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Final Report Summary - TR1 CELLS (Molecular mechanisms leading to generation of Type 1 regulatory cells and their role in autoimmunity)

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Around 2.5 million people worldwide have MS. Although the etiology of MS is still unclear, there is a good evidence suggesting that T cells are the main players during initiation of the pathogenic process. The myelin proteins on the myelin sheath are targeted by activated auto-reactive lymphocytes causing repeated inflammation, subsequent demyelination and symptoms of MS. It has been described, that generation of Type I regulatory (Tr1) cells is impaired in MS patients. Furthermore, in Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of human disease multiple sclerosis (MS), transfer of IL-10 producing Tr1 cells prevents CNS (central nervous system) inflammation in an IL-10 dependent manner. Together, these point to the crucial role of Tr1 cells during the course of MS. Tr1 cells were first described as a distinct subset of T cells that were induced in vitro by activating naïve T cells in the presence of IL-10 and were shown to regulate immune and autoimmune responses. They lack FoxP3 expression, the master transcription factor that induces Treg cells, but production of copious amounts of IL10 – their signature cytokine, makes them a subset of regulatory T cells that is distinct from FoxP3+ regulatory T cells. Their strong immunosuppressive capacity allows them to restrain autoimmune responses and alleviate pathology in several models of infectious and autoimmune diseases. IL-27 is the key inducer of Tr1 cells. Yet, the mechanism by which IL-27 mediates its suppressive effects has not been fully elucidated. The “Tr1 project” aims to undertake a detailed analysis of IL-27 mediated Tr1 induction with a view to identify targets that could be manipulated to suppress autoimmunity of the CNS.
Using a microarray analysis of cells treated in Tr1 conditions, the fellow identified two transcription factors, IRF1 and BATF, that are expressed early during differentiation of Tr1 cells and that are strictly required for Tr1 differentiation, as CD4+ T cells deficient in either of these factors fail to secrete IL-10. The fellow used an EAE model to assess whether IRF1 plays a role in vivo during development of autoimmunity. Batf-/- mice have been previously described as resistant to EAE due to failure to generate Th17 cells. Interestingly, as the disease in the wild type mice ameliorates after reaching its peak, the Irf1-/- mice do not undergo recovery but progressively develop more severe disease, and cells isolated from the knockout mice produce increased amounts of IL-17A, as well as decreased IL-10. Furthermore, Irf1-deficient 2D2 transgenic mice, which bear a transgenic MOG specific T cell receptor, developed spontaneous EAE at around 6 weeks of age, whereas the wild type 2D2 littermates remained free of clinical signs of disease. Furthermore, cells from Irf1-/- and Batf-/- mice treated in vitro with IL-27 and transferred into wild-type sick mice (immunized to induce EAE) failed to suppress the disease, while wild-type cells did reduce the EAE score. It has been previously reported that repeated injections of an anti-CD3 antibody lead to induction of Tr1 cells in vivo and that induction of IL-10 in these settings is mediated by IL-27. This model was used to assess the role of IRF1 and BATF in generation of Tr1 cells in vivo. An anti-CD3 antibody or an isotype control antibody were repeatedly administered to wild type, Irf1-/- and Batf-/- mice and the frequency of IL-10+ cells was assessed in mesenteric lymph nodes. Administration of the anti-CD3 antibody resulted in induction of IL-10+ cells in the lymph nodes of wild-type but not in Irf1-/- or Batf-/- mice.
The fellow further worked on elucidation of the molecular mechanisms used by IRF1 and BATF to control IL-10 expression. IRF1 and BATF were found to bind to multiple regulatory elements within the Il10 locus in a co-operative manner, meaning that binding of IRF1 is dependent on the presence of BATF, and vice versa. Moreover, IRF1 could directly transactivate multiple regulatory elements of the Il10 locus, whereas BATF could not. Transcription factors cMaf and AhR were previously shown to bind to and to transactivate the Il10 promoter. The study showed that both IRF1 and BATF are required for binding of cMaf and AhR to the Il10 locus.
Furthermore, deficiency of IRF1 or BATF induced changes in the epigenetic marks of CD4+ T cells from the knockout mice isolated from the knockout mice (reduction in the activating epigenetic marks within the Il10 locus (H3K9Ac, H3K4me3), as well as an increase in the inhibitory marks (H3K27me3)).
Those results pointed to the possibility of IRF1 and BATF being pioneering factors for differentiation of Tr1 cells. For this reason, the fellow analyzed chromatin accessibility using ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) in cells from WT, Irf1-/- and Batf-/- mice primed in Th0 (naïve) or Tr1 conditions collected at an early time point, 24 h, which coincides with induction of Il10 mRNA in cells treated with IL-27, and a late time point, 72 h, when the cells are already fully differentiated.
