Metabolic modeling of single Th17 cells reveals regulators of autoimmunity.

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.

Targeting PGLYRP1 promotes antitumor immunity while inhibiting autoimmune neuroinflammation.

Co-inhibitory and checkpoint molecules suppress T cell function in the tumor microenvironment, thereby rendering T cells dysfunctional. Although immune checkpoint blockade is a successful treatment option for multiple human cancers, severe autoimmune-like adverse effects can limit its application. Here, we show that the gene encoding peptidoglycan recognition protein 1 (PGLYRP1) is highly coexpressed with genes encoding co-inhibitory molecules, indicating that it might be a promising target for cancer immunotherapy. Genetic deletion of Pglyrp1 in mice led to decreased tumor growth and an increased activation/effector phenotype in CD8+ T cells, suggesting an inhibitory function of PGLYRP1 in CD8+ T cells. Surprisingly, genetic deletion of Pglyrp1 protected against the development of experimental autoimmune encephalomyelitis, a model of autoimmune disease in the central nervous system. PGLYRP1-deficient myeloid cells had a defect in antigen presentation and T cell activation, indicating that PGLYRP1 might function as a proinflammatory molecule in myeloid cells during autoimmunity. These results highlight PGLYRP1 as a promising target for immunotherapy that, when targeted, elicits a potent antitumor immune response while protecting against some forms of tissue inflammation and autoimmunity.

CD5L/AIM Regulates Lipid Biosynthesis and Restrains Th17 Cell Pathogenicity.

Th17 cells play a critical role in host defense against extracellular pathogens and tissue homeostasis but can induce autoimmunity. The mechanisms implicated in balancing "pathogenic" and "non-pathogenic" Th17 cell states remain largely unknown. We used single-cell RNA-seq to identify CD5L/AIM as a regulator expressed in non-pathogenic, but not in pathogenic Th17 cells. Although CD5L does not affect Th17 differentiation, it is a functional switch that regulates the pathogenicity of Th17 cells. Loss of CD5L converts non-pathogenic Th17 cells into pathogenic cells that induce autoimmunity. CD5L mediates this effect by modulating the intracellular lipidome, altering fatty acid composition and restricting cholesterol biosynthesis and, thus, ligand availability for Rorγt, the master transcription factor of Th17 cells. Our study identifies CD5L as a critical regulator of the Th17 cell functional state and highlights the importance of lipid metabolism in balancing immune protection and disease induced by T cells.

T cell deletional tolerance restricts AQP4 but not MOG CNS autoimmunity.

Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/ß usage revealed that AQP4-specific T cells from AQP4-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation.

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Induction and molecular signature of pathogenic TH17 cells.

Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are often present at the sites of tissue inflammation in autoimmune diseases, which has led to the conclusion that T(H)17 cells are main drivers of autoimmune tissue injury. However, not all T(H)17 cells are pathogenic; in fact, T(H)17 cells generated with transforming growth factor-ß1 (TGF-ß1) and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we found that the production of TGF-ß3 by developing T(H)17 cells was dependent on IL-23, which together with IL-6 induced very pathogenic T(H)17 cells. Moreover, TGF-ß3-induced T(H)17 cells were functionally and molecularly distinct from TGF-ß1-induced T(H)17 cells and had a molecular signature that defined pathogenic effector T(H)17 cells in autoimmune disease.

T Follicular Regulatory Cell-Derived Fibrinogen-like Protein 2 Regulates Production of Autoantibodies and Induction of Systemic Autoimmunity.

T follicular regulatory (TFR) cells limit Ab responses, but the underlying mechanisms remain largely unknown. In this study, we identify Fgl2 as a soluble TFR cell effector molecule through single-cell gene expression profiling. Highly expressed by TFR cells, Fgl2 directly binds to B cells, especially light-zone germinal center B cells, as well as to T follicular helper (TFH) cells, and directly regulates B cells and TFH in a context-dependent and type 2 Ab isotype-specific manner. In TFH cells, Fgl2 induces the expression of Prdm1 and a panel of checkpoint molecules, including PD1, TIM3, LAG3, and TIGIT, resulting in TFH cell dysfunction. Mice deficient in Fgl2 had dysregulated Ab responses at steady-state and upon immunization. In addition, loss of Fgl2 results in expansion of autoreactive B cells upon immunization. Consistent with this observation, aged Fgl2-/- mice spontaneously developed autoimmunity associated with elevated autoantibodies. Thus, Fgl2 is a TFR cell effector molecule that regulates humoral immunity and limits systemic autoimmunity.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

