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Maintenance of peripheral tolerance by CD4+Foxp3+ regulatory T cells (Tregs) is essential for regulating autoreactive T cells. The loss of function of Foxp3 leads to autoimmune disease in both animals and humans. An example is the rare, X-linked recessive disorder known as IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy X-linked) syndrome. In more common human autoimmune diseases, defects in Treg function are accompanied with aberrant effector cytokines such as IFNγ. It has recently become appreciated that Tregs plays an important role in not only maintaining immune homeostasis but also in establishing the tissue microenvironment and homeostasis of non-lymphoid tissues. Tissue resident Tregs show profiles that are unique to their local environments which are composed of both immune and non-immune cells. Core tissue-residence gene signatures are shared across different tissue Tregs and are crucial to homeostatic regulation and maintaining the tissue Treg pool in a steady state. Through interaction with immunocytes and non-immunocytes, tissue Tregs exert a suppressive function via conventional ways involving contact dependent and independent processes. In addition, tissue resident Tregs communicate with other tissue resident cells which allows Tregs to adopt to their local microenvironment. These bidirectional interactions are dependent on the specific tissue environment. Here, we summarize the recent advancements of tissue Treg studies in both human and mice, and discuss the molecular mechanisms that maintain tissue homeostasis and prevent pathogenesis.

Genome-wide association studies (GWAS) have identified genetic susceptibility loci for a variety of autoimmune and inflammatory diseases. These studies confirm the fundamental genetic basis of individual autoimmune diseases, and also point to shared etiological mechanisms across the spectrum of autoimmunity. While hundreds of genetic loci have been implicated in autoimmune diseases, the translation of individual susceptibility loci into specific molecular mechanisms for individual diseases remains difficult. This review highlights recent advances in the genetics of autoimmune disease, and the emerging use of epigenetic techniques to identify pathogenic cell types and causal molecular mechanisms of autoimmunity.

The T cell receptor (TCR) endows T cells with antigen specificity and is central to nearly all aspects of T cell function. Each naïve T cell has a unique TCR sequence that is stably maintained during cell division. In this way, the TCR serves as a molecular barcode that tracks processes such as migration, differentiation, and proliferation of T cells. Recent technological advances have enabled sequencing of the TCR from single cells alongside deep molecular phenotypes on an unprecedented scale. In this review, we discuss strengths and limitations of TCR sequences as molecular barcodes and their application to study immune responses following Programmed Death-1 (PD-1) blockade in cancer. Additionally, we consider applications of TCR data beyond use as a barcode.

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV-2 infection. We profiled MIS-C, adult COVID-19, and healthy pediatric and adult individuals using single-cell RNA sequencing, flow cytometry, antigen receptor repertoire analysis, and unbiased serum proteomics, which collectively identified a signature in MIS-C patients that correlated with disease severity. Despite having no evidence of active infection, MIS-C patients had elevated S100A-family alarmins and decreased antigen presentation signatures, indicative of myeloid dysfunction. MIS-C patients showed elevated expression of cytotoxicity genes in NK and CD8+ T cells and expansion of specific IgG-expressing plasmablasts. Clinically severe MIS-C patients displayed skewed memory T cell TCR repertoires and autoimmunity characterized by endothelium-reactive IgG. The alarmin, cytotoxicity, TCR repertoire, and plasmablast signatures we defined have potential for application in the clinic to better diagnose and potentially predict disease severity early in the course of MIS-C., Graphical abstract, Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening and unpredictable condition of unknown etiology. Ramaswamy et al. use peripheral blood single-cell transcriptomic profiling along with other techniques to define key innate and adaptive signatures that characterize MIS-C.

Protective immunity against COVID-19 likely depends on the production of SARS-CoV-2–specific plasma cells and memory B cells postinfection or postvaccination. Previous work has found that germinal center reactions are disrupted in severe COVID-19. This may adversely affect long-term immunity against reinfection. Consistent with an extrafollicular B cell response, patients with severe COVID-19 have elevated frequencies of clonally expanded, class-switched, unmutated plasmablasts. However, it is unclear whether B cell populations in individuals with mild COVID-19 are similarly skewed. In this study, we use single-cell RNA sequencing of B cells to show that in contrast to patients with severe COVID-19, subjects with mildly symptomatic COVID-19 have B cell repertoires enriched for clonally diverse, somatically hypermutated memory B cells ∼30 d after the onset of symptoms. This provides evidence that B cell responses are less disrupted in mild COVID-19 and result in the production of memory B cells.

