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A subset of human regulatory T cells (Tregs) can secrete IFN-γ or IL-17 and thus share features of TH1 or TH17 effector cells, and lose suppressive function. The main factors driving this differentiation of Tregs towards a pro-inflammatory phenotype include IL-12 for TH1-like and IL-6 for TH17-type Tregs. In this study we show that Tregs of patients with de novo autoimmune hepatitis (dAIH) display increased frequencies of pro-inflammatory IFN-γ and IL-17 cytokines. Irrespective of a fully demethylated FOXP3 locus, Tregs of subjects with de novo autoimmune hepatitis are functionally impaired. In line with the observed Treg phenotype, we detected the presence of two dominant cytokines (IL-12 and IL-6) clustering with CD68+ monocyte/macrophage cells in livers of subjects with de novo autoimmune hepatitis and isolated monocytes of subjects with de novo autoimmune hepatitis secrete high levels of pro-inflammatory IL-12 and IL-6 suggesting that this inflammatory milieu is key for functional impairment of Tregs. Importantly, the blockade of IFN-γ partially restores suppressive function of Tregs of subjects with de novo autoimmune hepatitis indicating that monocyte/macrophage derived triggers might play a central role in Treg dysfunction and pathogenesis of de novo autoimmune hepatitis.

CD226 costimulatory signals strongly promote Th1 differentiation, enhancing IFN-γ production by naive T cells. We recently reported that knockdown of CD226 on human T cells resulted in a decrease in T-bet and IFN-γ expression. However, the role of CD226 on Th2 and Th17 cells remains unknown. In this study, we found that CD226 and its ligand CD155 were decreased on Th2-polarized naive T cells, whereas both were highly expressed under Th17 conditions. Most IFN-γ– and IL-17–producing cells expressed high levels of CD226, but production of IL-13 did not correlate with CD226 expression. CD226 knockdown by lentiviral transduction resulted in increased STAT-6 phosphorylation, enhanced GATA3 expression, and consequently higher production of IL-4 and IL-13. Under Th17 conditions, CD226-depleted cells showed slightly impaired IL-17 secretion, suggesting that CD226 contributes, in part, to IL-17 production but is dispensable for Th17 cell generation. In line with these results, CD226 blockade with neutralizing Abs efficiently inhibited T cell activation and proliferation and production of IFN-γ and IL-17, whereas IL-13 secretion remained functional. Taken together, our results establish an important role for CD226 in differentially regulating the proinflammatory (Th1/Th17)/anti-inflammatory (Th2) balance, suggesting that the CD226/CD155 interaction could potentially be targeted in therapeutic approaches to human autoimmune diseases.

T cell Ig and ITIM domain (TIGIT) is a newly identified receptor expressed on T cells that binds to CD155 on the dendritic cell surface, driving them to a more tolerogenic phenotype. Given that TIGIT contains an ITIM motif in its intracellular domain and considering the potential importance of the TIGIT/CD226 pathway in human autoimmune disease, we investigated the specific role of TIGIT in human CD4+ T cells. Using an agonistic anti-TIGIT mAb, we demonstrate a direct inhibitory effect on T cell proliferation with a decrease in expression of T-bet, GATA3, IFN regulatory factor 4, and retinoic acid-related orphan receptor c with inhibition of cytokine production, predominantly IFN-γ. Knockdown of TIGIT expression by short hairpin RNA resulted in an increase of both T-bet and IFN-γ mRNA and protein expression with concomitant decrease in IL-10 expression. Increases in IFN-γ with TIGIT knockdown could be overcome by blocking CD226 signaling, indicating that TIGIT exerts immunosuppressive effects by competing with CD226 for the same CD155 ligand. These data demonstrate that TIGIT can inhibit T cell functions by competing with CD226 and can also directly inhibit T cells in a T cell-intrinsic manner. Our results provide evidence for a novel role of this alternative costimulatory pathway in regulating human T cell responses associated with autoimmune disease.