IL-11+CD4+ cells accumulate in the cerebrospinal fluid of patients with early relapsing-remitting multiple sclerosis (MS) and in active brain MS lesions. Mouse studies have confirmed a causal role of IL-11 in the exacerbation of relapsing-remitting experimental autoimmune encephalomyelitis (RREAE). Administration of IL-11 at the time of clinical onset of RREAE induced an acute exacerbation and increased clinical scores, which persisted during the entire course of the disease. IL-11 increased the numbers of spinal cord inflammatory foci, as well as the numbers of peripheral and CNS-infiltrating IL-17+CD4+ cells and IL-17A serum levels. Ag recall assays revealed that IL-11 induces IL-17A+, GM-CSF+, and IL-21+CD4+ myelin Ag-reactive cells. Passive transfer of these encephalitogenic CD4+ T cells induced severe RREAE with IL-17A+CCR6+ CD4+ and B cell accumulation within the CNS. Furthermore, passive transfer of nonmanipulated CNS-derived mononuclear cells from mice with RREAE after a single dose of IL-11 induced severe RREAE with increased accumulation of IL-17A+ and CCR6+ CD4+ cells within the CNS. These results suggest that IL-11 might serve as a biomarker of early autoimmune response and a selective therapeutic target for patients with early relapsing-remitting MS.

IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells.

Transcription factor Foxp3 is critical for generating regulatory T cells (T(reg) cells). Transforming growth factor-beta (TGF-beta) induces Foxp3 and suppressive T(reg) cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible T(reg) cells. Here we show that IL-4 blocked the generation of TGF-beta-induced Foxp3(+) T(reg) cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9(+)IL-10(+) T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9(+)IL-10(+) T cells into recombination-activating gene 1-deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9(+)IL-10(+) T cells were transferred with CD45RB(hi) CD4(+) effector T cells. Thus IL-9(+)IL-10(+) T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.

Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation.

Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögren's syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.

Dendritic Cell PD-L1 Limits Autoimmunity and Follicular T Cell Differentiation and Function.

The programmed death (PD)-1 coinhibitory receptor regulates the balance between T cell activation and tolerance. Although the PD-1 ligands, PD-L1 and PD-L2, are expressed on a variety of cell types, the cell type-specific functions of PD-1 ligands in inducing signals through PD-1 are unknown. In this study, we use PD-L1 conditional knockout mice to investigate the cell type-specific functions of PD-L1. We demonstrate that PD-L1 expressed on dendritic cells (DCs), and to a lesser extent on B cells, attenuates the progression of experimental autoimmune encephalomyelitis and inhibits naive and effector T cells. PD-1 is highly expressed on effector populations, including T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells, which reside in germinal centers. We also show that DC PD-L1 is essential for limiting Tfh and Tfr cell differentiation. In addition, we find that PD-1 suppresses Tfh cell differentiation and help for Ig class switching, even in the presence of wild-type Tfr cells. Our work points to critical roles for PD-L1 expressed on DCs in mediating PD-1 functions.

Functional Anti-TIGIT Antibodies Regulate Development of Autoimmunity and Antitumor Immunity.

Coinhibitory receptors, such as CTLA-4 and PD-1, play a critical role in maintaining immune homeostasis by dampening T cell responses. Recently, they have gained attention as therapeutic targets in chronic disease settings where their dysregulated expression contributes to suppressed immune responses. The novel coinhibitory receptor TIGIT (T cell Ig and ITIM domain) has been shown to play an important role in modulating immune responses in the context of autoimmunity and cancer. However, the molecular mechanisms by which TIGIT modulates immune responses are still insufficiently understood. We have generated a panel of monoclonal anti-mouse TIGIT Abs that show functional properties in mice in vivo and can serve as important tools to study the underlying mechanisms of TIGIT function. We have identified agonistic as well as blocking anti-TIGIT Ab clones that are capable of modulating T cell responses in vivo. Administration of either agonist or blocking anti-TIGIT Abs modulated autoimmune disease severity whereas administration of blocking anti-TIGIT Abs synergized with anti-PD-1 Abs to affect partial or even complete tumor regression. The Abs presented in this study can thus serve as important tools for detailed analysis of TIGIT function in different disease settings and the knowledge gained will provide valuable insight for the development of novel therapeutic approaches targeting TIGIT.