While inhibition of T cell co-inhibitory receptors has revolutionized cancer therapy, the mechanisms governing their expression on human T cells have not been elucidated. Type 1 interferon (IFN-I) modulates T cell immunity in viral infection, autoimmunity, and cancer, and may facilitate induction of T cell exhaustion in chronic viral infection1,2. Here we show that IFN-I regulates co-inhibitory receptors expression on human T cells, inducing PD-1/TIM-3/LAG-3 while surprisingly inhibiting TIGIT expression. High-temporal-resolution mRNA profiling of IFN-I responses enabled the construction of dynamic transcriptional regulatory networks uncovering three temporal transcriptional waves. Perturbation of key transcription factors on human primary T cells revealed both canonical and non-canonical IFN-I transcriptional regulators, and identified unique regulators that control expression of co-inhibitory receptors. To provide direct in vivo evidence for the role of IFN-I on co-inhibitory receptors, we then performed single cell RNA-sequencing in subjects infected with SARS-CoV-2, where viral load was strongly associated with T cell IFN-I signatures. We found that the dynamic IFN-I response in vitro closely mirrored T cell features with acute IFN-I linked viral infection, with high LAG3 and decreased TIGIT expression. Finally, our gene regulatory network identified SP140 as a key regulator for differential LAG3 and TIGIT expression. The construction of co-inhibitory regulatory networks induced by IFN-I with identification of unique transcription factors controlling their expression may provide targets for enhancement of immunotherapy in cancer, infectious diseases, and autoimmunity.

Combining transcriptomic and TCRαβ repertoire analysis of circulating and tumor-infiltrating CD8 T cells from patients with metastatic melanoma, Lucca et al. identify a blood-based population with effector properties that reflect those of clonally related tumor-resident T cells., Understanding the relationship between tumor and peripheral immune environments could allow longitudinal immune monitoring in cancer. Here, we examined whether T cells that share the same TCRαβ and are found in both tumor and blood can be interrogated to gain insight into the ongoing tumor T cell response. Paired transcriptome and TCRαβ repertoire of circulating and tumor-infiltrating T cells were analyzed at the single-cell level from matched tumor and blood from patients with metastatic melanoma. We found that in circulating T cells matching clonally expanded tumor-infiltrating T cells (circulating TILs), gene signatures of effector functions, but not terminal exhaustion, reflect those observed in the tumor. In contrast, features of exhaustion are displayed predominantly by tumor-exclusive T cells. Finally, genes associated with a high degree of blood–tumor TCR sharing were overexpressed in tumor tissue after immunotherapy. These data demonstrate that circulating TILs have unique transcriptional patterns that may have utility for the interrogation of T cell function in cancer immunotherapy.

Foxp3+ regulatory T cells (Treg cells) are the central component of peripheral immune tolerance. Whereas a dysregulated Treg cytokine signature has been observed in autoimmune diseases, the regulatory mechanisms underlying pro- and anti-inflammatory cytokine production are elusive. Here, we identify an imbalance between the cytokines IFN-γ and IL-10 as a shared Treg signature present in patients with multiple sclerosis and under high-salt conditions. RNA-sequencing analysis on human Treg subpopulations revealed β-catenin as a key regulator of IFN-γ and IL-10 expression. The activated β-catenin signature was enriched in human IFN-γ+ Treg cells, as confirmed in vivo with Treg-specific β-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional Treg phenotype. Moreover, we identified prostaglandin E receptor 2 (PTGER2) as a regulator of IFN-γ and IL-10 production under a high-salt environment, with skewed activation of the β-catenin-SGK1-Foxo axis. Our findings reveal a novel PTGER2-β-catenin loop in Treg cells linking environmental high-salt conditions to autoimmunity.

In recent years, the understanding of regulatory T cell (Treg cell) biology has expanded considerably. Key observations have challenged the traditional definition of Treg cells and have provided insight into the underlying mechanisms responsible for the development of autoimmune diseases, with new therapeutic strategies that improve disease outcome. This Review summarizes the newer concepts of Treg cell instability, Treg cell plasticity and tissue-specific Treg cells, and their relationship to autoimmunity. Those three main concepts have changed the understanding of Treg cell biology: how they interact with other immune and non-immune cells; their functions in specific tissues; and the implications of this for the pathogenesis of autoimmune diseases.