ATACseq analysis detected ~180,000 accessible regions (peaks) across samples. Chromatin landscape was most altered by time, polarization condition, and BATF deficiency, with each perturbation leading to upwards of 20,000 altered peak intensities. Principal component analysis (PCA) at 24h showed that all samples displayed relatively similar chromatin landscapes, with small differences between Tr1 and TH0 polarizations for Batf–/–, Irf1–/–, and control cells. At 72h, the TH0- vs. Tr1- dependent trends dominate, although the Batf–/– chromatin landscape resembles neither Tr1 nor TH0. At 72h, Batf–/– TH0 and Tr1 cells have a distinct chromatin accessibility pattern, whereas Tr1 cells derived from mice deficient in IRF1, AhR and cMaf show relatively similar chromatin accessibility patterns. Differential analysis with DESeq2 was performed to quantify the number of transcription factor-dependent accessible peaks per transcription factor in Tr1 conditions. BATF deficiency led to altered accessibility at over twenty thousand loci. IRF1 deficiency resulted in ~100 peaks with increased accessibility and 1100 peaks with decreased accessibility. In contrast, AhR- and cMaf-dependent alterations were minor. Moreover, IRF1 and BATF affected unique regions. Additionally, BATF-dependent chromatin regions with increased accessibility are visible in Batf–/– cells, suggesting that, while much of the Tr1 landscape is being “turned off”, new regions are also being ‘turned on’. This further supports a role for IRF1 and BATF as Tr1 pioneering factors. IRF1 is a smaller-scale/focused pioneer factor, whereas BATF acts globally.
Next, RNAseq analysis was employed to analyze the transcriptome of WT, Irf1-/- and Batf-/- CD4+ T cells differentiated in Tr1 conditions for 72hrs. The Tr1 expression signatures were strikingly different among Batf–/–, Irf1–/–, and control cells. In agreement with the ATAC-seq analysis, BATF deficiency led to the greatest number of differentially expressed genes (~500 genes, compared to ~110 for Irf1–/–). Deficiency of IRF1 and BATF resulted in large differences in transcription factor expression profiles, with down-regulation of the majority of transcription factors affected by BATF deficiency. Of these, only a few were also down-regulated in the IRF1-deficient cells and these shared transcription factors are reported to regulate IL-10 (e.g. Hif1a and Prdm1). Although Rorc is down-regulated in both IRF- and BATF-deficient cells, Rora is actually up-regulated in Irf1–/– samples. Consistent with the loss of suppressive function in Irf1–/– and Batf–/– cells differentiated into Tr1 cells, reduced levels of cytokines associated with Tr1 functions, such as Il10, and of Perforin, were observed under both conditions.
Finally, RNA-seq and ATAC-seq data sets were integrated to derive transcriptional regulatory networks for both IRF1- and BATF-deficient Tr1 cells. The changes induced by IRF1 deficiency are relatively limited. IRF1 putatively represses Klf7, as Klf7 is over-expressed in Irf1–/– cells and an accessible IRF1 motif is found cis to the Klf7 locus. The Klf7 motif, in turn, is enriched cis to genes with increased expression under IRF1-deficient conditions, such as Id3. Stat1 is also IRF1-dependent with an accessible IRF1 motif in cis, but Stat1 is a part of the Tr1 network that ‘turns off’ in Irf1-/- cells. Irf1 might also directly regulate expression of a number of genes, such as Il10, Ccr5, and Prf1. The BATF Tr1 network is more complex, involving sixteen transcription factors. Explanations for the part of the Tr1 BATF network that is ‘turned on’ can only be explained indirectly, by several transcription factors regulated downstream of BATF. The part of the Tr1 network that is ‘turned off’ is explained not only by putatively direct BATF targets but likely also involves secondary regulatory interactions, mediated by MAF, HIF1A, FOSL2, ETS1, RUNX2, KLF10, RORG, RORA. BATF induces expression of Klf10, Runx2, Prdm1, Hif1a, Fols2 and Ets1, whose expression is reduced in the Batf–/– cells. They, in turn, might regulate a number of other cytokines or receptors that we found differentially expressed in the Batf–/– T cells, such as Il10, Prf1, Icos, Il2, Csf2, Cxcr5, and Il1rn. The ‘off” network is connected to the ‘on’network by Runx2 and Klf10, which putatively repress the ‘on’ transcription factors Eomes, Csr2, Sp6, and several members of the NF-κB family. The Batf–/– Tr1 transcriptional regulatory model suggests a dramatically altered transcriptional state for BATF-deficient Tr1 cells, mediated by several layers of altered transcriptional regulatory interactions.
Overall, this study identifies the critical role of IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation.

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