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Although implicated in antagonistic functions, both regulatory T cells (Tregs) and Th17 effector cells play an important role in controlling autoimmune pathogenesis. Paradoxically, recent studies indicate that Tregs have the capacity to produce interleukin-17 (IL-17), although the ability of these cells to retain their suppressive function remains unknown. Here we report that human Tregs within the CD4+CD45RA−CD25highCCR6+HLA-DR−FoxP3+ population produce IL-17 when activated in the presence of the proinflammatory cytokines IL-1β and IL-6, whereas IL-17 secretion was inhibited by TGFβ. To assess the ability of a single Treg to secrete IL-17 and to suppress in vitro immune function, we isolated clones from this population. We found that IL-17+/FoxP3+ Treg clones retain suppressive function and exhibit the plasticity to secrete IL-17 or suppress depending on the nature of the stimulus provided. IL-17 production by these Treg clones was accompanied by sustained FoxP3 expression and concomitant, but reversible, loss of suppressive activity. Our data demonstrate that at the single cell level a subset of in vitro suppressive FoxP3+ cells can be driven to secrete IL-17 under inflammatory conditions. These findings suggest a new mechanism by which inflammation can drive Tregs to secrete IL-17, thereby dampening suppression and promoting an inflammatory milieu.

Familial clustering of autoimmune disease is well recognized and raises the possibility that some susceptibility genes may predispose to autoimmunity in general. In light of this observation, it might be expected that some of the variants of established relevance in one autoimmune disease may also be relevant in other related conditions. On the basis of this hypothesis, we tested seven single nucleotide polymorphisms (SNPs) that are known to be associated with type I diabetes in a large multiple sclerosis data set consisting of 2369 trio families, 5737 cases and 10 296 unrelated controls. Two of these seven SNPs showed evidence of association with multiple sclerosis; that is rs12708716 from the CLEC16A gene (P=1.6 x 10(-16)) and rs763361 from the CD226 gene (P=5.4 x 10(-8)). These findings thereby identify two additional multiple sclerosis susceptibility genes and lend support to the notion of autoimmune susceptibility genes. ispartof: Genes and immunity vol:10 issue:1 pages:11-14 ispartof: location:England status: published

As part of a biomarker discovery effort in peripheral blood, we acquired an immunological profile of cell-surface markers from healthy control and untreated subjects with relapsing-remitting MS (RRMS). Fresh blood from each subject was screened ex vivo using a panel of 50 fluorescently labelled monoclonal antibodies distributed amongst 56 pools of four antibodies each. From these 56 pools, we derived an immunological profile consisting of 1018 'features' for each subject in our analysis using a systematic gating strategy. These profiles were interrogated in an analysis with a screening phase (23 patients) and an extension phase (15 patients) to identify cell populations in peripheral blood whose frequency is altered in untreated RRMS subjects. A population of CD8(low)CD4(-) cells was identified as being reduced in frequency in untreated RRMS subjects (P = 0.0002), and this observation was confirmed in an independent sample of subjects from the Comprehensive Longitudinal Investigation of MS at the Brigham & Women's Hospital (P = 0.002). This reduction in the frequency of CD8(low)CD4(-) cells is also observed in 38 untreated subjects with a clinically isolated demyelination syndrome (CIS) (P = 0.0006). We also show that these differences may be due to a reduction in the CD8(low)CD56(+)CD3(-)CD4(-) subset of CD8(low) cells, which have a natural killer cell profile. Similarities between untreated CIS and RRMS subjects extend to broader immunological profiles: consensus clustering of our data suggests that there are three distinct populations of untreated RRMS subjects and that these distinct phenotypic categories are already present in our sample of untreated CIS subjects. Thus, our large-scale immunophenotyping approach has yielded robust evidence for a reduction of CD8(low)CD4(-) cells in both CIS and RRMS in the absence of treatment as well as suggestive evidence for the existence of immunologically distinct subsets of subjects with a demyelinating disease.

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.