The introduction of immunotherapy with checkpoint receptor blockade has changed the treatment of advanced cancers, at times inducing prolonged remission. Nevertheless, the success rate of the approach is variable across patients and different tumor types, and treatment is often accompanied by severe immune-related side effects, suggesting the importance of co-inhibitory pathway for both prevention of autoimmunity and failure of tumor rejection. A better understanding of how to uncouple anti-tumor activity from loss of self-tolerance is necessary in order to increase the therapeutic efficacy of checkpoint immunotherapy. In this review, we describe basic concepts of T cell exhaustion that occur in cancer, highlighting the role of co-inhibitory receptors in contributing to this process while preventing immunopathology. By providing an overview of the current therapeutic success and immune-related burden of secondary effects of checkpoint immunotherapy, we illustrate the “double-edged sword” related to interference with immune-regulatory pathways. Finally, since achieving tumor rejection while preserving self-tolerance is particularly important for the central nervous system, we analyze the case for checkpoint immunotherapy in glioblastoma, the most common adult brain tumor.

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.

Multiple sclerosis (MS) is a genetically mediated autoimmune disease characterized by inflammation in the central nervous system (CNS). Disease onset is thought to occur when autoreactive T cells orchestrate a cascade of events in the CNS resulting in white and grey matter inflammation and axonal degeneration. It is unclear what triggers the activation of CNS-reactive T cells and their polarization into inflammatory subsets. Mounting evidence from animal and human studies supports the hypothesis that the gut microbiome affects MS pathogenesis. We investigated the association between the gut microbiome and inflammatory T cell subsets in relapsing-remitting MS patients and healthy controls. Gut microbiome composition was characterized by sequencing the V4 region of the 16S rRNA gene from fecal DNA, and inflammatory T cell subsets were characterized by flow cytometry. We identified an altered gut microbiome in MS patients, including decreased abundance of Coprococcus, Clostridium, and an unidentified Ruminococcaceae genus. Among circulating immune cells, patients had increased expression of CXCR3 in both CD4 and CD8 T cells, and both CD4+CXCR3+ and CD8+CXCR3+ populations expressing the gut-homing α4β7 integrin receptor were increased. Finally, we show that alpha diversity inversely correlated with a CXCR3+ Th1 phenotype in MS. These findings indicate the presence of an aberrant gut-immune axis in patients with MS., Highlights • Patients with MS exhibited decreased alpha diversity in the composition of their gut bacteria compared to healthy controls. • Circulating lymphocytes from MS patients were enriched for Th1 and Th1-17 memory CD4+ cells and for CXCR5+ memory CD4+ cells. • A larger proportion of Th1 and Th1–Th17 ​cells expressed the gut homing receptor α4β7 compared to Th17 ​cells. • The frequency of Th1, Th1-17 and CD8 T cells co-expressing α4β7 and CXCR3 wereincreased in patients compared to controls. • Reduced alpha diversity correlated significantly with expression of CXCR3+ on CD8+ T-cells and CXCR5— memory T-cells.

Fingolimod is an approved therapeutic option for patients with relapsing-remitting multiple sclerosis that primarily functions by sequestering T cells in lymph nodes inhibiting their egress to the central nervous system. However, recent data suggests that Fingolimod may also directly affect the immune cell function. Here we examined the in vivo effects of Fingolimod in modulating the phenotype and function of T cell and Foxp3 regulatory T cell populations in patients with multiple sclerosis under Fingolimod treatment. Besides decreasing the cell numbers in peripheral blood and sera levels of pro-inflammatory cytokines, Fingolimod inhibited the expression of Th1 and Th17 cytokines on CD4(+) T cells and increased the expression of exhaustion markers. Furthermore, treatment increased the frequency of regulatory T cells in blood and inhibited the Th1-like phenotype that is characteristic of patients with multiple sclerosis, augmenting the expression of markers associated with increased suppressive function. Overall, our data suggest that Fingolimod performs other important immunomodulatory functions besides altering T cell migratory capacities, with consequences for other autoimmune pathologies characterized by excessive Th1/Th17 responses and Th1-like regulatory T cell effector phenotypes.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications. This work was supported by a grant from the European Cooperation in Science and Technology (COST) for the AFACTT project (Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies; BM1305). COST is part of the EU Framework Programme Horizon 2020. peer-reviewed

Substantial effort has been made over the last six decades to identify biomarkers for multiple sclerosis that can improve disease diagnosis, predict disease progression, and improve clinical outcomes. However, to date, few of these findings have proven clinically useful. In this review, we address the current state of MS biomarker research. We start by discussing biomarkers currently in clinical use including Oligoclonal bands, MRI, and JC viral titers. We go on to discuss other potential biomarkers from MS serum and cerebrospinal fluid including Markers of neurodegeneration including neurofilament and GFAP, the monocyte macrophage marker CD163, the glial activation marker YKL-40, the B cell chemoattractant CXCL13, miRNA and mRNA, myelin reactive t cells, Kir4.1 antibodies, osteopontin, and microbiome associated lipopeptides. Finally, we discuss the current state of MS genetic studies and how genetics may offer simple, reliable testing for MS susceptibility and progression.

