment plots for Tph gene signature in T cells stimulated with or without TGF-P, under indicated conditions of AHR agonist TCDD, AHR inhibitor (AHRinh) CH223191, or DMSO control.
  • Figures lla-lle Effects of AHR and TGF-p on CD4+ T-cell subsets.
  • ELISA measurement for IL-22 in supernatants of indicated CD4 + T cell subsets stimulated under indicated conditions in the presence (orange) or absence (teal) of TGF-P (n 10).
  • statistical comparisons shown compare AHR agonist/inhibitor to DMSO within presence or absence of TGF-P, and TGF-P versus no TGF-P within each treatment, p-values from left to right in each subset is as follows, Naive: 0.0123, CD96hi: 0.0188, 7.24e-4, 1.96e-3, Thl7; 4.31e-4, 0.028, 3.17e-3, 0.0188, Thl : 0.002.
  • p-value from left to right in each subset is as follows, Naive: 0.0188, 8.98e-3, 0.0264, 3.57e-3, Tph: 2.73e-3, 5.21e-3, 5.57e-3, 0.0104, 0.0109, 0.0293, Tfh: 2.20e-4, 1.54e-3, 3.34e-4, 8.89e-4, 5.49e-5, 1.31e-3, CD96 hi : 5.17e-3, 6.66e-3, 4.52e-4, 0.0154, 2.10e-3, 3.14e-7, 4.39e-3.
  • p- value from left to right in each subset is as follows, Naive: 0.0112, Tph: 0.0312, 0.0205, 6.75e-3, 5.57e-3, Tfh: 0.0315, CD96 hi : 0.0463, 5.93e-3, 0.0296, 0.0224.
  • TIGIT p-value from left to right in each subset is as follows, Naive: 0.0160, 0.0203, Tph: 2.41e-3, 0.0115, 0.0165, 1.69e-3, Tfh: 3.26e-3, 0.0238, 0.0321, 4.35e-3, 8.51e-3, 6.84e-3, CD96 hi : 0.0105, 0.0157.
  • p-value from left to right in each subset is as follows, Naive: 9.17e-3, 4.63e-3, 1.04e-3, 2.33e-3, 8.55e-4, 0.0331, Tph: 0.0133, 0.0119, 3.49e-3, 0.0104, Tfh: 8.49e- 4, 5.38e-3, 3.73e-4, 1.03e-3, 1.75e-3, 0.0292, 6.66e-3, CD96 hi : 9.75e-3, 0.0485, 0.0129, 0.0197, 2.94e-3, 2.07e-3, 0.0328. Data shown as min/max/median for Figs. Ila and lid, and as mean ⁇ S.D for Fig. 11c and lie.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor, CH-223191.
  • Figures 12a-12f ATAC-seq analysis of Tph cells, Tfh cells, and AHR inhibitor- treated cells.
  • Figs. 12a, 12b Example flow cytometry cell sorting of CD4 + T cell populations from RA synovial fluid (Fig. 12a) or tonsil (Fig. 12b) mononuclear cells.
  • Fig. 12c PCA plot of ATAC-seq data from CD4 T cell populations sorted from RA synovial fluid or from tonsil based on PD-1 expression level.
  • Fig. 12a, 12b Example flow cytometry cell sorting of CD4 + T cell populations from RA synovial fluid (Fig. 12a) or tonsil (Fig. 12b) mononuclear cells.
  • Fig. 12c PCA plot of ATAC-seq data from CD4 T cell populations sorted from RA synovial fluid or from tonsil based on PD-1 expression level.
  • Fig. 12a, 12b Example flow cytometry cell sorting of CD4
  • FIG. 12d PCA plot of ATAC-seq data from blood CD4 + T cells of healthy donors cultured with DMSO, AHR agonist TCDD or AHR inhibitor (AHRinh) CH-223191 in the presence of TGF-p.
  • Fig. 12e GSEA plots of annotated genes of DARs from synovial fluid Tph cells (top) and tonsil Tfh cells (bottom) in CD4 + T cells treated with AHRinh versus TCDD in presence of TGF-P for 1 week.
  • Fig. 12f Differentially accessible regions (red square) near the CXCL13 gene locus from ATAC-seq of each indicated cell type/culture condition.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor CH-223191.
  • Figure 13 Detection of PD-1 + Tph cells in systemic lupus erythematosus peripheral blood mononuclear cells (PBMC). Gating strategy for flow cytometry detection of PD-1 + CXCR5' Tph cells and CD96 hl cells in PBMC from SLE patient after treatment with AHR inhibitor (AHRinh) CH-223191.
  • Figures 14a-14h Transcriptomic and epigenetic evaluation of AHR activation in T cells and association with AP-1 family members.
  • Fig. 14a Schematic of RNA-Seq time course experiment to identify early transcriptomic events of AHR modulation.
  • Fig. 14a Schematic of RNA-Seq time course experiment to identify early transcriptomic events of AHR modulation.
  • FIG. 14b PCA plots of RNA-seq samples after 12 hours (left) and 48 hours (right) of stimulation with TGF- P and either AHR agonist (TCDD) or AHR inhibitor (AHRinh) CH-223191.
  • Figs. 14c, 14d Volcano plots of DESeq2 results from RNA-Seq analysis of memory CD4 + T cells cultured for 12 hours (Fig. 14c) and 48 hours (Fig. 14d) in TGF-P and either TCDD or AHRinh.
  • the samples used for DESeq2 analysis correspond with the PCA plots in Figs. 14b.
  • Fig. 14e Pathway enrichment analysis of genes upregulated in TCDD-treated CD4 + T cells at 48 and 72 hours of culture, based on Elsevier pathway collection.
  • Fig. 14f Transcription factor enrichment analysis using EnrichR databases TRRUST Transcription factors 2019 (left) and EnrichR Transcription factor Co-occurrence (right).
  • Fig. 14g Volcano plot of AHR CUT&RUN Diffbind analysis comparing samples with and without AHR CRISPR knockout (left) and HOMER motif analysis of all upregulated peaks found in AHR WT samples (right).
  • Fig. 14h Venn diagram of overlapped genes bound by AHR with Th22 signature genes as shown in Fig. 1g, hypergeometric P-value is shown.
  • TCDD AHR agonist
  • AHRinh AHR inhibitor, CH-223191.
  • Figures 15a-15f Overexpression of JUN in human CD4+ T cells.
  • Fig. 15a, 15b Verification of AHR and JUN as interactors. Immunoblot (WB) analysis of HA (Fig. 15a) or Flag (Fig. 15b) immunoprecipitates from the indicated cell lysates probed with the indicated antibodies.
  • Fig. 15c Cytoplasmic or nuclear extracts (as indicated on bottom) from HEK293T cells stably expressing HA-AHR or vector control treated with AHRinh, TCDD or vehicle control (DMSO) were immunoblotted for AHR, JUN and respective controls (P- tubulin for cytoplasmic extract, Histone H3 for nuclear extract).
  • Fig. 15a, 15b Verification of AHR and JUN as interactors. Immunoblot (WB) analysis of HA (Fig. 15a) or Flag (Fig. 15b) immunoprecipitates from the indicated cell lysates probed with the indicated antibodies.
  • FIG. 15d JUN expression by Western blot in T cells transduced with JUN overexpression construct or control vector.
  • Fig. 15e Example of flow cytometry sorting to obtain JUN-overexpressing cells based on GFP positivity.
  • Fig. 15f JUN overexpression (JUN OE) CUT&RUN assessment by peak density on total JUN bound peaks compared to vector control as in Fig. 4f and top HOMER motifs for each respective condition.
  • Figs. 16a-16c Increased IFN in systemic lupus erythematosus patients inhibit AHR signaling.
  • Fig. 16a-16c Increased IFN in systemic lupus erythematosus patients inhibit AHR signaling.
  • Fig. 16a IFN signature score in RNA-seq data of CD4 + T cell subsets from systemic lupus erythematosus and control patients as in Fig. 1g.
  • Fig. 16b Elsevier pathway enrichment from annotated genes of DAR in No IFN-P control treated CD4 + T cells.
  • TCDD AHR agonist.
  • the term “about” placed before a specific numeric value may mean ⁇ 20% of the numeric value; ⁇ 18% of the numeric value, ⁇ 15% of the numeric value; ⁇ 12% of the numeric value; ⁇ 8% of the numeric value; ⁇ 5% of the numeric value; ⁇ 3% of the numeric value; ⁇ 2% of the numeric value; ⁇ 1% of the numeric value or ⁇ 0.5% of the numeric value.
  • compositions and methods are intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others.
  • Consisting essentially of when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
  • a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
  • ORF open reading frame
  • the term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins.
  • expression refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the term “overexpress” intends a level of expression of the mRNA, the protein or the polypeptide” that is greater than or exceeds the level of expression of the mRNA, the protein or the polypeptide in a native, wild-type or cell that has not been engineered to increase expression.
  • a “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
  • the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide or a fragment thereof, and optionally translated to produce the polypeptide or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • an amino acid sequence coding sequence refers to a nucleotide sequence encoding the amino acid sequence.
  • a regulatory sequence intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide.
  • a promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. Polymerase II and III are examples of promoters.
  • a regulatory sequence is bidirectional, i.e., acting as a regulatory sequence for the coding sequences on both sides of the regulatory sequence.
  • Such bidirectional regulatory sequence may comprise, or consists essentially of, or consists of a bidirectional promoter (see for example Trinklein ND, et al. (2004) An abundance of bidirectional promoters in the human genome. Genome Res. Jan;14(l):62-6).
  • the term “protein,” “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • antibody collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins.
  • the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10 3 M' 1 greater, at least 10 4 M' 1 greater or at least 10 5 M' 1 greater than a binding constant for other molecules in a biological sample).
  • the term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies).
  • T cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T- cells may either be isolated or obtained from a commercially available source. “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gammadelta T cells.
  • CD4+ cells T-helper cells
  • CD8+ cells cytotoxic T-cells
  • Reg T-regulatory cells
  • gammadelta T cells gammadelta T cells.
  • sample refers to clinical samples obtained from a subject.
  • a sample is obtained from a biological source (i.e., a “biological sample”), such as tissue, bodily fluid, or microorganisms collected from a subject.
  • Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.
  • stimulation refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory molecule e.g., a TCR/CD3 complex
  • Stimulation can mediate altered expression of certain molecules, such as downregulation of TGFP, and/or reorganization of cytoskeletal structures, and the like.
  • TGF-P Transforming growth factor beta
  • a “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
  • therapeutically effective amount refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.
  • the therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. As used in the methods provided herein, the terms “therapeutically effective amount” and “effective amount” are used interchangeably.
  • Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • Treatment of cancer or an infection, immune disorder, or autoimmune response, disorder or disease can be at any time during the cancer or an infection, immune disorder, or autoimmune response, disorder or disease.
  • Certain embodiments of the present disclosure can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the methods described herein as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by cancer or an infection, immune disorder, or autoimmune response, disorder or disease, or an adverse symptom, condition or complication associated with or caused by cancer or an infection, immune disorder, or autoimmune response, disorder or disease.
  • a method can be practiced one or more times (e.g., 1-10, 1-5 or 1-3 times) an hour, day, week, month, or year.
  • times e.g., 1-10, 1-5 or 1-3 times
  • a non-limiting dosage schedule is 1-7 times per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.
  • contacting means direct or indirect binding or interaction between two or more.
  • a particular example of direct interaction is binding.
  • a particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity.
  • Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096): 816-21 (2012)).
  • compositions for use in genome editing using the CRISPR/Cas systems are described in detail in US Pub. No. 2016/0340661, US Pub. No. 20160340662, US Pub. No. 2016/0354487, US Pub. No. 2016/0355796, US Pub. No. 20160355797, and WO 2014/018423, which are specifically incorporated by reference herein in their entireties.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer”, “guide RNA” or “gRNA” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a “spacer”, “guide
  • One or more tracr mate sequences operably linked to a guide sequence can also be referred to as “pre-crRNA” (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.