Genome-wide association studies have revealed a large number of genetic associations with autoimmune diseases. Despite this progress, the mechanisms underlying the contribution of allelic variants to the onset of immune-related diseases remain mostly unknown. Our recent meta-analysis of genome-wide association studies of multiple sclerosis (MS) identified a new susceptibility locus tagged by a single nucleotide polymorphism, rs17824933 (p = 3.8 × 10−9), that is found in a block of linkage disequilibrium containing the CD6 gene. Because CD6 plays an important role in maintenance of T cell activation and proliferation, we examined the biologic phenotypes of the risk-associated allele. In this article, we report that the MS susceptibility allele in CD6 is associated with decreased expression of full-length CD6 in CD4+ and CD8+ T cells. As a consequence, proliferation is diminished during long-term activation of CD4+ T cells from subjects with the risk allele. Selective knockdown of full-length CD6 using exon 5-specific small interfering RNA induces a similar proliferation defect of CD4+ T cells from subjects homozygous for the protective allele. Exon 5 encodes for the extracellular binding site of the CD6 ligand ALCAM, which is required for CD6 stimulation. In CD4+ T cells from subjects with the risk allele, exon 5 is consistently underexpressed, thereby providing a mechanism by which the allele affects proliferation of CD4+ T cells. These findings indicate that the MS risk allele in the CD6 locus is associated with altered proliferation of CD4+ T cells and demonstrate the influence of a disease-related allelic variant on important immunological characteristics.

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG97–109(107E-S) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG97–109-reactive CD4+ T cells, isolated with DRB1*0401/MOG97–109 tetramers, and after a short-term culture of PMBCs with MOG97–109 peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG97–109(107E-S) tetramer+ cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG97–109-reactive T cells is greater in patients with MS as compared with healthy controls.

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-β and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-β during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-β to the Th17-inducing cytokines (IL-1β, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-β–dependent coexpression of IL-9 and IL-17 was identified to be IL-1β. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1β. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9+IL-17+ cells. These data demonstrate the presence of IL-17+IL-9+ CD4 cells induced by IL-1β that may play a role in human autoimmune disease.

Autoimmune diseases including type 1 diabetes (T1D) are thought to have a Th1/Th17 bias. The underlying mechanisms driving the activation and differentiation of these proinflammatory T cells are unknown. We examined the monocytes isolated directly from the blood of T1D patients and found they spontaneously secreted the proinflammatory cytokines IL-1β and IL-6, which are known to induce and expand Th17 cells. Moreover, these in vivo-activated monocytes from T1D subjects induced more IL-17-secreting cells from memory T cells compared with monocytes from healthy control subjects. The induction of IL-17-secreting T cells by monocytes from T1D subjects was reduced in vitro with a combination of an IL-6-blocking Ab and IL-1R antagonist. In this study, we report a significant although modest increase in the frequency of IL-17-secreting cells in lymphocytes from long-term patients with T1D compared with healthy controls. These data suggest that the innate immune system in T1D may drive the adaptive immune system by expanding the Th17 population of effector T cells.

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c+ and CD11b+ cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.

The inflammatory myopathies are putative autoimmune disorders characterized by muscle weakness and the presence of intramuscular inflammatory infiltrates. Although inclusion body myositis and polymyositis have been characterized as cytotoxic CD8+ T cell-mediated diseases, we recently demonstrated high frequencies of CD138+ plasma cells in the inflamed muscle tissue of patients with these diseases. To gain a deeper understanding of the role these B cell family members play in the disease pathology, we examined the molecular characteristics of the H chain portion of the Ag receptor. Biopsies of muscle tissue were sectioned and tissue regions and individual cells were isolated through laser capture microdissection. Ig H chain gene transcripts isolated from the sections, regions, and cells were used to determine the variable region gene sequences. Analysis of these sequences revealed clear evidence of affinity maturation in that significant somatic mutation, isotype switching, receptor revision, codon insertion/deletion, and oligoclonal expansion had occurred within the B and plasma cell populations. Moreover, analysis of tissue regions isolated by laser capture microdissection revealed both clonal expansion and variation, suggesting that local B cell maturation occurs within muscle. In contrast, sequences from control muscle tissues and peripheral blood revealed none of these characteristics found in inflammatory myopathy muscle tissue. Collectively, these data demonstrate that Ag drives a B cell Ag-specific response in muscle in patients with dermatomyositis, inclusion body myositis, and polymyositis. These findings highlight the need for a revision of the current paradigm of exclusively T cell-mediated intramuscular Ag-specific autoimmunity in inclusion body myositis and polymyositis.