Little is known regarding the functional effects of common autoimmune susceptibility variants on human immune cells. The SNP CT60 (rs3087243; A/G) located in the 3′ UTR of theCTLA4gene has been associated with autoimmune diseases. We examined a cohort of healthy individuals stratified by genotypes atCTLA4to gain insight into the functional effects of allelic variation on T cell signaling. Using phospho-site-specific mAbs, we tested the hypothesis that the CT60 genotype atCTLA4is associated with altered T cell antigen receptor (TCR) signaling in naive and/or memory T cells. By normalizing for the extent of the initial TCR signaling event at CD3ζ, we observed that the relative responsiveness to TCR stimulation as assessed by phosphorylation levels of downstream signaling molecules was altered in naive (CD4+CD45RAhigh) and memory (CD4+CD45RAlow) T cells obtained from individuals with the disease-susceptibility allele atCTLA4. Thus, allelic variation associated with autoimmune disease can alter the signaling threshold of CD4+T cells. These experiments provide a rational approach for the dissection of T cell-susceptibility genes in autoimmune diseases.

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.

αB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease. ©2007 Nature Publishing Group.

The intestinal flora has long been thought to play a role either in initiating or in exacerbating the inflammatory bowel diseases (IBD). Host defenses, such as those mediated by the Toll-like receptors (TLR), are critical to the host/pathogen interaction and have been implicated in IBD pathophysiology. To explore the association of genetic variation in TLR pathways with susceptibility to IBD, we performed a replication study and pooled analyses of the putative IBD risk alleles in NFKB1 and TLR4, and we performed a haplotype-based screen for association to IBD in the TLR genes and a selection of their adaptor and signaling molecules. Our genotyping of 1539 cases of IBD and pooled analysis of 4805 cases of IBD validates the published association of a TLR4 allele with risk of IBD (odds ratio (OR): 1.30, 95% confidence interval (CI): 1.15 - 1.48; P = 0.00017) and Crohn's disease (OR: 1.33, 95% CI: 1.16 - 1.54; P = 0.000035) but not ulcerative colitis. We also describe novel suggestive evidence that TIRAP (OR: 1.16, 95% CI: 1.04 - 1.30; P = 0.007) has a modest effect on risk of IBD. Our analysis, therefore, offers additional evidence that the TLR4 pathway - in this case, TLR4 and its signaling molecule TIRAP - plays a role in susceptibility to IBD. ispartof: Genes and immunity vol:8 issue:5 pages:387-397 ispartof: location:England status: published

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.

Although several therapies exist for multiple sclerosis (MS), the most common inflammatory demyelinating disease of the central nervous system (CNS), there remains a large unmet clinical need for more effective immunomodulatory treatments in this category of diseases and for interventions that address their neurodegenerative component, which is currently untreated. Progress in our understanding of the immunology of MS over the past 30 years has recently synergized with novel computational methods and emerging high-throughput technologies that characterize variations in DNA, RNA, proteins, and metabolites to usher in a period of intense pathophysiologic investigation. These efforts are beginning to define subsets of patients with different forms of demyelinating disease. This partitioning of patients will prove valuable as we begin to tailor immunotherapy to the underlying pathophysiologic processes of individual patients using current therapies, emerging treatments, and rational combinations of all of these treatments. Preventing the entry of lymphocytes into the CNS and modifying the nature of the immune response are treatment approaches that work in the inflammatory component of MS but have little or no effect on neurodegeneration. Two challenges confront us: to develop cocktails of therapies that shift the immune homeostasis of patients with MS toward a healthy profile, and to identify and modulate the activity of targets within the neurodegenerative component of MS.

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.