  • pre-crRNA pre-CRISPR RNA
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. While the specifics can be varied in different engineered CRISPR systems, the overall methodology is similar.
  • a practitioner interested in using CRISPR technology to target a DNA sequence can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid.
  • the sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells.
  • Such vectors are commercially available (see, for example, Addgene). Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in transfected cells results in a single or double strand break (depending of the activity of the Cas enzyme) at the desired target site.
  • C-X-C motif chemokine 13 is a cytokine that can act as a B cell chemoattractant.
  • the human CXCL13 has an amino acid sequence according to Uniprot 043927.
  • the CXCL13 gene has an mRNA sequence according to NCBI NM_001371558.1.
  • Described herein are a number of transcription factors which can play a role in T cell differentiation, such as through enhancement or suppression of CXCL13.
  • Aryl hydrocarbon receptor is a transcription factor encoded by the AHR gene.
  • the human AHR protein has an amino acid sequence according to Uniprot P35869.
  • the AHR gene has an mRNA sequence according to NCBI NM_001621.5.
  • Transcription factor JUN is a transcription factor that is a part of the AP-1 transcription factor family.
  • the human JUN protein has an amino acid sequence according to Uniprot P05412.
  • the JUN gene has an mRNA sequence according to NCBI NM_002228.4.
  • Protein c-FOS is a nuclear phosphoprotein that forms a complex with the JUN/AP-1 transcription factor.
  • FOS is a transcription factor part of the AP-1 transcription factor family.
  • the human FOS protein has an amino acid sequence according to Uniprot P01100.
  • the FOS gene has an mRNA sequence according to NCBI NM_005252.4.
  • Cyclic AMP-dependent transcription factor ATF3 (ATF3) is a transcription factor that binds the cAMP response element. It is a part of the AP-1 transcription factor family.
  • the human ATF3 protein has an amino acid sequence according to Uniprot Pl 8847.
  • the ATF3 gene has an mRNA sequence according to NCBI NM_001030287.4.
  • Fos-related antigen 1 is a transcription factor that is a part of the AP-1 transcription factor family.
  • the human FOSL1 protein has an amino acid sequence according to Uniprot Pl 5407.
  • the FOSL1 gene has an mRNA sequence according to NCBI NM_001300844.2.
  • Fos-related antigen 2 is a transcription factor that is a part of the AP-1 transcription factor family.
  • the human FOSL2 protein has an amino acid sequence according to Uniprot Pl 5408.
  • the FOSL2 gene has an mRNA sequence according to NCBI NM_005253.4.
  • BATF Basic leucine zipper transcriptional factor ATF-like
  • the human BATF protein has an amino acid sequence according to Uniprot Q16520.
  • the BATF gene has an mRNA sequence according to NCBI NM_006399.5.
  • E3 ubiquitin-protein ligase is a transcription factor that has an amino acid sequence according to Uniprot Q13191.
  • the CBLB ene has an mRNA sequence according to NCBI NM_001321786.1.
  • STAT5B Signal transducer and activator of transcription 5B
  • STAT5B is a transcription factor that has an amino acid sequence according to Uniprot P51692.
  • the STAT5B gene has an mRNA sequence according to NCBI NM_012448.4.
  • STAT5A Signal transducer and activator of transcription 5 A
  • STAT5A is a transcription factor that has an amino acid sequence according to Uniprot P42229.
  • the STAT5A gene has an mRNA sequence according to NCBI NM_001288718.2.
  • STAT1 Signal transducer and activator of transcription 1-alpha/beta
  • the STAT1 gene has an mRNA sequence according to NCBI NM_001384880.1.
  • Tyrosine-protein kinase (JAK1) is a transcription factor that has an amino acid sequence according to Uniprot P23458.
  • the JAK1 gene has an mRNA sequence according to NCBI NM_001320923.2.
  • JUNB Transcription factor JunB
  • the JUNB gene has an mRNA sequence according to NCBI NM_002229.3.
  • Mineralocorticoid receptor is a transcription factor that can have an amino acid sequence according to Uniprot P08235.
  • the NR3C2 gene has an mRNA sequence according to NCBI NM_000901.5.
  • T-cell surface glycoprotein CD3 delta chain is a transcription factor that has an amino acid sequence according to Uniprot P04234.
  • the CD3D gene has an mRNA sequence according to NCBI NM_000732.6.
  • Lymphocyte cytosolic protein 2 is a transcription factor that has an amino acid sequence according to Uniprot Q13094.
  • the LCP2 gene has an mRNA sequence according to NCBI NM_005565.5.
  • Cyclic AMP-dependent transcription factor ATF-4 (ATF4) is a transcription factor that has an amino acid sequence according to Uniprot Pl 8848.
  • the ATF4 gene has an mRNA sequence according to NCBI NM_001675.4.
  • Interferon alpha/beta receptor 2 is a transcription factor that has an amino acid sequence according to Uniprot P48551.
  • the IFNAR2 gene has an mRNA sequence according to NCBI NM_000874.5.
  • STAT2 Signal transducer and activator of transcription 2
  • STAT2 is a transcription factor that has an amino acid sequence according to Uniprot P52630.
  • the STAT2 gene has an mRNA sequence according to NCBI NM_001385110.1.
  • the present disclosure provides regulators that control the differentiation of CXCL13 + Tph (T peripheral helper) and Tfh (T follicular helper) cells, which may place this phenotype in the context of other differentiated T cell states.
  • the present disclosure provides the ligand gated receptor aryl hydrocarbon receptor (AHR) as a central regulator of an unexpected axis (“T cell differentiation axis”) of T cell differentiation with CXCL13 + and IL-22 + states at opposing ends of this polarization (see Fig. 6k) Manipulation of AHR can regulate the CXCL13 + and IL-22 + T cell states.
  • AHR ligand gated receptor aryl hydrocarbon receptor
  • This T cell differentiation axis can be markedly skewed in patients with systemic lupus erythematosus (see e.g. Fig. li), in part due to actions of type I interferon, which opposes AHR and JUN to drive T cells towards autoimmunity-associated CXCL13 + Tph and Tfh cell states.
  • AHR can reduce the B cell-helper function of Tfh cells by regulating B cell chemoattractant CXCL13.
  • AHR aryl hydrocarbon receptor
  • AHR agonists can include FICZ, TCDD, and chemokines including IL-2.
  • the present disclosure identifies the aryl hydrocarbon receptor as a potent negative regulator of Tfh/Tph cell differentiation, and demonstrates that at least two AHR agonists (FICZ, TCDD) inhibit T cell ability to acquire Tph/Tfh cell features including inhibiting production of the B cell chemoattractant CXCL13 (chemokine (C-X-C) motif ligand 13) (see e.g. Fig. 2d, Figs. 9f and 9i).
  • FICZ (6-formylindolo(3,2-b)carbazole) is commercially available, for example from InvivoGen.com.
  • TCDD (2,3,7,8-tetrachlorodibenzodioxin, also referred to as 2, 3,7,8- Tetrachlorodibenzo-/?-dioxin) is also commercially available, for example at caymanchem .com .
  • AHR Agonist compositions and methods
  • the present disclosure provides an AHR agonist for treatment of autoimmune disease.
  • the present disclosure provides a T cell-directed AHR agonist, optionally capable of more specific targeting of AHR to T cells (e.g. anti-CD5 or other anti-T cell antibody conjugated to AHR agonist).
  • the AHR agonist is FICZ, TCDD, and/or chemokines including IL-2.
  • the AHR agonist is selected from TCDD, FICZ, ITE, tapinarof, TEACOP270, kynurenic acid, indole[3,2-b]carbazole, l,3-di(lH-indol-3-yl) propan-2-one, and 1 -( lH-indol-3 -y l)-3 -(3H-indol-3 -ylidene)propan-2-one.
  • the present disclosure provides a vector including the AHR agonist.
  • the vector may be comprised of, or derived from, a plasmid, an adenovirus, and adeno-associated virus (AAV), a retrovirus, a herpes simplex virus, a human immunodeficiency virus (HIV), a lentivirus, or a synthetic vector.
  • AAV adeno-associated virus
  • the AHR agonist or vector are part of a composition.
  • the composition also includes a carrier, which may be a pharmaceutically acceptable carrier.
  • AHR activation strongly drives T cells away from a CXCL13 + phenotype and towards an IL-22 + phenotype (see e.g. Fig. 2b, which shows deletion of the AHR gene upregulated CXCL13 production in Memory CD4+ T cells).
  • the present disclosure provides a method of preventing CXCL13+ Tph/Tfh cell generation or differentiation.
  • the method comprises, consists essentially of, or consists of contacting a cell or cell population (i.e. a T cell population) with one or more AHR agonists, or vectors or compositions including the AHR agonist, thereby preventing CXCL13+ Tph/Tfh cell generation or differentiation.
  • the T cells are CD4+ or CD8+ cells.
  • the T cells may be a CD4+ subset selected from Thl, Thl7, Tfh, and/or Tph cells.
  • the T cells may be a CD8+ subset selected from TCM, TEM, and/or TEMRA.CCIIS.
  • the T cell populations may be antigen specific.
  • an AHR agonist, or vector or composition including the AHR agonist is contacted with the T-cell populations. Contacting may occur in vitro, in vivo, or ex vivo. In other embodiments these methods may be used in situ, for example via delivery through lipid nanoparticles, retroviruses, lentiviruses, or other agents to specifically target T cell subsets.
  • In situ routes of administration or contacting may be systemic or targeted.
  • the in situ administration or contacting is intravenous, subcutaneous, or topical.
  • the T-cell populations are reprogrammed towards an IL-22+ phenotype and/or away from a B-cell helper phenotype.
  • the AHR agonist may be used in combination with inhibition of proteins critical for T-cell receptor (TCR) signaling such as the CD3 protein complex or LCP2.
  • TCR T-cell receptor
  • LCP2 proteins critical for T-cell receptor
  • the present disclosure also provides methods of using AHR agonists to inhibit functions of autoimmunity-associated T cell populations — including T follicular helper (Tfh) cells and T peripheral helper (Tph) cells — in a subject in need or a cell population.
  • the method comprises, consists essentially of, or consists of contacting a cell or cell population (i.e. a T cell population) with one or more AHR agonists or vectors or compositions including the AHR agonist, thereby inhibiting functions of autoimmunity-associated T cell populations.
  • the method comprises, consists essentially of, or consists of administering one or more AHR agonists or vectors or compositions including an AHR agonist to a subject in need, thereby inhibiting functions of autoimmunity-associated T cell populations.
  • the contacting is in vivo, in vitro, or ex vivo. In other embodiments the method may be used in situ.
  • the subject in need has an autoimmune disease.
  • administering is intravenous, subcutaneous, oral, or topical.
  • the function is Tph/Tfh differentiation or B cell recruitment (see e.g. Figs. 2d and 2n, Figs. 9f and 9i).
  • the present disclosure provides a method directed to preventing the expansion of pathological T cells found in autoimmune diseases using AHR agonists.
  • the method comprises, consists essentially of, or consists of administering one or more AHR agonists or vectors or compositions including an AHR agonist to a subject in need, thereby preventing the expansion of pathological T cells.
  • administering is intravenous, subcutaneous, oral, or topical.
  • T cells with the capacity to help B cells, Tfh cells and Tph cells are highly expanded in patients with autoantibody-associated autoimmune diseases such as lupus and rheumatoid arthritis.
  • the AHR agonist can target key biological markers specific to the pathological T cells, and inhibiting either function or fit of these cells (see e.g. Fig. 8).
  • the present disclosure provides a method of using one or more AHR agonists, or vectors or compositions including an AHR agonist, to prevent B cell differentiation in a cell or cell population or subject in need.
  • the method comprises, consists essentially of, or consists of contacting the cell or cell population with an AHR agonist, thereby preventing B cell differentiation. Contacting may occur in vivo, in vitro, or ex vivo, or in situ.
  • the method comprises, consists essentially of, or consists of administering the AHR agonist to a subject in need, thereby preventing B cell differentiation.
  • the AHR agonist may be administered intravenously, subcutaneously, orally, or topically.