Epitope Mapping of SERCA2a Identifies an Antigenic Determinant That Induces Mainly Atrial Myocarditis in A/J Mice.

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.

Liver-specific knockdown of long-chain acyl-CoA synthetase 4 reveals its key role in VLDL-TG metabolism and phospholipid synthesis in mice fed a high-fat diet.

Long-chain acyl-CoA synthetase 4 (ACSL4) has a unique substrate specificity for arachidonic acid. Hepatic ACSL4 is coregulated with the phospholipid (PL)-remodeling enzyme lysophosphatidylcholine (LPC) acyltransferase 3 by peroxisome proliferator-activated receptor δ to modulate the plasma triglyceride (TG) metabolism. In this study, we investigated the acute effects of hepatic ACSL4 deficiency on lipid metabolism in adult mice fed a high-fat diet (HFD). Adenovirus-mediated expression of a mouse ACSL4 shRNA (Ad-shAcsl4) in the liver of HFD-fed mice led to a 43% reduction of hepatic arachidonoyl-CoA synthetase activity and a 53% decrease in ACSL4 protein levels compared with mice receiving control adenovirus (Ad-shLacZ). Attenuated ACSL4 expression resulted in a substantial decrease in circulating VLDL-TG levels without affecting plasma cholesterol. Lipidomics profiling revealed that knocking down ACSL4 altered liver PL compositions, with the greatest impact on accumulation of abundant LPC species (LPC 16:0 and LPC 18:0) and lysophosphatidylethanolamine (LPE) species (LPE 16:0 and LPE 18:0). In addition, fasting glucose and insulin levels were higher in Ad-shAcsl4-transduced mice versus control (Ad-shLacZ). Glucose tolerance testing further indicated an insulin-resistant phenotype upon knockdown of ACSL4. These results provide the first in vivo evidence that ACSL4 plays a role in plasma TG and glucose metabolism and hepatic PL synthesis of hyperlipidemic mice.

Dimethyl fumarate treatment induces adaptive and innate immune modulation independent of Nrf2.

Dimethyl fumarate (DMF) (BG-12, Tecfidera) is a fumaric acid ester (FAE) that was advanced as a multiple sclerosis (MS) therapy largely for potential neuroprotection as it was recognized that FAEs are capable of activating the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, DMF treatment in randomized controlled MS trials was associated with marked reductions in relapse rate and development of active brain MRI lesions, measures considered to reflect CNS inflammation. Here, we investigated the antiinflammatory contribution of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE). C57BL/6 wild-type (WT) and Nrf2-deficient (Nrf2(-/-)) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (p35-55) for EAE induction and treated with oral DMF or vehicle daily. DMF protected WT and Nrf2(-/-) mice equally well from development of clinical and histologic EAE. The beneficial effect of DMF treatment in Nrf2(-/-) and WT mice was accompanied by reduced frequencies of IFN-γ and IL-17-producing CD4(+) cells and induction of antiinflammatory M2 (type II) monocytes. DMF also modulated B-cell MHC II expression and reduced the incidence of clinical disease in a B-cell-dependent model of spontaneous CNS autoimmunity. Our observations that oral DMF treatment promoted immune modulation and provided equal clinical benefit in acute EAE in Nrf2(-/-) and WT mice, suggest that the antiinflammatory activity of DMF in treatment of MS patients may occur through alternative pathways, independent of Nrf2.

Tolerance checkpoint bypass permits emergence of pathogenic T cells to neuromyelitis optica autoantigen aquaporin-4.