It is well established that the major histocompatibility complex (MHC) on chromosome 6p21 contains at least one gene that influences susceptibility to multiple sclerosis.1–6 Although this association was first identified more than 30 years ago1 through the study of class I human leukocyte antigens (HLAs), it was quickly realized that this signal was predominantly, if not exclusively, the result of linkage disequilibrium (LD) with class II HLA genes, and that these exert the primary effect on susceptibility.7, 8 The complex nature of the MHC, especially its high gene content, extreme polymorphism, and extensive LD,9 has confounded efforts to resolve the nature of the MHC association in multiple sclerosis, although progress and useful clarifications have been made, especially in recent years. In virtually every population studied, multiple sclerosis is found to be associated with the DRB1*1501 allele.10 The only exceptions are those populations where this allele has a low frequency, and analysis is therefore underpowered; but even in these situations, DRB1*1501 is generally overrepresented in cases.11 The DRB1*1501 allele is carried on a particularly extensive haplotype,12 the most common DR15 haplotype found in white Europeans. As a result, many variants from flanking genes, even some located quite a distance from DRB1, have sufficient LD with DRB1*1501 that they invariably also show evidence for association with the disease in any population where association with DRB1*1501 can be demonstrated.11, 13–17 This extensive LD has made it difficult to establish which of the variants making up this haplotype is primarily responsible for the association. The distinction between DRB1*1501 and DQB1*0602 has been particularly taxing because the LD between these closely mapped genes is especially tight in those populations where the disease is frequent, that is, white Europeans and their migrant descendants. However, recent studies in the admixed African American population indicate the supremacy of the DRB1*1501 allele.18 In the presence of one susceptibility allele it is difficult to identify effects attributable to a second allele,19 especially if the second allele exerts a more modest effect or has a low frequency, or both. However, by analyzing populations where DR15 haplotypes are less common, and by using large cohorts, it has been possible to demonstrate that the DRB1*0301 allele also confers susceptibility to multiple sclerosis, thereby confirming allelic heterogeneity at the DRB1 locus.4, 6, 18, 20 Furthermore available evidence suggests that the susceptibility effects of the DRB1*1501 allele may be modulated by other DRB1 alleles.6, 20 The relation between the MHC and multiple sclerosis is further complicated by the accumulating evidence suggesting that MHC loci mapping outside DRB1 also influence susceptibility to the disease.11, 13–16 Work in animal models suggests that clustering of susceptibility loci is a common phenomenon in complex disease,21 and it therefore appears reasonable to expect that other genes from the MHC region may influence susceptibility to multiple sclerosis. The observation of positive logarithm of odds scores in the MHC region in linkage studies stratified for the effects of DRB1 supports the existence of secondary loci,22–24 although none of these data reaches a level providing statistical confidence. In considering these linkage data, it is important to remember that early linkage studies in multiple sclerosis25–27 were significantly underpowered,28 to the point that they could not even convincingly demonstrate evidence for linkage resulting from the effects of DRB1. Confirmation of linkage in this region has been established only in more recent studies involving many hundreds of families.23, 24, 29 Given the inherently limited resolution of linkage-based studies,28 the absence of statistically significant linkage in the MHC region after exclusion of primary effects attributable to DRB1 does not exclude the presence of secondary loci. Several authors have attempted to identify secondary loci using more powerful association-based methods. Two groups have typed dense microsatellite maps of the region and both found evidence for a secondary locus maximal in a region close to HLA-A: one group identifying the marker D6S1683 just telomeric of HLA-A,11 and the second group implicating a region including HLA-A extending from MOGCA to D6S265 marker.14 Follow-up studies in Norway also found evidence implicating the D6S265 marker.16 In another smaller study, a microsatellite marker close to HLA-C (marker C1_3_2) also showed evidence for an independent effect.15 In contrast, a systematic effort to screen the MHC and flanking regions using single nucleotide polymorphisms (SNPs) found no evidence for association beyond that attributable to DRB1*1501, although this study was limited by a high genotyping failure rate (40%) and, more importantly, a distribution of markers leaving regions close to the classical loci essentially unexplored.17 In all of these studies, statistical power has inevitably been reduced by the processes required to filter out the primary effect attributable to DRB1 and the large correction required for multiple testing. Unfortunately, none of the published studies has used sufficient samples to compensate for these statistical penalties, and thus none is able to provide unequivocal evidence supporting any particular secondary locus.