  • the B cell-helper function of T cells (Tfh cells) treated with an AHR agonist is reduced, preventing the induction of B cell differentiation into plasmablasts (Fig. 2n).
  • the present disclosure provides a method of treating an autoimmune disease using AHR agonists.
  • the method comprises, consists essentially of, or consists of obtaining a population of T cells from the subject, treating the population of T cells with an AHR agonist, or vector or composition including an AHR agonist, and administering the treated population of T cells to the subject, thereby treating the autoimmune disease.
  • the T cells populations are CD4+ or CD8+ cells.
  • the T cells may be a CD4+ subset selected from Thl, Thl7, Tfh, and/or Tph cells.
  • the T cells may be a CD8+ subset selected from TCM, TEM, and/or TEMRA.CCIIS.
  • the T cell populations may be antigen specific.
  • the T-cell populations may be reprogrammed towards an IL-22+ phenotype and/or away from a B-cell helper phenotype.
  • treatment of a cell population with an AHR agonist reduces the frequency of PD-1+ CXCR5- Tph cells in the treated T cell population (Fig. 2m.)
  • treatment of a cell population with an AHR agonist can stabilize the transcription factor JUN function.
  • AHR supports expression of AP-1 family member JUN, which directly suppresses CXCL13 and supports IL-22 expression.
  • AHR supports expression of other AP-1 family members including FOSL2, FOSL1, ARID5B, and HIF1A (Fig. 3b, Fig. 14f).
  • the AHR agonist is administered to the T cells or T cell populations (i.e. contacted with the cells) at a rate of about l-500nM, or about l-250nM, or about 1- lOOnM, or about l-50nM, or about l-10nM, or about l-5nM, or about 0.1 -5nM.
  • the AHR agonist is applied at a rate of about lOpM.
  • the AHR agonist is applied at a rate of about 3-5nM.
  • the autoimmune disease may be selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • ANCA anti-neutrophil cytoplasmic antibody
  • the present disclosure also identifies transcription factors including — AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and BATF — which can regulate Tfh and Tph cell function in autoantibody driven autoimmune disease.
  • JUN is identified as a key regulator of Tfh and Tph function, including but not limited to the production of CXCL13 (see e.g. Fig. 3g, which shows expression in a CRISPR screen).
  • the present disclosure demonstrates AHR antagonists and/or inhibitors reduces JUN functions.
  • the present disclosure provides JUN as a targetable biomolecule for treatment of autoantibody driven autoimmune disease. JUN activation drives T cells away from a CXCL13 + phenotype and towards an IL-22 + phenotype (see e.g. Fig. 3i and Fig. 3k, which shows deletion of the JUN gene upregulates CXCL13 production and downregulates IL-22 production).
  • the present disclosure provides a transcription factor — AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and/or BATF — for treatment of autoimmune disease.
  • the transcription factor can suppress the generation of Tph/Tfh cells and may inhibit disease activity in autoantibody-associated autoimmune diseases.
  • the present disclosure provides a vector including the transcription factor.
  • the vector may be comprised of, or derived from, a plasmid, an adenovirus, and adeno-associated virus (AAV), a retrovirus, a herpes simplex virus, a human immunodeficiency virus (HIV), a lentivirus, or a synthetic vector.
  • the vector is a VSV-G pseudotyped lentivirus.
  • the transcription factor or vector are part of a composition.
  • the composition may also include a carrier, which may be a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of preventing CXCL13+ Tph/Tfh cell generation or differentiation.
  • the method comprises, consists essentially of, or consists of contacting a cell or cell population (i.e. a T cell population) with one or more transcription factor, vectors, or compositions including the transcription factor, thereby preventing CXCL13+ Tph/Tfh cell generation or differentiation.
  • the transcription factors may be selected from AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and BATF.
  • the transcription factor, or vector or composition including the transcription factor is contacted with the T cell populations. Application of the transcription factors results in the transcription factor to be overexpressed in the cell population.
  • the T cells populations are CD4+ or CD8+ cells.
  • the T cells may be a CD4+ subset selected from Thl, Thl7, Tfh, and/or Tph cells .
  • the T cells may be a CD8+ subset selected from TCM, TEM, and/or TEMRA.CC11S.
  • the T cell populations may be antigen specific. With the treatment of the T-cell populations with a transcription factor described herein, the T-cell populations may be reprogrammed towards an IL-22+ phenotype and/or away from a B-cell helper phenotype.
  • overexpression of select transcription factors decreases CXCL13 when overexpressed in a cell or population of cells.
  • Transcriptions factors which decrease CXCL13 when overexpressed are found below in Table 1, which includes DNA sequences for AHR and JUN, and mutants of each. As shown in Table 1, the mutants can be mutants with constitutive activity, or mutants with loss of a transactivation domain (TAD). In still other embodiments, the transcription factors may have other mutants which decrease CXCL13 activity.
  • the present disclosure also provides a method of using transcription factors to inhibit functions of autoimmunity-associated T cell populations — T follicular helper (Tfh) cells and T peripheral helper (Tph) cells — in a subject in need.
  • the transcription factors are selected from AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and BATF
  • the present disclosure also provides a method of using the transcription factor mutants found in Table 1 to inhibit functions of autoimmunity-associated T cell populations in a subject in need.
  • other mutants of transcription factors AHR, JUN, FOS, AFT3. FOSL1, FOSL2, and/or BATF are used.
  • the present disclosure provides a method directed to preventing the expansion of pathological T cells found in autoimmune diseases using the transcription factors.
  • These methods comprise, consist essentially of, or consist of administering a transcription factor, vectors, or compositions including a transcription factor mutant to a subject in need, thereby inhibiting functions of autoimmunity-associated T cell populations or preventing the expansion of pathological T cells.
  • Application of the transcription factors results in the transcription factor to be overexpressed in the subject.
  • Administration may be systemic or targeted. In some embodiments, administration is intravenous, subcutaneous, oral, or topical.
  • T cells with the capacity to help B cells, Tfh cells and Tph cells, are highly expanded in patients with autoantibody-associated autoimmune diseases such as lupus and rheumatoid arthritis.
  • the transcription factor can target key biological markers specific to the pathological T cells, and inhibiting either function or fit of these cells.
  • the present disclosure provides a method to treating an autoimmune disease using transcription factors (AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and/or BATF).
  • the method comprises, consists essentially of, or consists of obtaining a population of T cells from the subject, treating the population of T cells with a transcription factor selected from AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and/or BATF, or vector or composition including the transcription factor, and administering the treated population of T cells to the subject, thereby treating the autoimmune disease.
  • transcription factors AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and/or BATF
  • the present disclosure also provides a method to treating an autoimmune disease using the transcription factor mutants found in Table 1, which includes obtaining a population of T cells from the subject, treating the population of T cells with a transcription factor mutant, or vector or composition including the transcription factor mutant, and administering the treated population of T cells to the subject, thereby treating the autoimmune disease.
  • other mutants of the transcription factors are used.
  • the present disclosure also provides a method of using both an AHR agonist and transcription factor (AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and/or BATF and/or transcription factor mutant) to treat an autoimmune disease in a subject in need.
  • the method comprises, consists essentially of, or consists of administering both an AHR agonist and transcription factor to the subject, thereby treating the autoimmune disease.
  • transcription factor is JUN.
  • the AHR agonist and the transcription factor may be administered consecutively, or they may be administered sequentially.
  • the AHR and/or transcription factor may be administered intravenously, subcutaneously, orally, or topically.
  • the autoimmune disease may be selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • ANCA anti-neutrophil cytoplasmic antibody
  • crRNP complexes compositions and methods
  • gRNA targeting a gene selected from CD3D, LCP2, ATF4, IFNAR2, or STAT2 wherein the gRNA has a sequence according to one of the sequences in Table 5.
  • the gRNAs are designed for use in a CRISPR system.
  • the CRISPR system is a CRISPR-Cas9-gRNA ribonucleoprotein (crRNP) complex comprising the gRNA for targeting at least one of CD3D, LCP2, ATF4, IFNAR2, and/or STAT2.
  • crRNP CRISPR-Cas9-gRNA ribonucleoprotein
  • the CRISPR system includes other RNAs, for example crRNAs for targeting at least one of CD 3D, LCP2, ATF4, IFNAR2, and/or STAT2.
  • crRNAs for example crRNAs for targeting at least one of CD 3D, LCP2, ATF4, IFNAR2, and/or STAT2.
  • the genes in Table 5 can decrease CXCL13 production in T cells
  • Another embodiment is a vector comprising a crRNP complex as described herein, optionally wherein the vector is, comprises, or is derived from a plasmid, an adenovirus, and adeno-associated virus (AAV), a retrovirus, a herpes simplex virus, a human immunodeficiency virus (HIV), a lentivirus, or a synthetic vector.
  • the vector is a VSV-G pseudotyped lentivirus.
  • the vector is a lipid nanoparticle.
  • the vector is a gamma retrovirus.
  • Another embodiment is a composition comprising a crRNP complex as described herein, or the vector as described herein, and a carrier.
  • the carrier is pharmaceutically acceptable.
  • a method of decreasing CXCL13 production in a cell comprises, consists essentially of, or consists of contacting a T-cell or cell population with the crRNP complex, vector, or composition as described herein, thereby decreasing CXCL13 production. Contacting may occur ex vivo, in situ, in vitro or in vivo.
  • the crRNP complex comprises a gRNA for targeting at least one of CD3D, LCP2, ATF4, IFNAR2, and/or STAT2 and the target gene in the cell is knocked out upon introduction of the crRNP complex.
  • the T cells populations are CD4+ or CD8+ cells.
  • the T cells may be a CD4+ subset selected from Thl, Th2, TH17, Tfh, Tph, iTreg, Th9, Tri, and/or Th22 cells.
  • the T cells may be a CD8+ subset selected from TCM, TEM, and/or TEMRA.CCIIS.
  • the T cell populations may be antigen specific. With the treatment of the T-cell populations with a crRNP complex as described herein, the T-cell populations may be reprogrammed towards an IL-22+ phenotype.
  • a method of treating an autoimmune disease in a patient in need comprises, consists essentially of, or consists of administering an effective amount of the crRNP complex, vector, or composition as described herein to a subject in need, thereby treating the autoimmune disease.
  • the target gene is knocked out.
  • the administering is done intravenously, subcutaneously, orally, or topically.
  • the patient in need is human.
  • the autoimmune disease may be selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • ANCA anti-neutrophil cytoplasmic antibody
  • compositions Compositions, Subject Population and Methods of Administration
  • the term “subject” refers includes but is not limited to a subject at risk of an immune disorder, or autoimmune response, disorder or disease, as well as a subject that has already developed an immune disorder, or autoimmune response, disorder or disease.
  • Such subjects include mammalian animals (mammals), such as a nonhuman primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • mammalian animals such as a nonhuman primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs),
  • Subjects include animal disease models, for example, mouse and other animal models of immune disorders, or autoimmune response, disorder or disease known in the art.
  • the subject is an adult human. In other embodiments the subject is a juvenile human.
  • administering an AHR agonist, transcription factor, crRNP complex, vector, and/or composition can be accomplished by any method known in the art suitable for the particular type of agonist and formulation selected.
  • Suitable routes of administration include without limitation oral, parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intraarticular intramedullary and intrathecal), intraperitoneal, and topical (including dermal/epicutaneous, transdermal, mucosal, transmucosal, intranasal (e.g., by nasal spray or drop), intraocular (e.g., by eye drop), pulmonary (e.g., by inhalation), buccal, sublingual, rectal and vaginal).
  • AHR agonists can be formulated for administration by oral inhalation.
  • the administering to a subject in need can be done intravenously or subcutaneously. In other aspects, in the methods described herein, the administering to a subject in need can be topical.
  • an additional therapeutic agent is administered to the subject in need.
  • the additional therapeutic agent is anifrolumab.
  • Doses in the methods described herein can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies, e.g. a mouse, and the amount treatment or agent disclosed herein administered in an amount that is determined to be effective. Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg/kg to about 100 mg/kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01-500 mg/kg, and any numerical value or range or value within such ranges.