Emergent responses of the immune system result from the integration of molecular and cellular networks over time and across multiple organs. High-content and high-throughput analysis technologies, concomitantly with data-driven and mechanistic modeling, hold promise for the systematic interrogation of these complex pathways. However, connecting genetic variation and molecular mechanisms to individual phenotypes and health outcomes has proven elusive. Gaps remain in data, and disagreements persist about the value of mechanistic modeling for immunology. Here, we present the perspectives that emerged from the National Institute of Allergy and Infectious Disease (NIAID) workshop 'Complex Systems Science, Modeling and Immunity' and subsequent discussions regarding the potential synergy of high-throughput data acquisition, data-driven modeling, and mechanistic modeling to define new mechanisms of immunological disease and to accelerate the translation of these insights into therapies.

Influenza remains a significant cause of morbidity and mortality in older adults, with 90% of annual influenza-related deaths occurring in individuals older than 65 years [1]. Although the seasonal inactivated vaccine is recommended for prevention, a meta-analysis of 64 studies of influenza vaccine response in older adults (>65 years) revealed an efficacy of 23% for the prevention of influenzalike illness [2]. This poor vaccine response is a result in part of age-associated alterations in immune function [3]. Immunosenescence affects the adaptive immune system, resulting in impairments in B- and T-cell development, signaling, and antigen receptor repertoire [4–8]. The human innate immune system is also affected by aging, with chronic inflammation arising from sources including age-associated herpesvirus reactivation and increased non–cell-associated DNA reflected in elevated levels of cytokines and acute phase reactants; such inflammation contributes to dysregulated innate immune function, particularly of pattern recognition receptors such as Toll-like receptors (TLRs) [9]. However, how aging affects human innate immunity to influenza vaccination remains incompletely understood. Here, we evaluated monocyte function at baseline and after immunization with the inactivated influenza vaccine in a cohort of young and older adults.

West Nile virus (WNV) infection is usually asymptomatic but can cause severe neurological disease and death, particularly in older patients, and how individual variations in immunity contribute to disease severity is not yet defined. Animal studies identified a role for several immunity-related genes that determine the severity of infection. We have integrated systems-level transcriptional and functional data sets from stratified cohorts of subjects with a history of WNV infection to define whether these markers can distinguish susceptibility in a human population. Transcriptional profiles combined with immunophenotyping of primary cells identified a predictive signature of susceptibility that was detectable years after acute infection (67% accuracy), with the most prominent alteration being decreased IL1B induction followingex vivoinfection of macrophages with WNV. Deconvolution analysis also determined a significant role for CXCL10 expression in myeloid dendritic cells. This systems analysis identified markers of pathogenic mechanisms and offers insights into potential therapeutic strategies.

T-cell immunoglobulin and mucin domain 3 (TIM-3) is an Ig-superfamily member expressed on IFN-γ-secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM-3 is expressed by a subset of activated CD4+ T cells, and anti-CD3/anti-CD28 stimulation increases both the level of expression and the number of TIM-3+ T cells. In mice, TIM-3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4+ T-cell differentiation. Here, we examined TIM-3 expression on human Treg cells to determine its role in T-cell suppression. In contrast to mice, TIM-3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM-3+ Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM-3− Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT-3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM-3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1- and Th17-cell responses.

Regulatory T cells are the central element for the maintenance of peripheral tolerance. Several subtypes of regulatory T (Treg) cells have been described, and most of them belong to the CD4(+) T-helper (Th) cell lineage. These specific subtypes can be discriminated according to phenotype and function. Forkhead box protein 3 (FoxP3)-expressing natural Treg cells (Tregs) and IL-10-producing, T-regulatory type 1 cells (Tr1) are the best-studied types of CD4(+) regulatory T cells in humans and experimental animal models. It was shown that they play a crucial role during autoimmune neuroinflammation. Both cells types seem to be particularly important for multiple sclerosis (MS). Here, we discuss the role of CD4(+) regulatory T cells in autoimmune neuroinflammation with an emphasis on Tregs and Tr1 cells in MS.