Multiple sclerosis (MS) is postulated to be a T cell-mediated autoimmune disease characterized clinically by a relapsing-remitting (RR) stage followed by a secondary progressive (SP) phase. The progressive phase is felt to be secondary to neuronal degenerative changes triggered by inflammation. The status of the innate immune system and its relationship to the stages of MS is not well understood. Dendritic cells (DCs) are professional APCs that are central cells of the innate immune system and have the unique capacity to induce primary immune responses. We investigated circulating myeloid DCs isolated directly from the blood to determine whether there were abnormalities in myeloid DCs in MS and whether they were related to disease stage. We found that SP-MS subjects had an increased percentage of DCs expressing CD80, a decreased percentage expressing PD-L1, and an increased percentage producing IL-12 and TNF-α compared with RR-MS or controls. A higher percentage of DCs from both RR and SP-MS patients expressed CD40 compared with controls. We then investigated the polarization effect of DCs from MS patients on naive T cells taken from cord blood using a MLR assay. Whereas DCs from RR-MS induced higher levels of Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-13) cytokines compared with controls, DCs from SP-MS only induced a polarized Th1 response. These results demonstrate abnormalities of DCs in MS and may explain the immunologic basis for the different stages and clinical patterns of MS.

It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4+CD25high regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+CD25high T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II− CD4+CD25high Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25high Treg express higher levels of Foxp3 message and protein, compared with the DR−CD25high Treg population. Direct single-cell cloning of CD4+CD25high Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25high, and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25high identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and αB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD+CD25+ T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4+ population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4+CD25+ regulatory cells isolated in mice. With TCR cross-linking, CD4+CD25high cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4+CD25− responder T cells in a contact-dependent manner. The CD4+CD25high regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RBlow on murine CD4+CD25+ regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4+CD25high regulatory cells and CD4+CD25− responder cells. Whereas higher ratios of CD4+CD25high T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.

Antibiotic treatment-resistant Lyme arthritis is a chronic inflammatory joint disease that follows infection with Borrelia burgdorferi (Bb). A marked Ab and T cell response to Bb outer surface protein A (OspA) often develops during prolonged episodes of arthritis. Furthermore, cross-reaction between the bacterial OspA and human LFA-1αL at the T cell level and the inability to detect Bb in the joint implicate an autoimmune mechanism. To analyze the nature of response to OspA and LFA-1αL, we used OspA-specific T cell hybrids from DR4 transgenic mice, as well as cloned human cells specific for OspA165–184, the immunodominant epitope, from five DRB1*0401+ patients, using OspA-MHC class II tetramers. Although OspA165–184 stimulated nearly all OspA-specific human T cell clones tested to proliferate and secrete IFN-γ and IL-13, LFA-1αL326–345 stimulated ∼10% of these clones to proliferate and a greater percentage to secrete IL-13. Assays with LFA- or OspA-DR4 monomers revealed that higher concentrations of LFA-DR4 were needed to stimulate dual-reactive T cell hybrids. Our analysis at the clonal level demonstrates that human LFA-1αL326–345 behaves as a partial agonist, perhaps playing a role in perpetuating symptoms of arthritis.