  • the dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually.
  • a typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1.0-10 mg/kg, or any numerical value or range or value within such ranges.
  • the maximum tolerable dose of the methods described herein can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.
  • Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has previously had cancer or an infection, immune disorder, or autoimmune response, disorder or disease, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, the treatment protocol and compositions, the clinical endpoint desired, the occurrence of previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
  • the route, dose, number and frequency of administrations, treatments, and timing/intervals between treatment and disease development can be modified.
  • a desirable treatment of the present disclosure will elicit robust, long-lasting immunity against cancer or an infection, immune disorder, or autoimmune response, disorder or disease.
  • disclosure methods, uses and compositions provide long-lasting immunity to cancer or an infection, immune disorder, or autoimmune response, disorder or disease.
  • the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions i.e. pharmaceutical compositions described herein can be formulated to be compatible with a particular route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
  • Formulations suitable for parenteral administration comprise aqueous and nonaqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
  • Tfh T follicular helper
  • Tph T peripheral helper
  • Applicants identify a dramatic imbalance in CD4 T cell phenotypes in systemic lupus erythematosus patients, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells.
  • AHR aryl hydrocarbon receptor
  • Transcriptomic, epigenetic, and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ Tph/Tfh cell differentiation and promote an IL-22+ phenotype.
  • Type I interferon IFN
  • SLE Type I interferon
  • AHR activation drives T cells away from a CXCL13 + phenotype and towards an IL-22 + phenotype
  • Treatment of T cells from SLE patients with an AHR agonist reduced the frequency of PD-1+ Tph cells.
  • type I interferon (IFN-I) a central mediator in SLE, represses AHR activation in T cells and synergizes with AHR inhibition to boost CXCL13 production and promote a Tph cell phenotype.
  • the cells were clustered by Louvain-based clustering. Two clusters were identified as significantly enriched in SLE patients and 1 cluster was identified as significantly depleted in SLE patients (odds ratio (OR) >2 or ⁇ 0.5).
  • PCA principal component analysis
  • CD96 hl cells showed the highest enrichment score for a Th22-associated gene set 32 (Fig. Ih). This pattern was validated in PBMC from additional SLE patients, showing that CD96 hl cells expressed higher transcript levels of IL22 compared to PD-l hl Tfh and Tph cells, while Tfh and Tph cells expressed significantly higher levels of CXCL13 (Fig. li). Similarly, there was also a pattern of distinct localization of cells with signatures of CD96 hl cells or Tph/Tfh cells in a published scRNA-Seq dataset of PBMC from pediatric SLE patients and controls 91 (Fig. 8d).
  • CD96 hl cells sorted from PBMC of healthy donors frequently produced IL-22 protein. While total IL-22 production was comparable in CD96 hl cells and CD96 low CCR6 + CXCR3' Thl7 cells, CD96 hl cells were more often single producers of IL-22 (IL-22 + IL- 17 A') and less often single producers of IL-17A (IL-22' IL17A + ) compared to traditional CD96 low Thl7 cells (Fig. Ij). IFN-y and TNF production were comparable in CD96 hl cells and Tfh/Tph cells (Fig. 8e).
  • AHR and other transcription factors are negative regulators of CXCL13 production and Tph differentiation
  • CXCL13 + versus IL-22 + T cells in SLE patients suggested that CXCL13 and IL-22 may exist on opposite sides of an axis of T cell differentiation. Without wishing to be bound by any particular theory, Inventors hypothesized that specific transcriptional regulators may control such a differentiation axis. To identify molecular drivers of these distinct phenotypes, a CRISPR depletion screening assay in memory CD4 + T cells was conducted to identify regulators of CXCL13 and IL22 production.
  • gRNA guide RNA
  • 10 controls were synthesized in arrayed format in a 96-well plate and complexed with purified Cas9 protein in vitro to form CRISPR-Cas9 ribonucleoproteins (crRNPs). Multiplexed pools of 4 gRNAs per gene were chosen to improve depletion efficiency and minimizes off-target effects 33 ' 35 .
  • These 86 target genes were selected from genes upregulated in Tph/Tfh cells in the current RNA-Seq dataset (Fig.
  • Table 2 further includes gRNAs for AP-1 family member which was also a CRISPR array screen target.
  • the genes of particular interest are those that resulted in increased CXCL13 levels when knocked out. This indicates genes that could reduce CXCL13 levels when overexpressed in a cell.
  • Memory CD4 + T cells from 2 healthy donors were nucleofected with the arrayed crRNP libraries and cultured for 4 days in the presence or absence of TGF-0, a known inducer of CXCL13 production 36 (Fig.
  • CXCL13 in supernatants from the two replicate screens were quantified.
  • Cbl Proto-Oncogene B CBLB an E3 ubiquitin ligase, and the aryl hydrocarbon receptor (AHR) were the top regulators of CXCL13 production; deletion of either of these genes strongly upregulated CXCL13 production, with similar results in the absence or presence of TGF-0 (Fig. 2b; Fig. 9a).
  • AHR has a recognized role in regulating IL-22 production and Th22 differentiation 25 ’ 37 ’ 38 .
  • CRISPR deletion of AHR in multiple donors confirmed that AHR deletion increases memory T cell CXCL13 production (Fig. 9b; Fig. 2c). This increase was further enhanced by the presence of TGF-b (Fig. 9c).
  • deletion of AHR decreased production of IL-22, consistent with prior studies 38 (Fig. 2c).
  • the AHR inhibitor CH- 223191 also increased CXCL13 and decreased IL-22 production from total CD4 T cells, with similar effects in purified naive and memory CD4 + cells (Figs.
  • AHR agonism did not suppress in vitro polarization of CD4 T cells into IFN-g + Thl cells, IL-4 + Th2 cells, or IL-17A + Thl7 cells, indicating that AHR selectively suppresses CXCL13 production (Fig. 9h).
  • T cell differentiation into Tfh cells or Thl7 cells can be influenced by multiple cytokines or extrinsic factors 10,39 ’ 40 . Therefore, Inventors evaluated whether other cytokines or signals associated with Tfh or Thl7/Th22 differentiation similarly influenced CXCL13 production by CD4 + T cells. Among these factors, only TGF-b and modulation of AHR activity altered CXCL13 and IL-22 production (Fig. 9i). TGF-0 increased CXCL13 production and decreased IL-22 and IFNy production. In addition to cytokine production, AHR and TGF-P also controlled key features of the Tfh/Tph cell surface phenotype.
  • AHR inhibition with CH-223191 increased ICOS expression and decreased CD96 expression in CD4 + T cells from control donors, while AHR activation with TCDD decreased ICOS expression and increased CD96 (Fig. 2e).
  • AHR modulation altered similar functional and phenotypic features in human CD8 + T cells.
  • Both CRISPR deletion and pharmacologic inhibition of AHR increased CXCL13 production and decreased IL-22 production by CD8 + T cells, while AHR activation decreased CXCL13 production and increased IL-22 production, with no effect on IFN-y production (Figs. 9j, 9k).
  • AHR inhibition also inhibited CD96 expression and upregulated ICOS expression in CD8 + T cells (Fig. 91). Together, these observations suggest that CXCL13 production and IL-22 production lie on opposites ends of a T cell differentiation axis that is regulated by AHR and TGF-p.
  • TGF-P and AHR control CXCL13-associated transcriptomic features
  • RNA-Seq single cell RNA-Seq analysis was performed on memory CD4 + T cells from 3 donors after 2 weeks of culture with DMSO, TGF-p+DMSO, TGF-p+AHR agonist (TCDD), or TGF-p+AHR inhibitor (CH- 223191).
  • TCDD TGF-p+AHR agonist
  • CH- 223191 TGF-p+AHR inhibitor
  • TGF-P+AHR agonist TCDD clustered together with cells that were not treated with TGF-P, suggesting that AHR agonism inhibited the transcriptomic program associated with chronic TGF-P treatment in these cultures.
  • Results indicate T cells cultured with TGF-P+AHR inhibitor or TGF-P+DMSO co-localized in a specific region of the UMAP demonstrating the highest Tph signature (Fig. 2g).
  • the RA synovial T cell dataset contained 2 clusters marked by high expression of CXCL13 that represented synovial Tph cells (cluster 9) and Tfh+Tph cells (cluster 5) 42 , with high expression of genes including CXCL13, PDCD1, ICOS, CTLA4, 11.21. n MAF (Figs. 2h-2j).
  • T cells treated with TGF-P+AHR inhibitor or TGF-P+DMSO preferentially mapped to the synovial Tph cell cluster (cluster 9), as well as one proliferating cluster (cluster 12) and a cluster with high IFN signature (cluster 14), while DMSO-treated cells and TGF-P+AHR agonist-treated cells preferentially mapped to a different T cell cluster (cluster 3) separate from the CXCL13 + region (Figs. 2i, 2j).
  • RNA-Seq data was generated.
  • CXCL13 expression was strongly induced by TGF-P+AHR inhibitor, especially after 2 weeks (Fig. 10b).
  • scRNA-Seq data bulk RNA-Seq analyses of naive CD4 + T cells stimulated in the presence of TGF-P+AHR inhibitor were enriched for a Tph signature compared to cells stimulated with TGF-P+TCDD (Fig. 10c).
  • Memory T cells showed a similar direction of enrichment though a less robust change, consistent with the greater plasticity of naive T cells.
  • TGF-P+AHR inhibitors for 2 weeks Comparing transcriptomes of memory CD4 + T cells from the strongest CXCL 13 -inducing condition (TGF-P+AHR inhibitors for 2 weeks) versus the weakest CXCL 13 -inducing condition (TCDD only for 1 week) showed a significant enrichment of Tph-associated genes in the TGF-P+AHR inhibitor condition and an enrichment for CD96 hl -associated genes in the AHR agonist condition (Fig. 2k).
  • the ability of TGF-P to promote a Tph cell signature was strongly influenced by AHR activity in that TGF-P treatment strongly induced a Tph signature when AHR was inhibited yet could not induce a Tph signature when AHR was activated, consistent with scRNA-Seq results (Fig. lOd).
  • TGF-P A strong interaction between TGF-P and AHR at the protein level across diverse T cell subsets was confirmed.
  • both CRISPR deletion of AHR and AHR inhibition induced CXCL13 production from multiple sorted T cell subsets, including Tph cells, Tfh cells, Thl7 cells, Thl cells, and naive CD4 + T cells (Figs. Ila, 11b).
  • the ability of AHR inhibition to induce CXCL13 production depended on the presence of TGF-P for most cell subsets; however, Tph cells showed a unique ability to produce CXCL13 with AHR inhibition alone without co-treatment with TGF-0.
  • Tph cells may have been exposed to TGF-P in vivo before isolation, obviating the need for exogenous TGF-0.
  • Tph cells isolated from the blood of systemic lupus erythematosus patients showed upregulation of a TGF-P gene signature compared to other T cell subsets, suggesting that these cells may have recently responded to TGF-P in vivo (Fig. 11c).
  • Both TGF-P and AHR inhibition reduced IL-22 production from CD96 hl and Thl7 cells, yet AHR activation did not induce IL-22 production from Tph or Tfh cells (Fig. lid).
  • AHR inhibition also increased ICOS and PD-1 expression in stimulated Tph, Tfh, CD96 hl , and naive cell subsets while suppressing CD96 and TIGIT expression, generally with stronger effects in the absence of TGF-p (Fig. He).
  • ATAC-Seq was used to evaluate areas of open chromatin in CD4 + T cells stimulated with TGF-P plus either AHR agonist or inhibitor. This ATAC-Seq was compared data to ATAC-Seq data from sorted PD-l hl CXCR5' Tph cells from RA synovial fluid (SF) and PD-l hi CXCR5 + Tfh cells from tonsil (Figs. 12a, 12b). PD-l mid and PD-1 10 CD4 T cells from each respective tissue were sorted as comparator cell populations.
  • SF Tph cells and tonsil Tfh cells clustered together, separated from PD-l mid and PD-1 10 CD4 + T cells (Fig. 12c).