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.

Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual’s unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.

Regulatory T cells (Tregs) are critical to the human immune system, providing appropriately scaled immune responses and mediating peripheral tolerance. A central role for forkhead box protein 3 (FoxP3)(+) Tregs has been shown in the pathogenesis of mechanistically diverse central nervous system (CNS) diseases from autoimmune diseases such as multiple sclerosis to glioblastomas. Understanding how tumors induce Treg function to escape immune surveillance in marked contrast to autoimmune diseases, where there is loss of Treg function, will provide valuable lessons regarding Treg biology and potential therapeutic targets for CNS 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.

We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.

Using retrospectively collected outcome data for treatment naive subjects treated with either glatiramer acetate (GA) ( n = 332) or interferon beta (IFN β) ( n = 424), we replicated the lack of a significant difference in efficacy between these treatments. Further, for both treatments, we observed a decline in the hazard of a relapse over time, which may suggest the existence of subsets of subjects with differential responses to each treatment. The HLA DRB1*1501 allele explained some of this variation in event-free survival while on GA, and we found suggestive evidence that an IRF8 polymorphism influences event-free survival in IFN β treated subjects.

Human regulatory T cells (Tregs) play a critical role in preventing autoimmunity, and their failure contributes to autoimmune diseases. In recent years, our understanding of human Tregs has been greatly enhanced by improvements in the definition and isolation of pure human Tregs, as well as by the discovery of phenotypically and functionally distinct human Treg subsets. This progress has also yielded a better understanding of the mechanisms of human Treg suppression and the role of human Tregs in autoimmune diseases. An unexpected discovery is that human Tregs have considerable plasticity that allows them to produce the pro-inflammatory cytokine IL-17 under certain conditions. These recent advances highlight the importance of studying the roles of both mouse and human Tregs in autoimmunity.

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.

While suppressor T cells might have been the so-called “baby thrown out with the bath water” when molecular biology strongly influenced immunology in the 1980s, it was clear to investigators of autoimmune disease that adoptively transferred suppression by T cells could influence these diseases. Here, we will present an overview of T cells as regulators of immune responses from a human species point of view, beginning with a history of T cells as regulatory cells, followed by a discussion of how these cells critically influence human autoimmune disease. Twenty-five years ago, immunologists determined that two cellular mechanisms of dominant tolerance could mediate peripheral regulation and the phenomena of induced CD4+ T cell non-responsiveness. These two mechanisms were MHC-restricted T cell presentation of antigen to other T cells [1] and suppression via thymically derived ‘suppressor’ T cells [2]. Shortly after the discovery of MHC class II+ CD4+ T cells in human peripheral blood (these cells are present in most mammalian species excluding mice [3]) [4, 5], Kunkel and his colleagues observed that autoimmune patients have a higher percentage of these cells in peripheral blood than healthy patients [6]. Using the newly developed technique of CD4+ T cell cloning, researchers determined that MHC class II+ CD4+ T cell clones induce anergy when they present antigen to other CD4+ T cells [7]. Evidence from these studies was used to support the argument for the existence of anti-idiotypic T cell networks. ‘Suppressor’ T cells, too, were thought to recognize the antigen receptor of autologous CD4+ cells and prohibit activation with specificity directed against the suppressed cell. In the years to follow, the molecular or biochemical processes underlying these mechanisms could not be determined; MHC class II was relegated to a biomarker of late CD4+ T cell activation; many early studies of suppressor T cells were discredited or disproved; and the paradigm of idiotypic networks fell from fashion. Unfortunately, as Rolf Zinkernagel writes, “science (particularly immunology) undergoes periods of democratic, if not demagogic decisions … while populistic or experimentally biased views within science will eventually be corrected, if proven wrong this can take a long time” [8]. Although relegated to the list of immunological “not-so-good ideas” for almost fifteen years, ‘suppressor’ T cells have now been shown to play an essential role in preventing autoimmune disease and immunologists have begun to better characterize the now-called ‘regulatory’ T cells that can mediate suppression of autoimmunity. Not surprisingly, MHC class II+ CD4+ T cell have re-emerged as well, as expression of this immunomodulatory receptor was found to identify a functionally distinct subset of human ‘regulatory’ T cells (Tregs) expressing FoxP3. In this review we discuss recent advances in the characterization of human CD4+CD25high Tregs, with a focus on the impairment of these cells in human autoimmune disease.

