Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3fl/fl ) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses. Th1 cells | M1 polarization

Regulatory T cells (Treg) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Treg (tTreg) and CD4+FOXP3+Helios− T cells, followed by comparison to CD4+FOXP3−Helios− T conventional (Tconv) cells. These analyses revealed that the coinhibitory receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) was highly expressed on tTreg. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4+CD25+CD127lo/− T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Treg following activation and in vitro expansion and is associated with lineage stability and suppressive capacity. Conversely, the CD226+TIGIT− population was associated with reduced Treg purity and suppressive capacity following expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Treg in health and disease.

The ability to alter antigen specificity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inflammation by redirecting the specificity of regulatory T cells (Tregs). Herein, we demonstrate efficient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction of antigen-specific tolerance to halt β-cell destruction. We generated human Tregs expressing a high-affinity GAD555–567-reactive TCR (clone R164), as well as the lower affinity clone 4.13 specific for the same peptide. We demonstrated that de novo Treg avatars potently suppress antigen-specific and bystander responder T-cell (Tresp) proliferation in vitro in a process that requires Treg activation (P < 0.001 versus unactivated Tregs). When Tresp were also glutamic acid decarboxylase (GAD)-reactive, the high-affinity R164 Tregs exhibited increased suppression (P < 0.01) with lower Tresp-division index (P < 0.01) than the lower affinity 4.13 Tregs. These data demonstrate the feasibility of rapid expansion of antigen-specific Tregs for applications in attenuating β-cell autoimmunity and emphasize further opportunities for engineering cellular specificities, affinities, and phenotypes to tailor Treg activity in adoptive cell therapies for the treatment of type 1 diabetes.

The costimulatory molecule CD226 is highly expressed on effector/memory T cells and natural killer cells. Costimulatory signals received by T cells can impact both central and peripheral tolerance mechanisms. Genetic polymorphisms in CD226 have been associated with susceptibility to type 1 diabetes and other autoimmune diseases. We hypothesized that genetic deletion of Cd226 in the non-obese diabetic (NOD) mouse would impact type 1 diabetes incidence by altering T cell activation. CD226 knockout (KO) NOD mice displayed decreased disease incidence and insulitis in comparison to wild-type (WT) controls. Although female CD226 KO mice had similar levels of sialoadenitis as WT controls, male CD226 KO mice showed protection from dacryoadenitis. Moreover, CD226 KO T cells were less capable of adoptively transferring disease compared to WT NOD T cells. Of note, CD226 KO mice demonstrated increased CD8 + single positive (SP) thymocytes, leading to increased numbers of CD8 + T cells in the spleen. Decreased percentages of memory CD8 + CD44 + CD62L − T cells were observed in the pancreatic lymph nodes of CD226 KO mice. Intriguingly, CD8 + T cells in CD226 KO mice showed decreased islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-tetramer and CD5 staining, suggesting reduced T cell receptor affinity for this immunodominant antigen. These data support an important role for CD226 in type 1 diabetes development by modulating thymic T cell selection as well as impacting peripheral memory/effector CD8 + T cell activation and function.

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The transcription factor Tbet is critical for the differentiation of Th1 CD4 T cells and is associated with the induction of multiple autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate that Tbet suppresses IL-17A and Th17 differentiation both in vitro and in vivo in a cell-intrinsic manner, and that in fact, Tbet is not necessary for EAE induction. Moreover, we find that IFNγ inhibits the production of IL-17A and IL-17F in a STAT1-dependent, Tbet-independent manner. These findings illustrate multiple mechanisms utilized by developing Th1 cells to silence the Th17 program.

The inheritance of one defective BRCA1 or BRCA2 allele predisposes an individual to developing breast and ovarian cancers. BRCA1 is a multifunctional tumor suppressor protein, which through interaction with a vast array of proteins has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. Conversely, the oncogene, cyclin D1 is overexpressed in about 35% of all breast cancer cases. In this study, we provide detailed analyses on the phosphorylation state of BRCA1 by cyclin D1/cdk4 complexes. In particular, we have identified Ser 632 of BRCA1 as a cyclin D1/cdk4 phosphorylation site in vitro. Using chromatin immunoprecipitation assays, we observed that the inhibition of cyclin D1/cdk4 activity resulted in increased BRCA1 DNA binding at particular promoters in vivo. In addition, we identified multiple novel genes that are bound by BRCA1 in vivo. Collectively, these results indicate that cyclin D1/cdk4-mediated phosphorylation of BRCA1 inhibits the ability of BRCA1 to be recruited to particular promoters in vivo. Therefore, cyclin D1/Cdk4 phosphorylation of BRCA1 could provide a mechanism to interfere with the DNA-dependent activities of BRCA1.

Many autoimmune chronic inflammatory diseases, including multiple sclerosis, are associated with the presence of Th1 and Th17 effector CD4 T cells. Paradoxically, the principal Th1 cytokine IFNγ does not appear necessary for disease, but the key Th1-associated transcription factor Tbet has been reported to be essential for disease development. This conundrum propelled us to investigate the regulation of this transcription factor during autoimmunity. Following the onset of experimental autoimmune encephalomyelitis (EAE), we observed a preferential upregulation of Tbet by CD4 T cells within the CNS, but not the secondary lymphoid organs. These Tbet-positive CD4 T cells were capable of producing the cytokine IFNγ, and a proportion of these cells produced both IFNγ and IL-17A. Interestingly, these Tbet-positive cells were present in high frequencies during disease in IFNγ-deficient mice. Moreover, we found that CD4 T cells from IFNγ-deficient/IFNγ reporter mice upregulated the Thy1.1 reporter, indicating the presence of Th1 or “Th1-like”, Tbet-positive CD4 T cells even in the absence of the cardinal Th1 cytokine, IFNγ. These IFNγ-deficient “Th1-like” cells not only maintain multiple Th1 properties but also exhibit increased expression of genes associated with the Th17 phenotype. We further examined the requirement of other Th1-associated molecules in controlling Tbet expression during EAE, and noted that STAT1, IL-12, and IFNγ were dispensable for the induction of Tbet in vivo. Hence, this study highlights the complex regulation of Tbet and the potential unrecognized role for Th1 cells during autoimmunity.