  • Blood CD4 + T cells stimulated in vitro with the AHR inhibitor CH-223191 clustered separately from cells treated with TCDD and DMSO control, consistent with scRNA-Seq results (Fig. 12d).
  • DESeq2 43 was used to identify differentially accessible regions (DAR) in Tph and Tfh cells from SF and tonsil tissues, respectively, by comparing PD-l hl versus PD- l 10 cells from each tissue.
  • aberrant AHR activation may be a systemic feature in systemic lupus erythematosus patients that allows for expansion of Tph and Tfh cells.
  • the effects of serum from SLE patients on AHR activation were tested using a HepG2 cell line with a luciferase reporter driven by AHR response elements 44 .
  • Serum from either new-onset SLE patients or controls showed comparably low intrinsic AHR agonist activity; however, treatment of HepG2 reporter cells with serum from SLE patients significantly inhibited TCDD-induced activation of the AHR reporter, as compared to serum samples from anti-nuclear antibody (ANA + ) control patients without SLE (Fig. 21).
  • PBMC of SLE or healthy donor controls were treated with AHR agonists and/or inhibitors for 4 days without further TCR stimulation, and the frequency of T cell subsets within memory CD4 T cells was evaluated (Fig. 13).
  • the AHR agonist TCDD significantly reduced the frequency of PD-1 + CXCR5' Tph cells among memory CD4 + T cells from SLE patients (Fig. 2m).
  • the AHR inhibitor CH-223191 reduced the frequency of CD96 hl cells in both control and SLE patients.
  • Tfh cells circulating PD-l hl CXCR5 + Tfh cells were separated from blood of control donors, and Tfh cells were pretreated with an AHR agonist, AHR inhibitor, or control prior to co-culture with memory B cells. Tfh cells treated with an AHR agonist were less able to induce B cell differentiation into plasmablasts in vitro (Fig. 2n), which indicates that AHR activation can reduce the B cell-helper function of circulating Tfh cells.
  • AHR controls CXCL13-IL22 polarization through AP-1 family member JUN
  • RNA-Seq of CD4 + memory T cells treated with TGF-0 and either an AHR agonist or inhibitor for 12, 24, 48 or 72 hours was conducted (Fig. 14a). These early time points were chosen to capture proximal transcriptional events induced by AHR agonism. Expression of canonical AHR target genes (CYP1A1, CYP1B1, TIPARP, AHRR) 45 ' 47 increased within 12 hours of culture with TCDD, and PCA analysis revealed clear separation of samples based on AHR activation status by 48 hours (Figs. 14b, 14c; Table 3). AHR agonism significantly downregulated expression of CXCL13 and other Tph genes such as ICOS, MAF and IL21 at 48 hours and significantly upregulated CD96 expression (Fig.
  • Pathway analysis showed AHR signaling pathways first enriched at 48 hours in CD4+ memory T cells cultured with TCDD (AHR agonist), followed by co-enrichment of T cell activation and differentiation pathways at 72 hours (Elsevier Pathway collection database, padj ⁇ 0.05, Fig. 14e).
  • Analysis of transcription factor signatures for each time points revealed additional transcription factors (TF) altered by AHR agonism, including upregulation of genes enriched for putative cis-binding sites for AP-1 transcription factors such as JUN, FOS, ATF3, FOSL1, and FOSL2 in cells treated with TCDD after 12 hours (Fig. 14f).
  • CUT&RUN was used to identify the sites directly bound by AHR using an anti-AHR antibody or anti-IgG control 49 .
  • AHR ChlP-Seq and/or CUT&RUN has not been previously reported in primary human T cells.
  • CUT&RUN was done for AHR on cells in which AHR had been targeted by CRISPR and on control cells (Fig. 3c).
  • CUT&RUN yielded 2736 AHR-bound peaks in AHR- intact cells and 1379 peaks in AHR-depleted cells.
  • Pathway analysis utilizing the Kyoto Encyclopedia of Genes and genomes (KEGG) database for T cell specific pathways showed that AHR peak-associated genes were enriched in a Thl7 signature (Fig. 3f); however, there were no AHR peaks in the IL17A or IL17F gene loci, consistent with a Th22 phenotype.
  • AP-1 transcription factors act as hetero- or homodimers with highly similar DNA binding motifs, such that it is not possible to identify the specific AP-1 family member(s) responsible for this effect based on motif analysis alone 51,52 .
  • a targeted, arrayed CRISPR screen in human memory CD4 + T cells targeting 22 AP-1 family members was conducted using an approach similar to Fig. 2a, but in the presence of TGF-0 plus AHR agonist TCDD or AHR inhibitor CH-223191.
  • Results of the CRISPR screen with AP-1 family members indicate deletion of JUN strongly upregulated T cell CXCL13 (Fig. 3g) and downregulated IL-22 production, showing the largest effect of any AP-1 family member (Fig. 3h).
  • Validation experiments using an independent gRNA confirmed that JUN deletion upregulated CXCL13 production and downregulated IL-22 production in CD4 + memory T cells from multiple healthy donors (Figs. 3i-3k).
  • RNA-Seq on JUN-deleted cells and JUN-intact cells treated with AHR agonist or inhibitor was conducted.
  • GSEA analysis showed significant enrichment of Tph signatures in JUN-deleted cells stimulated with an TCDD (FDR ⁇ 0.001), and also a trend towards depletion of Th22 gene signatures (Fig. 31).
  • JUN also bound to the CXCL13 gene locus, which suggests that it inhibits CXCL13 expression directly (Fig. 4d).
  • AHR inhibition also had broader effects on JUN binding peaks, with 60% of the total 7181 JUN peaks lost in AHR inhibitor-treated cells, including 87% of peaks that are not cobound by AHR (Fig. 4f), which suggests that AHR inhibitors do not modulate JUN- dependent genes merely by abrogating recruitment of JUN to AHR-bound sites.
  • AHR may modulate JUN expression.
  • JLWRNA time course RNA-Seq data of AHR activation
  • JNK c-JUN N-terminal kinases
  • AHR inhibition (with or without TGF-0) moderately but significantly reduced levels of native JUN protein and its Ser73 phosphorylated proteoform in 13 healthy donors, which suggests that AHR inhibition may reduce JUN protein stability (Figs. 4g, 4h). This effect was not dependent on TGF-0. Taken together, these results suggest that AHR inhibition may moderately inhibit JUN protein stability, leading to reduced DNA binding to enforce an AHR-induced transcriptional program.
  • JUN overexpression significantly decreased CXCL13 production and increased IL-22 expression, even in cells treated with AHR inhibitor (Fig. 4i).
  • CUT&RUN analysis showed that JUN overexpression restored DNA binding in CXCL13 and IL22 gene loci at regions suppressed by AHR inhibition (Fig. 4j; Figs. 15e, 15f).
  • JUN overexpression restored JUN binding to other peaks in the Thl7/Th22 differentiation pathways that are otherwise lost in AHR inhibitor-treated cells (Fig. 4k).
  • RNA-Seq analysis of JUN-overexpressing CD4 + T cells demonstrated enrichment of Th22 signatures and suppression of Tph signatures compared to control cells (Fig. 41).
  • Type I IFN promotes CXCL13 + Tph cells at the expense of Th22 cells in a JUN- dependent manner
  • extrinsic factors that may skew the CXCL13 + Tph/Tfh versus IL-22 + Th22 axis polarization in systemic lupus erythematosus patients were identified.
  • Differential expression analyses comparing T cell populations from SLE patients and controls highlighted a prominent upregulation of IFN-inducible genes in cells from SLE patients, consistent with a well-recognized IFN signature in SLE 56 (Fig. 16a).
  • RNA-Seq data on T cells from a larger cohort of SLE patients stratified in to IFN-high and IFN-low subgroups Inventors found that CXCL13 was significantly higher in T cells from IFN-high SLE patients compared to IFN-low patients 57 (Fig. 5a).
  • IFNAR IFN-a/p receptor
  • the effect of blockade of the IFN-a/p receptor (IFNAR) on circulating CXCL13 levels was then evaluated in SLE patients treated with either anifrolumab (anti-IFNAR) or placebo in the TULIP- 1 randomized controlled trial 58 .
  • Clustering of memory CD4 + T cells yielded clusters with distinct T helper subset gene signatures, including a Tph cluster (cluster 0), which was enriched for a Tph signature (p ⁇ 2.2e-16, Linear Mixed Model [LMM]), and a Th22 cluster (cluster 11), which was enriched for both Th22 and CD96 hl gene signatures (p ⁇ 2.2e-16, Fig.
  • IFN-a increased T cell CXCL13 production in all cases and significantly decreased IL-22 production in T cells treated with AHR agonist.
  • IFN-a and AHR inhibition or deletion further amplified CXCL13 production, particularly in the presence of TGF-0 (Fig. 5j).
  • RNAseq data from naive CD4 + T cells treated with IFN-P was analyzed 59 .
  • IFN was sufficient to induce Tph genes such as CXCL13, ICOS and MAF (data not shown), similar to AHR inhibition in memory CD4 + T cells.
  • CRISPR deletion of IFN alpha/beta receptor 2 obviated IFN induced CXCL13.
  • IFNAR2 IFN alpha/beta receptor 2
  • the genes of particular interest are those that resulted in decreased CXCL13 levels when knocked out.
  • CXCL13 levels decrease when select transcription factors were knocked out in the CRISPR screen, including CD3D, LCP2, ATF4, IFNAR2, and STAT2. This is indicative of genes that could increase CXCL13 levels when overexpressed in a cell or genes that play a role in increasing levels of CXCL13 production in a cell.
  • Described herein is an imbalance in T cell differentiation in patients with systemic lupus erythematosus, which manifests not as an isolated expansion of activated population, but as a dysregulated balance of distinct effector T cell states.
  • Expansion of Tph and Tfh cells has been consistently observed across multiple SLE cohorts 9,66 ' 68 , yet the deficiency in a counterbalanced CD96 hl Th22 T cell subset has not been previously recognized.
  • a disrupted Thl7/Treg balance in certain autoimmune diseases herein it is shown through multiple, orthogonal assays that Tph/Tfh and Th22 developmental pathways are reciprocally interconnected.
  • AHR+JUN and type I IFN as critical, opposing regulators of a T cell differentiation axis, which were identified using CRISPR screening, pharmacologic interventions, and cytokine assays.
  • AHR blunts T cell acquisition of several phenotypic, transcriptomic, and epigenetic marks of B cell-helper function.
  • Boosting Th22 cell generation may also have benefits in mucosal barrier integrity in systemic lupus erythematosus given reports of compromised gut barrier integrity in both SLE patients and murine lupus models 70 ' 72 , though potential inflammatory consequences are possible 73 .
  • Collectively, these data support the therapeutic potential of AHR agonism for autoimmune diseases, either systemically or directed towards T cells 74 ' 77 .
  • AHR acts in concert with JUN to limit CXCL13 + Tph/Tfh cell generation and usher T cells towards IL-22 production.
  • AHR activation increases JUN protein levels and promotes JUN nuclear localization, which may occur through direct protein-protein interaction.
  • CUT&RUN analyses indicated that AHR and JUN coregulate a shared gene transcriptional program. While the roles of AP-1 in T cell differentiation and cytokine production have been investigated for decades, these results add a previously unappreciated layer to the regulation and function of JUN in controlling T cell phenotypes. Although AP-1 factors appear to bind to similar DNA-binding motifs, there are repeated examples of functional specificity 78,79 . Applicants have shown here that JUN is shown to be necessary and sufficient to induce the Th22 phenotype and antagonize CXCL13 expression.
  • IFN an endogenous inhibitor of AHR activation that may contribute to inadequate AHR activation and function in systemic lupus erythematosus through multiple mechanisms.
  • IFN inhibits AHR-induced gene upregulation and induces epigenetic changes that resemble those caused by AHR inhibition, pushing T cells towards CXCL13 production and away from IL-22 production.