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.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). In the recent years, accumulating evidence has supported an immunosuppressive role for regulatory T cells (Tregs). Most studies in the context of autoimmunity have focused on the defects of the CD4+CD25 high Tregs. However, we recently demonstrated an altered function of Tr1 Treg cells in MS, characterized by a lack of IL-10 secretion. Therefore, several major regulatory T cell defects are involved in human autoimmune disease. Hence, the induction of Tregs or the stimulation of Treg activity may be beneficial for the treatment of such diseases.

A recent workshop discussed the recognition of multiple distinct ligands by individual T cell and B cell receptors and the implications of this discovery for lymphocyte biology. The workshop recommends general use of the term polyspecificity because it emphasizes two fundamental aspects, the inherent specificity of receptor recognition and the ability to recognize multiple ligands. Many different examples of polyspecificity and the structural mechanisms were discussed, and the group concluded that polyspecificity is a general, inherent feature of TCR and antibody recognition. This review summarizes the relevance of polyspecificity for lymphocyte development, activation and disease processes.

The paradox of autoreactivity to self-peptides in physiological as opposed to pathological immune responses is not well understood. Here, we directly examined the human T cell response to endogenous self-peptides in a series of healthy subjects. CFSE-labeled T cells were stimulated with unmanipulated antigen-presenting cells containing endogenous self-antigen, and the resulting CD4+ populations entering into cell cycle (CFSE(low)) or non-proliferating CD4+ cells (CFSE(high)) were single-cell sorted, cloned and screened against a panel of self-antigens and microbial recall antigens to interrogate their antigen reactivity. The percentage of CD4+ T cells entering cell cycle in response to self-peptide/MHC was calculated to be 0.04%, and entry into cell cycle was dependent upon CD28 costimulation. Clones derived from CFSE(low) T cells exhibited significantly greater cross-reactivity to multiple antigens than CFSE(high) clones or other CD4+ clones generated after microbial antigen stimulation. Sequencing the TCRbeta chains indicated that CFSE(low) clones were indeed clonal. These data demonstrate that T cell clones generated on stimulation by endogenous self-peptides exhibit a high degree of multispecificity, and we speculate that their multispecificity is based upon recognition of shared-backbone MHC determinants.

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.

T cell responses are initiated by ligation of their cognate T cell receptor by MHC loaded with antigenic peptide, but their response is carefully controlled by a myriad of environmental cues, including co-activation receptors, cytokines, nutrients, growth factors, local oxygen levels, salt concentrations, and microbiome. The complexity of the integration of signals received by T cells is only beginning to be fully understood (1). This research topic in T cell biology aims at highlighting some of the latest research on intrinsic and extrinsic signals regulating T cell responses. The ebook contains 10 articles that encompass key pathways that modulate T cell function and discuss how T cells coordinate their response to environmental cues. One of the first components regulating T cell activation is the expression and subsequent activation of surface receptors. Notably, T cell activation is governed by the co-activation of the TCR and of co-stimulatory or co-inhibitory molecules (2, 3). Expression and activation of co-inhibitory molecules, such as CTLA-4 and PD1 play a key part in turning off effector responses and the balance of expression of co-stimulatory and co-inhibitory receptors needs to be tightly regulated to ensure a proper level of T cell activation (4, 5). The review by Schneider and Rudd nicely illustrates how expression of the co-inhibitory molecule CTLA-4 is regulated, and how that affects T cell activation (6). In recent years, the importance of cellular metabolism in the activation of immune cells, in particular T cells, has been reported. T cell activation requires a change in cellular metabolism to face increased energetic demand. This is an exciting area of research showing that not only changes in metabolism are necessary for cell activation but that they are also actively involved in regulating T cell function and differentiation. This is discussed in several reviews. Craig Byersdorfer focuses on the role of fatty acid oxidation for T cell functions, and notably on its role in graft versus host disease (7). Ramsay and Cantrell discuss the importance of glucose metabolism for T cell function and highlight the role of hypoxia-inducible factor alpha and mTOR in coordinating the responses to the environmental cues (8). They also summarize the importance of the microbiome in regulating T cells, and the key role of the aryl hydrocarbon receptor in sensing microbes. Clovis Palmer and collaborators review glucose metabolism in T cells and also discuss how HIV infection modulates T cell metabolism (9). One of the central questions is how do T cells integrate the multitude of signals received? In their comprehensive review, Chapman and Chi discuss the central role of mTOR in the integration of the many signals received by the T cells that ultimately shape their response (10). Another related aspect of the topic is the impact of infection on T cells, whereby pathogens control the host response, mostly to their advantage, and are able to switch T cell responses to favor their own survival. A study by the group of Francisca Mutapi describes a novel mechanism by which helminth infection downregulates T cell activation, by lowering the level of CD3zeta chain in infected individuals (11). A review from Zaunders further illustrates how HIV infection affects the balance of the various T cell subsets and promotes regulatory T cells and T helper follicular cells (12). Diet can also influence immune homeostasis, with recent studies showing, for instance, how salt can affect Th differentiation (13). The effects of vitamins and their metabolites on T cells are also well described. Colleen Hayes’ group summarizes the latest data on the effects of Vitamin D on T cell responses, and how this can be modulated in autoimmune diseases (14). A review by Leving’s group focuses more particularly on the environmental factors that affect regulatory T cells. This includes cytokines, vitamin A and vitamin D, metabolism, and microbiome (15). Finally, can we therapeutically manipulate the environmental milieu to modulate T cell responses in humans? The study by the group of David Klatzmann highlights the effects of IL-2 in modulating T cell responses in type 1 diabetes (16). While the functions of regulatory CD4+ Tregs have been described years ago, the characterization of CD8+ Tregs is more recent. The authors notably report how injection of low doses of IL-2 modulates the levels of CD8+ Tregs in vivo, in both mice and in patients with type 1 diabetes, following their numbers and phenotype. This study illustrates how exogenous cytokines can modulate in vivo T cell responses, which may prove beneficial for autoimmune diseases. We hope that this compilation of reviews and research that provides an overview on environmental regulators of T cells will give the readers a flavor on the latest development in T cell biology. We would also like to thank all the contributors to this topic and the reviewers for their time in making this ebook possible.