Aquaporin-4 (AQP4)-specific T cells are expanded in neuromyelitis optica (NMO) patients and exhibit Th17 polarization. However, their pathogenic role in CNS autoimmune inflammatory disease is unclear. Although multiple AQP4 T-cell epitopes have been identified in WT C57BL/6 mice, we observed that neither immunization with those determinants nor transfer of donor T cells targeting them caused CNS autoimmune disease in recipient mice. In contrast, robust proliferation was observed following immunization of AQP4-deficient (AQP4-/-) mice with AQP4 peptide (p) 135-153 or p201-220, peptides predicted to contain I-Ab-restricted T-cell epitopes but not identified in WT mice. In comparison with WT mice, AQP4-/- mice used unique T-cell receptor repertoires for recognition of these two AQP4 epitopes. Donor T cells specific for either determinant from AQP4-/-, but not WT, mice induced paralysis in recipient WT and B-cell-deficient mice. AQP4-specific Th17-polarized cells induced more severe disease than Th1-polarized cells. Clinical signs were associated with opticospinal infiltrates of T cells and monocytes. Fluorescent-labeled donor T cells were detected in CNS lesions. Visual system involvement was evident by changes in optical coherence tomography. Fine mapping of AQP4 p201-220 and p135-153 epitopes identified peptides within p201-220 but not p135-153, which induced clinical disease in 40% of WT mice by direct immunization. Our results provide a foundation to evaluate how AQP4-specific T cells contribute to AQP4-targeted CNS autoimmunity (ATCA) and suggest that pathogenic AQP4-specific T-cell responses are normally restrained by central tolerance, which may be relevant to understanding development of AQP4-reactive T cells in NMO.

Anti-CD48 Monoclonal Antibody Attenuates Experimental Autoimmune Encephalomyelitis by Limiting the Number of Pathogenic CD4+ T Cells.

CD48 (SLAMF2) is an adhesion and costimulatory molecule constitutively expressed on hematopoietic cells. Polymorphisms in CD48 have been linked to susceptibility to multiple sclerosis (MS), and altered expression of the structurally related protein CD58 (LFA-3) is associated with disease remission in MS. We examined CD48 expression and function in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We found that a subpopulation of CD4+ T cells highly upregulated CD48 expression during EAE and were enriched for pathogenic CD4+ T cells. These CD48++CD4+ T cells were predominantly CD44+ and Ki67+, included producers of IL-17A, GM-CSF, and IFN-γ, and were most of the CD4+ T cells in the CNS. Administration of anti-CD48 mAb during EAE attenuated clinical disease, limited accumulation of lymphocytes in the CNS, and reduced the number of pathogenic cytokine-secreting CD4+ T cells in the spleen at early time points. These therapeutic effects required CD48 expression on CD4+ T cells but not on APCs. Additionally, the effects of anti-CD48 were partially dependent on FcγRs, as anti-CD48 did not ameliorate EAE or reduce the number of cytokine-producing effector CD4+ T cells in Fcεr1γ-/- mice or in wild-type mice receiving anti-CD16/CD32 mAb. Our data suggest that anti-CD48 mAb exerts its therapeutic effects by both limiting CD4+ T cell proliferation and preferentially eliminating pathogenic CD48++CD4+ T cells during EAE. Our findings indicate that high CD48 expression is a feature of pathogenic CD4+ T cells during EAE and point to CD48 as a potential target for immunotherapy.

Identification of an Epitope from Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice.

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

Tim-1 is essential for induction and maintenance of IL-10 in regulatory B cells and their regulation of tissue inflammation.

T cell Ig and mucin domain (Tim)-1 identifies IL-10-producing regulatory B cells (Bregs). Mice on the C57BL/6 background harboring a loss-of-function Tim-1 mutant showed progressive loss of IL-10 production in B cells and with age developed severe multiorgan tissue inflammation. We demonstrate that Tim-1 expression and signaling in Bregs are required for optimal production of IL-10. B cells with Tim-1 defects have impaired IL-10 production but increased proinflammatory cytokine production, including IL-1 and IL-6. Tim-1-deficient B cells promote Th1 and Th17 responses but inhibit the generation of regulatory T cells (Foxp3(+) and IL-10-producing type 1 regulatory T cells) and enhance the severity of experimental autoimmune encephalomyelitis. Mechanistically, Tim-1 on Bregs is required for apoptotic cell (AC) binding to Bregs and for AC-induced IL-10 production in Bregs. Treatment with ACs reduces the severity of experimental autoimmune encephalomyelitis in hosts with wild-type but not Tim-1-deficient Bregs. Collectively, these findings suggest that in addition to serving as a marker for identifying IL-10-producing Bregs, Tim-1 is also critical for maintaining self-tolerance by regulating IL-10 production in Bregs.