  • the synergistic effects of IFN plus AHR inhibition indicates that IFN has additional roles in promoting a CXCL13 + phenotype beyond inhibiting AHR. For one, IFN inhibited JUN expression and disrupted JUN binding to multiple sites across the genome, indicating that IFN substantially alters AP-1 activation in T cells.
  • Inhibition of AP-1 activity is a key function of BCL6 in Tfh cells, enabling full Tfh cell differentiation and function 82,83 ; it is possible that IFN can similarly disrupt or alter AP-1 activity in BCL6 low Tph cells, helping to enable their B cell-helper function in the absence of high BCL6 expression.
  • IFN blocked IL-2-STAT5-mediated suppression of CXCL13 production.
  • IL-2 has been shown sustain AHR activation in tumor infiltrating lymphocytes 62 , highlighting the connected interactions between these regulators.
  • the ability of IFNAR blockade to reduce levels of Tph cells and CXCL13 in lupus patients strongly implicates IFN in this pathway in vivo.
  • CXCL13 + CD8 + T cells in tumors appear chronically TCR- activated, exposed to TGF-0 and/or type I IFN, and deprived of IL-2, a factor that can promote intrinsic AHR signaling.
  • the need for persistent TCR stimulation for CXCL13 production was highlighted by CRISPR deletion of CBLB and repeated TCR stimulation assays. Intriguingly, as for Tph cells, CXCL 13 -associated dysfunctional states in CD8 + T cells may critically rely on downregulation of JUN 78 .
  • the identified critical controlling factors including AHR, IFN, and JUN, may be manipulated therapeutically to alter this axis and dampen the production of pathologically expanded CXCL13 + Tph and Tfh cells in systemic autoimmune diseases.
  • Mononuclear cells from synovial fluid and peripheral blood were isolated by density centrifugation using Ficoll-Paque Plus (GE healthcare) and cryopreserved in FBS + 10% DMSO by slow freeze, followed by storage in liquid nitrogen for batched analyses.
  • FBS + 10% DMSO fetal bovine serum
  • cryopreserved samples were thawed into warm RPMI medium + 10% FBS.
  • Samples were processed in 3 batches, including balanced numbers of SLE and control samples per batch. Cryopreserved cells were thawed and trypan blue negative viable cells were counted by hemocytometry. Approximately 1 million live cells per sample were used for mass cytometry staining. All antibodies were obtained from the Longwood Medical Area CyTOF Core. Buffers were from Fluidigm. Cells were stained with rhodium (Fluidigm) for viability then washed. Cells were washed and stained with primary antibody cocktails using custom metal-conjugated antibodies obtained from the Longwood Medical Area CyTOF Antibody Resource Core (Boston, MA).
  • FCS files were uploaded in FlowJo v.10.4.2. Live singlet cells were determined by manual gating and normalization beads were excluded. FCS files including all manually gated T CD4 memory cells (CD3+CD8-CD4+CD45RO+) were uploaded and read in R (v.4.0.3) using the flowCore package. Marker expressions were arcsinh transformed using a co-factor of 5. The transformed matrix of expression was transposed and implemented into a Seurat object with the corresponding metadata. To check data quality, the distribution of the markers across the three batches and confirmed minimal batch effect was verified.
  • CNA co-varying neighborhood analysis
  • MASC Mixed-effects Association testing for Single Cells
  • CD4 + T cells were isolated from PBMC by negative selection using magnetic beads (Miltenyi Biotec). Alternatively, CD4 + T cells were isolated by magnetic positive selection using Dynabeads (Invitrogen #1133 ID). CD45RO+ memory CD4 T cells were further isolated from bulk CD4 + T cells by negative selection, depleting CD45RA+ naive CD4 T cells using CD45RA mouse IgG antibodies (Invitrogen #14-0458-82) and Pan IgG Dynabeads (Invitrogen #1153 ID).
  • RPMI-1640 medium Gibco #21875034
  • FBS fetal bovine serum
  • penicillin/ streptomycin 1% penicillin/ streptomycin
  • lOmM HEPES 1% L-glutamine.
  • the 1% L-glutamine was replaced with ImM sodium pyruvate.
  • T cells were cultured in complete RPMI medium with Dyna anti-CD3/CD28 T activator beads (Invitrogen #1113 ID) at 1 :5 bead:cell ratio with 2ng/mL human TGF-bl (Peprotech #100-21C R&D 7754-BH- 025), and either lOpM of CH-223191 (Sigma C8124) or 3-5nM of TCDD as indicated (Accu Standard, #1746-01-6).
  • IFN-a or IFN-P lOOOU/mL of IFN-a (R&D PHC4814) or IFN-P (Peprotech #300-02BC) were added.
  • IL-2 For experiments with IL-2, unless indicated, lOng/mL of IL-2 (Peprotech #200-02) was added to complete RPMI medium and cells were either stimulated with CD3/CD28 T activator beads or plate-bound anti-CD3 antibodies (Thermo #16-0036-81, clone SK7).
  • gRNAs guide RNAs
  • crRNAs were duplexed with tracrRNA (IDT #1072534) for 2 minutes at 95°C or 40minutes at 37°C in 5% CO2 incubator and complexed with Cas9 protein (Macrolab, Berkeley, 40pM stock) at 1 :2 or 1 : 1 molar ratio for up to 60 minutes at 37°C.
  • tracrRNA IDT #1072534
  • Cas9 protein Macrolab, Berkeley, 40pM stock
  • cells were collected, stripped from beads, pelleted, and resuspended in Lonza electroporation buffer P3 (Lonza #V4XP- 3032) at 0.2-2M 10 A 6 cells / 20pL.
  • Custom 96-well CRISPR array library plates were purchased from Horizon Discovery Ltd. Each well consisted of 4 individual gRNA guides targeting the same gene.
  • Ribonucleoprotein (crRNP) complexes were made from gRNA from each well as previously described 34 , and aliquoted and stored at -80°C in lo-bind plates (Eppendorf). Electroporation of pre-stimulated T cells was done as described above. Non-targeting guides CD8a, CD19 and OR1A1 sgRNAs were included as controls in the CRISPR-Cas9 screen. Roughly 60 million memory CD4+ T cells were stimulated with anti-CD3/CD28 Dyna activator beads for 72 hours prior to electroporation.
  • Cells were then transferred to 96-well plates containing complete RPMI media and anti-CD3/CD28 T activator beads and incubated at 37°C and 5% CO2 for 4 days, supplementing lOOpL of fresh complete RPMI on day 3. After 4 days, cells were resuspended with gentle pipetting, and 20pL was removed for cell count using CountBrightTM beads (Invitrogen #C36950). Roughly 0.3 million cells were removed from each well to make protein lysates for CRISPR knockout verification as previously described 34 . Remaining cells were split evenly between two new 96-well plates for culture conditions of anti-CD3/CD28 bead stimulation with or without TGF-pi (2ng/mL).
  • CUT&RUN was performed using the CUTANA ChIC / CUT&RUN Kit (EpiCypher #14-1048) and following manufacture protocol with minor modifications. Briefly, human CD4 memory T cells were isolated and stimulated for 72 hours with TGF-01 and either AHR agonist or AHR antagonist, or in a separate experiment with either PBS or IFN-a. T cells were then collected and washed in wash buffer provided by kit. Cells were then incubated with activated ConA beads for 10 minutes at room temperature.
  • Bead bound cells were resuspended in kit-provided antibody buffer supplemented with 0.1% bovine serum albumin, lOOnM trichostatin A, 0.1 U citrate synthase, and ImM oxaloacetic acid.
  • Cells in antibody buffer were incubated with antibodies targeting either AHR (CST #83200), JUN (CST #9165), or IgG control (Epicypher #13-0042) overnight on nutator at 4°C. The following day, cells were washed with cell permeabilization buffer twice and incubated with pAG-Mnase for 1 hour on nutator at 4°C.
  • DNA sample was measured using Qubit dsDNA HS assay kit (Invitrogen #Q32851) and Qubit 2.0 fluorometer (Invitrogen).
  • CUT&RUN library prep was performed using KAPA HyperPrep Kit (Roche), and paired-end DNA sequencing was performed on a HiSeq 2500 platform (Illumina) at Admera Health Inc.
  • CUT&RUN Data analysis [0202] Raw CUT&RUN Sequenced reads (FASTQ files) were processed using script adapted and modified from the following repository: https://github.com/wherrylab/jogiles_ATAC. Briefly, samples were aligned to human genome hg38 (GRCh38) using Bowtie 2 (v.2.2.6). Samtools was used to remove unmapped, unpaired and mitochondrial reads. ENCODE blacklist regions were also removed (https://sites.google.com/site/anshulkundaje/projects/blacklists). PCR duplicates were removed using Picard. Peak calling was performed using SEACR (v.1.3) in relaxed setting normalized to IgG control.
  • RNA from human memory CD4 T cells was isolated from cells (200,000 cells) stimulated with anti-CD3/CD28 Dyna beads and cultured in TGF-bl with either AHR antagonist or agonist for 12, 24, 48 and 72 hours. RNA was isolated with Rneasy Plus Micro Kit (Qiagen #74034) following manufacture protocol. RNA libraries were prepared using the QuantSeq FWD Kit for Illumina Sequencing (Lexogen) for 3 ’RNA-Seq. Sequencing was performed on Illumina NextSeq platform. Three biological replicates of each sample were sequenced.
  • JUN cDNA encoding c-JUN
  • lentiviral expression vector System Bioscience #CD51 lb-1
  • JUN open reading frame ORF
  • NEB Gibson Assembly
  • JUN overexpressing vector was transformed into NEB Stable (NEB #C3040H) chemically competent cells and purified ZymoPURE plasmid Midiprep kit (Zymo Research #D4201-A). Pantropic.
  • VSV-G pseudotyped lentivirus was produced via transfection of 293T cells with JUN overexpression vector and the viral packaging plasmids pCMVdr8.91 and pCMV-VSV- G using FuGENE (Promega #E2311).
  • Primary CD4 memory T cells were isolated as described above, on the same day of 293 T cell transfection. After 24 hours in culture, memory T cells were stimulated with CD3/CD28 T-activator Dynabeads (Life Technologies #1113 ID) at a 1 :2 bead:cell ratio. At 48 hours, viral supernatant was harvested, filtered, concentrated, and added to primary T cell culture for 24 hours. At day 5 post T cell stimulation, Dynabeads were removed and T cells were re-cultured in AHR modulating conditions with or without TGF-0 for 8 days while supernatants were collected on days 4, and 8 for ELISA assays.
  • Cryopreserved cells were thawed, washed and counted. Cells ranging from 0.1-2 million cells were stained in PBS with Aqua fixable live/dead dye (Invitrogen) for 20 minutes at 4 °C.
  • Aqua fixable live/dead dye Invitrogen
  • Panel #1 included anti-CD3 BV510, anti-CD4 Pe-Cy7, anti-CD45RA BV605, anti-CD25 FITC, anti-CD127 BV711, anti-PD-1 APC-Cy7, anti-CXCR5 BV421, anti-CD96 APC, anti-TIGIT PE.
  • Panel #2 included anti-CD8 BV510, anti-CD19 BV510, anti-CD56 BV510, anti-CD25 FITC, anti-CD127 Alexa Fluor 700, anti-CD96 APC, anti- CXCR5 BV421, anti-TIGIT PE, anti-CCR6 BV605, anti-PD-1 BV711, propidium iodide (all from BioLegend), and anti-CD45RA APC-efluor788, anti-CXCR3 PE-Cy7 (Invitrogen).
  • Memory B cells were identified with this panel: propidium iodide, anti-CD14 APC, anti-CD3 Alexa700, anti-CD19 PE, anti-CD27 BV421 and anti-IgD FITC.
  • Cells were incubated at 4°C with antibodies in PBS /1% BSA for 15-30 minutes. Cells were washed once in PBS/1% BSA, centrifuged and passed through a 70pM filter, and propidium iodide was added immediately prior to sorting. Cells were sorted on a 4-laser BD FACSAria Fusion cell sorter. Intact cells were gated according to forward scatter and side scatter area (FSC-A and SSC-A). Doublets were excluded by serial FSC-H/FSC-W and SSC-H/SSC-W gates (H, height; W, width). Non-viable cells were excluded based on propidium iodide uptake.