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.

As self-recognition is fundamental to the efficient operation of the immune system, a number of mechanisms have evolved to keep this potential pathologic self-reactivity in check. Thus, even though the majority of strongly self-reactive T cells are deleted in the thymus during T-cell maturation, a number of mature T cells that recognize self-antigens can be found in the peripheral circulation in healthy individuals as well as in patients with autoimmune disease. These self-reactive cells are kept in a non-responsive state in healthy individuals while they appear to be involved in the etiology of a number of autoimmune diseases in patients. The primary role of a relatively recently identified T-cell population, referred to as natural CD4+ CD25+ regulatory T cells, is to modulate the activity of these self-reactive cells. Although it is still unclear how these regulatory cells function, they can inhibit the activation of other potentially pathologic T cells in in vitro assays. Using such assays, regulatory T cells isolated from patients with a number of autoimmune diseases have been shown to exhibit reduced inhibitory function as compared with those isolated from healthy individuals. In this review, we discuss human natural regulatory T cells, what is known about their function, and their associations with specific autoimmune diseases.

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.

The importance of CD4+ CD25+ regulatory T cells (Treg) in maintaining immune homeostasis has been directly demonstrated in vivo by their manipulation in a number of autoimmune disease models in the mouse. In the study of human regulatory cells, we have found that the cells that consistently demonstrate the in vitro regulatory activity most similar to that described for murine cells in vitro are best identified by restricting the isolation of CD25+ CD4 T cells to those cells expressing only the highest levels of CD25, representing approximately 2-3% of total CD4 T cells. Thus, it is the CD4+ CD25high subset that exhibits the in vitro characteristics that are identical to the CD4+ CD25+ regulatory cells initially characterized in mice. Furthermore, the cells expressing medium to low levels of CD25 not only do not exhibit suppressive activity directly ex vivo, but also actually contain a significant proportion of CD62L- CD4 T cells which are believed to be in vivo activated T cells. Due to the inherent difficulties in using CD25 as a marker for the purification of Treg cells, the finding that selection of the CD25high subset of CD4+ CD25+ T cells minimizes the co-isolation of contaminating activated CD4 T cells is important for future studies of these Treg cells in human disease. In order to perform these studies, we first had to establish a highly reproducible 'micro in vitro co-culture' assay system to enable the functional analysis of high-purity, but low-yield regulatory populations derived from FACS sorting. With this system in place, we are poised to dissect the potential heterogeneity of mechanisms employed by highly specific subpopulations of CD4+ CD25+ cells.

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.