Rapamycin is a fungal macrolide that inhibits the proliferation of T cells. Studies in both animals and humans have found that rapamycin significantly reduces graft rejection. However, though CD8+ T cells are involved in graft infiltration and rejection, little is known regarding the effects of rapamycin on CD8+ human T cell responses. In this study, we examined the mechanism of rapamycin-induced inhibition of Ag-driven activation of CD8+ T cells. Surprisingly, a heterogeneous proliferative response in the presence of rapamycin was observed among different Ag-specific CD8+ T cell clones; this was also observed in CD8+ peripheral blood T cells activated with TCR cross-linking ex vivo. Inhibition of T cell proliferation by rapamycin was controlled by both the strength of signal delivered through the Ag receptor as well as the specific costimulatory signals received by the T cell. Rapamycin-resistant proliferation occurred despite inhibition of p70s6 kinase activity. Moreover, rapamycin-resistant proliferation of the CD8+ T cell clones was blocked by anti-IL-2 Abs, suggesting that while some of the parallel pathways triggered by IL-2R signaling are sensitive to the effects of rapamycin, others account for the Ag-driven rapamycin resistance. These data provide a new framework for examining the specific mechanism of action of rapamycin in human disease.

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11–19-reactive CD8+ T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11–19-reactive T cell clones by single cell sorting of HLA-A2/Tax11–19 tetramer binding CD8+ T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8+ T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.

NK T cells are a T cell subset in the human that express an invariant α-chain (Vα24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on Vα24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of Vα24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of Vα24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated Vα24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The Vα24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of Vα24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the Vα24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between Vα24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.

A novel population of both IFN-γ- and IL-10-secreting human T cells that differentiate in the presence of exogenous IL-12 in vitro has recently been described. Whether this T cell population exists in vivo is unknown. Borrelia burgdorferi, the etiologic agent of Lyme disease, can induce a chronic infection in the presence of a vigorous humoral immune response. We established T cell lines specific for B. burgdorferi and tetanus toxoid from subjects with chronic B. burgdorferi infection and healthy controls in limiting dilution experiments and assessed proliferation and cytokine secretion. As expected, higher frequencies of B. burgdorferi-specific precursor T cells were observed in Lyme patients compared with controls. In both groups of subjects, T cell lines specific for B. burgdorferi secreted high amounts of IFN-γ. However, in patients with Lyme disease, 27% of T cell lines secreted not only IFN-γ but also IL-10, which was only observed in 0.6% of B. burgdorferi-reactive T cell lines generated from controls and in none of the tetanus toxoid-reactive T cell lines generated from either Lyme patients and controls. Single cell PHA cloning confirmed that both cytokines were secreted from one clonally expanded precursor cell. Whole mononuclear cells from B. burgdorferi-infected individuals, but not from controls, secreted IL-12. Moreover, neutralizing anti-IL-12 mAbs inhibited the generation of the IFN-γ/IL-10 population. These data demonstrate that this novel population of IL-12-induced IFN-γ/IL-10-secreting T cells is generated in response to chronic B. burgdorferi infection.

Multiple sclerosis (MS) is an inflammatory disease of the myelinated central nervous system that is postulated to be induced by myelin-reactive CD4 T cells. T cell activation requires an antigen-specific signal through the TCR and a costimulatory signal, which can be mediated by B7–1 or B7–2 engagement of CD28. To directly examine the activation state of myelin-reactive T cells in MS, the costimulation requirements necessary to activate myelin basic protein (MBP) or tetanus toxoid (TT)-reactive CD4 T cells were compared between normal controls and MS patients. Peripheral blood T cells were stimulated with Chinese hamster ovary (CHO) cells transfected either with DRB1*1501/DRA0101 chains (t-DR2) alone, or in combination with, B7–1 or B7–2. In the absence of costimulation, T cells from normal subjects stimulated with the recall antigen TT p830–843 were induced to expand and proliferate, but stimulation with MBP p85–99 did not have this effect. In marked contrast, T cells from patients with MS stimulated with MBP p85–99 in the absence of B7–1 or B7–2 signals expanded and proliferated. Thus, MBP-reactive CD4 T cells in patients with MS are costimulation independent and have been previously activated in vivo. These experiments provide further direct evidence for a role of activated MBP-specific CD4 T cells in the pathogenesis of MS.

In the current issue of The Journal of Immunology , the letter from Swiatek-de Lange et al. ([1][1]) presents data confirming our observation that class II expressing CD25high human natural regulatory T cells (nTregs) express the highest levels of FoxP3. We welcome additional studies into this nTreg