  • FSC-A and SSC-A forward scatter and side scatter area
  • Doublets were excluded by serial FSC-H/FSC-W and SSC-H/SSC-W gates (H, height; W, width). Non-viable cells were excluded based on propidium iodide uptake.
  • RNA- Seq up to 2000 cells were collected from each cell subset directly into buffer TCL (Qiagen) with 1% P-mercaptoethanol (Sigma). Flow cytometric quantification of cell populations was performed using FlowJo (v.10.0.7).
  • Tfh cells were flow sorted with panel #2 and activated with anti-CD3/CD28 Dynabeads in the presence of either DMSO, TCDD, or CH-223191 for 48 hours. Tfh cells were then collected and stained with LIVE/DEAD, and live Tfh cells were flow sorted again. In parallel, B cells were isolated from PBMCs from the same donor, and CD27 + IgD' memory B cells were flow sorted. Tfh and memory B cells were co-cultured in the presence of SEB (lug/ml) for 5 days. Cells were then analyzed by flow cytometry to quantify CD38 hl CD27 + plasmablasts among B cells.
  • SEB lug/ml
  • Th 1 -polarizing conditions IL-12 (10 ng/ml), IL-2 (20ng/ml) and anti-IL-4 Abs (1 pg/ml MAB204)
  • Th2-polarizing conditions IL-4 (20 ng/ml), anti-IFNy (1/zg/ml MAB285)
  • Thl7-polarizing conditions IL-6 (50ng/ml), TGF-0 (2ng/mL), IL-23 (40ng/mL), IL-10 (lOng/mL), anti-IFNy ( I g/ml), anti-IL-4 Abs (1 pg/ml) (R&D Systems) in the presence of DMSO, TCDD (5nM) or CH-223191 (10//M).
  • DMSO TCDD
  • CH-223191 10///M
  • Cytokine levels from supernatant of T-cell culture or from patient serum were quantified by ELISA using Human DuoSet ELISA kits for CXCL13, IL-22, IFN-y.
  • HEPG2 cells were obtained from Dr. Gary Perdew, Penn State University. Cells were first cultured in DMEM, 15% FBS, 1% penicillin/streptomycin at 37°C until reaching 70% confluence. Cells were then washed, counted, and cultured at a density of 70,000 cells in 96- well plate wells overnight. Cells were then incubated with 10% serum from healthy or SLE patients in RPMI 1640 overnight. The following day, cells were treated with trypsin (Gibco), rinsed, and washed at 800 rpm for 8 minutes. Cells were resuspended in DMEM and lysed with Dual-Glo Luciferase Assay System (Promega#, E2920). The luciferase activity was read with GloMax® Explorer Multimode Microplate Reader, Promega).
  • Rabbit anti-JUN, anti-phospho-JUN (Ser73), anti-AHR, anti-0-actin, anti-tubullin, anti-cyclophilin B, anti-vincullin antibodies were purchased from Cell Signaling Technology. Cells were pelleted and lysed with Laemmli buffer IX (Biorad #1610747) or RIPA buffer (Thermofisher #89901) for Ih at 4 degrees using a micro-tube shaker. Lysed cells were then centrifuged at >14000rpm for 10 min, and lysates stored at -80 degrees. Protein was measured using Pierce BCA assay (ThermoSci entific #23225), according to manufacturer’s instructions.
  • Protein lysates were loaded in lOx Tris 10% or 12% CriterionTM TGXTM Precast Midi protein gels, transferred to Immun-Blot® PVDF membrane at 4C for 2 hours with 0.2 amps. Membranes were blocked either with 5% milk or 5% BSA for 1 hour, or EveryBlot Blocking Buffer (Bio-Rad) for 5 min and then incubated with primary antibody overnight (1 : 1000 for anti-AHR, 1 :50,000 for anti-tubulin, anti-cyclophilin B, anti-vinculin or anti-P- actin).
  • Membranes were then incubated with the horseradish peroxidase conjugate-labeled secondary antibody (goat anti -rabbit IgG H+L, Invitrogen) for 1-2 hours and then washed with TBS-T. Protein bands were detected by SuperSignalTM West Femto Maximum Sensitivity substrate (#34096; Thermo Fisher Scientific). Images were obtained and quantified via Chemi Doc and Image Lab Software (Bio-Rad).
  • HEK293T Human embryonic kidney 293T cells were separately transduced with GFP containing lentiviral construct either native or encoding HA-tagged AHR as described above. Transduced HEK293Ts were sorted based on GFP positivity. Cells were then cultured for 72 hours with either AHR agonist, AHR antagonist or vehicle control as above and then harvested for protein lysates. The Standard Cell Fractionation Kit (Abeam #abl09719) was used to collect the cytoplasmic and nuclear fractions, as per the manufacturer’s instructions. The Pierce BCA Protein Assay (ThermoFisher, #23225) was used to quantify the protein concentration within the cytoplasmic and nuclear fractions, as per the manufacturer’s instructions. Western blot was performed as described above.
  • HEK293T Human embryonic kidney 293T cells were transduced with GFP containing lentiviral constructs encoding either HA-tagged AHR or 3xFLAG-tagged c-Jun as described above. Transduced HEK293Ts were sorted based on GFP positivity. Cells were cultured for 72 hours with either AHR agonist, AHR antagonist or vehicle control as above and then harvested for protein lysates using Pierce IP Lysis Buffer (ThermoFisher, #87787) containing Halt Protease Inhibitor Cocktail (ThermoFisher, #78429).
  • the Pierce BCA Protein Assay (ThermoFisher, #23225) was used to quantify the protein concentration within the cytoplasmic and nuclear fractions, as per the manufacturer’s instructions.
  • the 3xFLAG co-immunoprecipitation was completed with the ANTI-FLAG M2 Affinity Gel (Millipore Sigma, #A2220) and the HA co-immunoprecipitation was completed using Pierce Anti-HA magnetic beads (Fisher, #PI88836). Briefly, 250pg of protein was incubated with washed agarose slurry or magnetic beads overnight at 4°C as per manufacturer instructions.
  • Isolated memory CD4 T cells were cultured at IxlO 5 cell per well in a 96 well plate with 200ul of RPMI/10% FBS and stimulated with Dynabeads (ThermoFisher). As indicated, cells were cultured with DMSO alone, TGF-P (2ng/mL) and DMSO, CH-223191 (lOpM), or TCDD (3nM). Cells were collected at day 6 cells, restimulated, and collected at day 13. Cell counts were normalized across conditions and stained with LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit (Invitrogen) and cell hashing antibodies specific for each condition and donor, pooled, and stained with TotalSeq-C Human Universal Cocktail (BioLegend). Viable cells were then flow sorted and subjected to encapsulation and library preparation at the BWH Center for Cellular Profiling via the 10X Genomics pipeline, with 24-30K cells loaded per run with 82-95% viability.
  • Cell Ranger (v.6.1.1) workflow. FASTQ files containing gene expression and feature barcodes were aligned to the human genome hg38 (GRCh38). The filtered features, barcodes, and matrix files were imported into R and used to generate a Seurat object with Seurat package (v.4.3.0). Quality control was first performed filtering out cells with more than 10% mitochondrial reads and with ⁇ 200 or >5,500 reads. Cells that passed the QC were log normalized and scaled. The Seurat object was then demultiplexed using the hashtag oligos. HTO reads were normalized and cells assigned a HTO ID using the HTODemux function from Seurat.
  • Doublets were removed and the filtered cells were used to run PCA using the Seurat Package.
  • the Seurat object for each timepoint was individually integrated utilizing Harmony (v.0.1.1), where each Seurat object was corrected by donor.
  • the top 10 harmony embeddings were then used to generate a UMP and clustering was performed with a resolution of 0.7.
  • Differentially expressed genes between the four culture conditions were identified using the FindMarkers function in the Seurat Package.
  • the Rheumatoid Arthritis reference dataset was generated though the Accelerating Medicines Partnership (AMP) 17 .
  • T cells were identified as described 17 , and filtered for cells with mitochondrial read ⁇ 20% and with reads >200 and ⁇ 5000.
  • Cells passing filtering were normalized, scaled, and run though Harmony (v.0.1.1) integration correcting for donor.
  • the top 15 harmony embeddings were used to generate a UMAP and clustered with a resolution of 0.8.
  • the harmonized object was then used to generate a reference object with the buildReferenceFromSeurat Function in the Symphony package (v.0.1.1).
  • Data from day 13 in vitro single cell data was then query mapped to the AMP reference.
  • the frequency at which cells mapped to a given cluster was extracted and compared between culture conditions by donor.
  • Quality control was first performed filtering out cells with more than 15% mitochondrial reads and with ⁇ 1,000 or >4,000 reads. Samples were demultiplexed and clustered as above, and memory CD4 T cells were selected for further analysis. Cells passing filtering were normalized, scaled, and run though Harmony (v.0.1.1) integration correcting for donor and lOx sequencing batch. The top 20 harmony embeddings were then used to generate a UMAP and clustering was performed with a resolution of 1.2. The signature scores are calculated using addmodulescore function from Seurat package.
  • RNA-Seq libraries were prepared at Broad Technology Labs at the Broad Institute of Harvard and MIT using the Illumina SmartSeq2 platform. Libraries were Sequenced to generate 38 base paired-end reads. FASTQ files from sequencing were examined with FastQC for quality control and trimmed with trimmomatic. Reads were aligned to human genome hg38 (GRCh38) using hisat2 alignment program. Lowly expressed genes (log2 FPKM ⁇ 10 in 10 samples) were filtered out for downstream analysis. Differentially expressed genes (DEGs) were identified using DESeq2 with an adjusted p- value threshold of ⁇ 0.05 . Principal component analysis (PCA) was performed using the prcomp function in R. The top 20% most variable genes were selected for this analysis.
  • PCA Principal component analysis
  • Heatmap is generated using the pheatmap package (v.1.0.12) in R using FPKM values that have been scaled by each gene.
  • the signature genes for Tregs, CD96hi cells, or Tph/Tfh cells are the common DEGs in individual comparisons with each of the other cell groups.
  • the ssgsea score is calculated by gsva package (GSVA v.1.38.2) in R.
  • the Th22 cell gene signature list and Tph cell gene signature list were derived from previous reports 16,32 . [0241] ATAC-Seq data analysis
  • Raw sequencing data were trimmed using cutadapt (v.1.18) with Python (v.2.7.15) to remove the adapter. Trimmed reads were aligned to the GRCh38 human reference genome with Bowtie2; Aligned reads were filtered to remove mitochondrial reads and PCR duplicates, then peaks were called using Genrich in ATAC-Seq mode on individual samples. The fragment size distribution is checked using deepTools (v.3.1.2). The intervals were set to the default length of lOObp, and the peak-calling significance threshold was set to -log(p) > 2.
  • a union peak list for each data set was created by combining all peaks in all samples, merging overlapping peaks using bedtools (v.2.26.0), and retaining only peaks that were called in more than one sample. Normalized read counts for consensus peaks were computed for each sample using Diffbind, and differential accessibility between different groups was determined using a matched pairs t-test with the edgeR package (v.3.30). Peaks were annotated using ChlPseeker (v.1.26.2). Enrichment analysis of peaks and Gene Sets Enrichment Analysis was conducted using the clusterProfiler (v.3.16) package with a threshold of FDR ⁇ 0.05 to define enriched pathways.
  • peaks were declared differentially accessible at the genome-wide level with a false discovery rate adjusted p-value ⁇ 0.05, and those exhibiting a log2 fold change of ⁇ 2 or greater were defined as PDl hl signature in tonsil and SF.
  • the PD-l hl signatures were used for GSEA analysis using the clusterProfiler(v3.16) package. Peak signal tracks were generated using the rtracklayer package (v.1.48) or IGV software.