CD4+CD25+ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4+ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4+CD25+ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4+CD25hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4+CD25hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4+CD25hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4+CD25hi regulatory T cell function in patients with MS.

In this report, we review studies of human CD4+CD25+ regulatory T cells (T-reg). Although lagging a few years behind the discovery of these cells in the mouse, the equivalent population of CD4+CD25+ regulatory T cells has also been isolated from human peripheral blood, thymus, lymph nodes and cord blood. In general, the characteristics of this T cell subset are strikingly similar between mouse and man. In the recent explosion of research reports on human CD4+CD25+ cells, although the majority of the characteristics ascribed to these cells appear to be consistent, contrasting results have been found primarily in regards to potential involvement of TGFbeta and production of IL-10. One explanation for this variability may reside in the fact that markedly different techniques are used to isolate human CD4+CD25+ T-reg cells and thus may result in the comparison of T-reg populations that differ in cellular composition and/or activation state. Another potential explanation for differences in human T-reg function may rest on the extreme variability of the culture conditions and TCR stimuli that have been used to test the functional properties of these cells in vitro. The strength of the TCR signal provided to the culture greatly affects the functional outcome of the co-culture and can result in the difference between suppression and full activation. Surprisingly, it appears that stronger stimulation has a greater and more rapid effect on the T-resp cell than on the T-reg cell as it causes T-resp cells to quickly become resistant to suppression. Thus, the details of in vitro culture conditions may at least partially account for disparate findings in regard to the functional characterization of human CD4+CD25+ cells. Here we review the evidence regarding the identification of human CD4+CD25+ regulatory T cells and their possible mechanism(s) of function.

Multiple sclerosis (MS) is a complex disease with a significant genetic contribution. Similar to other complex diseases, the genetic risk for MS results from a combination of many genetic variants which individually confer only modest effects. Here we review the results of the complementary genetic approaches of linkage and association in MS, highlighting the challenges of identifying loci of modest effect. We describe meta-analytical approaches that provide additional statistical power necessary for detecting such loci and further apply this approach to evaluate the association of a CTLA-4 variant with MS. Lastly, we review recent important advances in our understanding of the patterns of genetic variation in the human genome and speculate about how these advances will aid in future studies of the genetic causes of MS.

Glatiramer acetate (GA; Copaxone) is a random sequence polypeptide used in the treatment of relapsing remitting multiple sclerosis (RR MS). We have recently demonstrated that prior to treatment, GA induces proliferation of resting T cells and is not cross-reactive with myelin antigens. Daily GA injections induce a significant loss of this GA responsiveness, which is associated with the induction of highly cross-reactive Th2-type T cells potentially capable of suppressing inflammatory responses. The mechanism of action by which GA induces T cell nonresponsiveness leading to T cell receptor degeneracy in patients with RR MS is unknown. Here, we examined the effects of daily GA administration on the induction of T cell hyporesponsiveness. The frequency of GA-reactive T cells in peripheral blood of seven patients with RR MS was measured by limiting dilution analysis prior to and during 6 months of treatment. In addition, a model in which GA-reactive T cells were stimulated in vitro was developed to better characterize the selection of T cell populations over time. In vivo treatment with GA induced a decrease in GA-reactive T cell frequencies and hyporesponsiveness of CD4+ T cell reactivity to GA in vitro that was only partially reversed by the addition of IL-2. These data suggest that T cell peripheral tolerance to GA was achieved in vivo during treatment. Thus, our in vitro data suggest that the underlying changes in GA-reactive CD4+ T cell reactivity could be explained by the induction of T cell anergy and clonal elimination.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, thought to be mediated in part by an autoimmune response of T cells to protein components of the myelin sheath. The reaction of naïve T cells against these antigens requires co-stimulation through CD28. However, the proliferative response of peripheral blood mononuclear cells isolated from patients with MS and stimulated with myelin basic protein (MBP) has been shown to be relatively independent of B7-CD28 co-stimulation, suggesting that dysregulation of co-stimulatory pathways may be involved in the pathogenesis of MS. Here, the role of CTLA-4 engagement was investigated. As expected, blocking CTLA-4-mediated signaling during stimulation of MBP-reactive T cells from healthy controls enhanced the proliferative and cytokine responses. In contrast, CTLA-4 blockade had less effect in patients with MS, suggesting that at least two regulatory mechanisms may be impaired in these individuals. Understanding how co-stimulatory signals may be dysregulated in patients with MS is important at a time when targeting of these pathways is being developed.