  • Circulating plasma samples taken at baseline, Week 12, and Week 52 (end of trial) from 302 patients in the TULIP- 1 trial 58 were assessed for protein biomarkers by targeted high-multiplex immunoassay panels on the Olink platform; Olink Target 96 ImmunoOncology (v.3112).
  • Data for CXCL13 protein is presented as relative Normalized Protein expression (NPX), which is on a Log2 scale.
  • NPX Normalized Protein expression
  • Model was adjusted for the trial stratification factors; baseline oral corticosteroid dose ( ⁇ 10 mg/day, >10 mg/day), Systemic Lupus Erythematosus Disease Activity Index 2000 (SLED Al 2K) score at screening ( ⁇ 10 points, >10 points), and IFN 4-gene status 90 . Multiple testing was accounted for using the Benjamini -Hochberg (FDR) procedure with ⁇ 0.05 threshold.
  • FDR Benjamini -Hochberg
  • Tph cell CRISPR Array Screen Targets Each gene included four gRNA sequences.
  • the CRISPR screens initially screened 86 target genes were selected from genes upregulated in Tph/Tfh cells in a current RNA-Seq dataset, or in previously published RNA- Seq datasets of rheumatoid arthritis synovial T cellsl6, or from genes previously correlated with CXCL13 expression, or AP-1 family members. Included in Table 2 are the genes that increased CXCL13 in cells when knocked out and the corresponding CRISPR gRNA sequences for the gene.
  • Table 3 Differential transcript expression at 12 and 24 hours after stimulation with an AHR agonist or inhibitor.
  • Log2 fold changes is a comparison of T cells treated with an AHR inhibitor+ TGF-0 compared to an AHR agonist+ TGF-0.
  • a positive log2 fold change indicated higher expression in T cells treated with the AHR agonist.
  • Table 4 AHR modulated gene expression. Shown here are transcripts identified as part of cluster 6, which is progressively upregulated over time by AHR activation, as seen in Fig. 3a.
  • the default setting of maSigPro breaks down profiles into 9 clusters, however only the cluster shown below was observed by Applicant to be progressively upregulated.
  • Tph cell CRISPR Array Screen Targets Each gene included four gRNA sequences.
  • the CRISPR screens initially screened 86 target genes were selected from genes upregulated in Tph/Tfh cells in a current RNA-Seq dataset, or in previously published RNA- Seq datasets of rheumatoid arthritis synovial T cellsl6, or from genes previously correlated with CXCL13 expression, or AP-1 family members. Included in Table 5 are the genes that decreased CXCL13 in cells when knocked out and the corresponding CRISPR gRNA sequences for the gene.
  • Additional embodiments include the following numbered embodiments:
  • AHR aryl hydrocarbon receptor
  • Tfh T follicular helper
  • Tph T peripheral helper
  • TCDD 2,3,7,8-tetrachlorodibenzodioxin
  • FICZ 6-formylindolo(3,2-b)carbazole
  • a vector comprising the agonist of any of embodiments 1-5, optionally wherein the vector is, comprises, or is derived from a plasmid, an adenovirus, and adeno-associated virus (AAV), a retrovirus, a herpes simplex virus, a human immunodeficiency virus (HIV), a lentivirus, or a synthetic vector.
  • AAV adeno-associated virus
  • composition comprising the agonist of any of embodiments 1-5 or the vector of embodiment 6 or 7, and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.
  • a method of preventing CXCL13+ Tph/Tfh cell generation comprising contacting a cell population with one or more agonists of any of embodiments 1-5, the vector of embodiment 6 or 7, or the composition of embodiment 8, thereby preventing CXCL13+ Tph/Tfh cell generation, wherein the contacting is in vivo, in vitro, in situ, or ex vivo.
  • a method of treating an autoimmune disease in a subject in need thereof comprising administering an effective amount to the subject in need of one or more agonists of any one of embodiments 1-5, the vector of embodiment 6 or 7, or the composition of embodiment 8, thereby treating the autoimmune disease, optionally wherein the subject in need thereof is a human, and optionally wherein the administering is done subcutaneously, orally, and/or intravenously.
  • a method of inhibiting the function of one or more autoimmunity-associated T cells or T cell populations comprising administering an effective amount of one or more agonists of any one of embodiments 1-5, the vector of embodiment 6 or 7, or the composition of embodiment 8 to a subject in need thereof, thereby inhibiting the function of one or more autoimmunity-associated T cells or T cell populations, optionally wherein the function is Tph/Tfh differentiation or B cell recruitment, optionally wherein the subject in need thereof is a human, and optionally wherein the administering is done subcutaneously, orally, and/or intravenously.
  • autoimmune disease is selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti -neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti -neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • autoimmune disease is rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • a method of treating an autoimmune disease in a subject in need thereof comprising obtaining a population of T cells from the subject, treating the population of T cells with an AHR agonist, and administering an effective amount of the treated population of T cells to the subject, thereby treating the autoimmune disease, optionally wherein the subject in need thereof is a human, optionally wherein the administering is done subcutaneously, orally, and/or intravenously.
  • JUN function 19.
  • a transcription factor selected from AHR, JUN, FOS, ATF3, FOSL1, FOSL2, and BATF, wherein the transcription factor negatively regulates Tfh and Tph cell function in autoantibody driven autoimmune disease.
  • JUN The transcription factor JUN, wherein JUN negatively regulates Tfh and Tph cell function in autoantibody driven autoimmune disease.
  • AAV adeno-associated virus
  • composition comprising the transcription factor of embodiment 19 or 20 or the vector of embodiment 21 or 22, and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.
  • a method of preventing CXCL13+ Tph/Tfh cell generation comprising contacting a cell population with one or more transcription factors of embodiment 19 or 20, vectors of embodiment 21 or 22, or composition of embodiment 23, thereby preventing CXCL13+ Tph/Tfh cell generation, optionally wherein the contacting is in vivo, in vitro, in situ, or ex vivo.
  • a method of inhibiting the function of one or more autoimmunity-associated T cells or T cell populations comprising administering an effective amount of one or more transcription factors of embodiment 19 or 20, vectors of embodiment 21 or 22, or composition of embodiment 23 to a subject in need thereof, thereby inhibiting the function of one or more autoimmunity-associated T cells or T cell populations, optionally wherein the function is Tph/Tfh differentiation or B cell recruitment, and optionally wherein the subject in need thereof is a human.
  • a method of treating an autoimmune disease in a subject in need thereof comprising administering an effective amount of one or more transcription factors of embodiment 19 or 20, vectors of embodiment 21 or 22, or composition of embodiment 23 to the subject in need thereof, thereby treating the autoimmune disease, wherein the transcription factor causes a target gene in the subject to be overexpressed, optionally wherein the subject in need thereof is a human, and optionally wherein the administering is done subcutaneously, topically, orally, and/or intravenously.
  • autoimmune disease is selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti -neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti -neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • a method of treating an autoimmune disease in a subject in need thereof comprising administering to the subject in need thereof: a) one or more transcription factor of embodiment 19 or 20, vector of embodiment 21 or 22, or composition of embodiment 23, wherein the transcription factor causes a target gene in the subject to be overexpressed; and b) one or more AHR agonist of any of embodiment 1-5, the vector of embodiment 6 or 7, or the composition of embodiment 8, thereby treating the autoimmune disease, optionally wherein the transcription factor is JUN, optionally wherein the one or more transcription factor and one or more AHR agonist are administered consecutively or sequentially, optionally wherein the subject in need thereof is a human, and optionally wherein the administering is done subcutaneously, topically, orally, and/or intravenously.
  • autoimmune disease is selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • a method of inhibiting B cell differentiation in a population of cells comprising contacting the population of cells with at least one of the AHR agonists of any of embodiments 1-5, the vector of embodiment 6 or 7, the composition of embodiment 8, transcription factors of embodiment 19 or 20, vectors of embodiment 21 or 22, or compositions of embodiment 23, thereby inhibiting B cell differentiation, optionally wherein the contacting takes place in vivo, in vitro, in situ or ex vivo.
  • a method of inhibiting B cell differentiation in a subject in need thereof comprising administering to the subject in need thereof an effective amount of at least one of the AHR agonists of any of embodiments 1-5, the vector of embodiment 6 or 7, the composition of embodiment 8, transcription factor of embodiment 19 or 20, vector of embodiment 21 or 22, or compositions of embodiment 23, thereby inhibiting B cell differentiation, optionally wherein the administering is done subcutaneously, topically orally, and/or intravenously, , optionally wherein the subject in need thereof is a human.
  • a CRISPR-Cas9-gRNA ribonucleoprotein (crRNP) complex comprising at least one gRNA of embodiment 33.
  • a vector comprising the crRNP complex of embodiment 34 optionally wherein the vector is, comprises, or is derived from a plasmid, an adenovirus, and adeno-associated virus (AAV), a retrovirus, a herpes simplex virus, a human immunodeficiency virus (HIV), a lentivirus, or a synthetic vector.
  • AAV adeno-associated virus
  • a composition comprising the crRNP complex of embodiment 34, or the vector of embodiment 35, and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.
  • a method of decreasing CXCL13 production in a cell or cell population comprising contacting the cell or cell population with the crRNP complex of embodiment 34, the vector of embodiment 35, or the composition of embodiment 36, thereby decreasing CXCL13 production, optionally where the contacting is in vivo, in vitro, in situ, or ex vivo.
  • a method of treating an autoimmune disease in a subject in need thereof comprising administering to the subject in need thereof an effective amount of the crRNP complex of embodiment 34, the vector of embodiment 35, or the composition of embodiment 36, thereby treating the autoimmune disease, optionally wherein the subject in need thereof is a human, and optionally wherein the administering is done subcutaneously, orally, and/or intravenously.
  • autoimmune disease is selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren disease, auto-antibody driven autoimmune disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Sjogren disease auto-antibody driven autoimmune disease
  • anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis juvenile idiopathic arthritis, ulcerative colitis, or systemic sclerosis.
  • Bocharnikov A. V. et al. PD-lhiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21. JCI Insight 4 (2019).
  • JNK1 a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025-1037 (1994). Doi.org: 10.1016/0092-8674(94)90380-8
  • T follicular helper (Tfh) cells in lupus Activation and involvement in SLE pathogenesis. Eur J Immunol 46, 281-290 (2016).
  • Lin, L, Yu, Y., Ma, I, Ren, C. & Chen, W. PD-1+CXCR5-CD4+T cells are correlated with the severity of systemic lupus erythematosus.
  • Rheumatology (Oxford) 58, 2188-2192 (2019).
  • Doi.org 10.1084/jem.20211110 Crotty, S. Follicular Helper CD4 T Cells (TFH). Annual Review of Immunology 29, 621-663 (2011).
  • Doi.org 10.1146/annurev-immunol-031210-101400 Aringer, M. et al. 2019 European League against Rheumatism/ American College of Rheumatology Classification Criteria for Systemic Lupus Erythematosus. Arthritis Rheumatol 'll, 1400-1412 (2019).
  • Doi.org 10.1038/nbt.3437 Yu, G., Wang, L. G. & He, Q. Y. ChlPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31, 2382-2383 (2015).
  • Doi.org 10.1093/bioinformatics/btvl45 Morand, E. F. et al. Trial of Anifrolumab in Active Systemic Lupus Erythematosus. N Engl J et/ 382, 211-221 (2020). doi.org: 10.1056/NEJMoal912196 Nehar-Belaid, D. et al. Mapping systemic lupus erythematosus heterogeneity at the single-cell level. Nat Immunol 21, 1094-1106 (2020).

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Abstract

L'invention concerne un agoniste du récepteur aryl hydrocarbone (AHR), l'agoniste d'AHR régulant négativement la différenciation des lymphocytes T ainsi que des méthodes d'utilisation d'un tel agoniste d'AHR. L'invention concerne également des méthodes d'utilisation de facteurs de transcription et d'inactivation génique pour réguler négativement la différenciation des lymphocytes T.
PCT/US2024/031858 2023-06-07 2024-05-31 Ciblage de lymphocytes t associés à l'auto-immunité par régulation de facteurs de transcription Ceased WO2024253957A1 (fr)

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