BACKGROUND: The prognosis for patients with glioblastoma (GBM) remains poor despite the availability of first-line therapies including surgery and chemoradiation. Recent evidence suggests that there are varying degrees of lymphocyte infiltration in GBM, and the composition may have prognostic significance. Expansion of regulatory T cells (CD4+FoxP3+, Treg) at the tumor site has been observed and is implicated in cancer-associated immunity in GBM. Immune checkpoint inhibitors, such as the fully human anti-PD-1 monoclonal antibody nivolumab, have shown promising antitumor activity in both solid tumors and preclinical glioma models. The CHECKMATE-143 study is evaluating nivolumab as monotherapy and in combination with ipilimumab (anti-CTLA-4) in patients with recurrent GBM (Cohorts 1 and 1b). As part of the initial phase of this study, T-cell maturation, activation, exhaustion, and effector function, as well as Treg frequencies and function and myeloid-derived suppressor cell frequencies were examined. METHODS: Analyses were performed on peripheral blood drawn from patients at pre-determined time points and upon tumor tissue samples obtained if resection was performed at the time of progression or suspected progression. Baseline and on-treatment samples were obtained from patients (n = 20) randomized 1:1 to receive either nivolumab 3 mg/kg Q2W or nivolumab 1 mg/kg + ipilimumab 3 mg/kg Q3W followed by nivolumab 3 mg/kg Q2W. Phenotypic analysis was determined by CyTOF and flow cytometry; Treg suppressive function by thymidine incorporation; cytokine release by multiplex immunoassay; and gene expression by RNAseq. RESULTS: Treatment-related changes in lymphocyte populations (including but not limited to T, B, and NK cells, granulocytes, and memory and effector T-cell subsets) in peripheral blood and resected tumor will be reported. CONCLUSIONS: Peripheral and tumor biomarker analyses of GBM subjects treated with checkpoint inhibitors will provide insights into treatment-related changes versus tumor progression.

BACKGROUND: Foxp3+ regulatory T cells (Tregs) are central in ensuring protective immune responses while preventing immunopathology. While Tregs exhibit defective function in autoimmune disorders, they appear to be enriched in tumor immune infiltrates, suggesting a role in failure of immune-mediated tumor rejection. Tumor-infiltrating Tregs express higher levels of the co-inhibitory receptor PD-1 compared to circulating Tregs, especially in patients with glioblastoma. PD-1 is overexpressed by effector T cells in chronic immune responses and plays a pivotal role in driving T cell exhaustion in these conditions. METHODS: We examined the phenotype and function of CD4+CD25+CD127−Foxp3+ Tregs from peripheral blood and tumor infiltrates of patients with different grades of glioma, including glioblastoma (n = 20). Assessments included functional evaluation of Tregs in co-culture suppression assays with blood-derived T effector cells, and transcriptional profiling by RNA sequencing. RESULTS: By comparing the functionality of Tregs derived from blood and tumors of patients with glioblastoma, we established increased Treg expression of PD-1 is associated with impaired suppressive function and production of proinflammatory cytokines. Transcriptional profiling of PD-1+ Tregs revealed tumor-infiltrating PD-1+ Tregs are enriched for several exhaustion signatures, confirming PD-1 is a marker of exhaustion not only for effector T cells but also for Tregs. Finally, we investigated phenotype and function of PD-1+ Tregs in the circulation of healthy individuals (no known history of cancer, autoimmune disease, or other inflammatory conditions), and observed a similar loss of suppressive function and an exhausted transcriptional profile. CONCLUSIONS: Expression of PD-1 marks a population of dysfunctional Tregs that occurs in normal immunity and is enriched in cancer, specifically in the tumor. That Tregs from the tumor fail to suppress effector immune responses suggests that loss of effector T-cell function is more likely to result from chronic stimulation by the tumor than from active regulation by Tregs.

BACKGROUND: The PD-1/PD-L1 immunomodulatory pathway plays a central role in regulating T-cell effector function and has been implicated in immune evasion in glioblastoma. It is also known that populations of tumor-infiltrating lymphocytes may be responsible for disease progression and poor prognosis. A substantial number of these lymphocytes are T effector cells (CD4+CD25−CD127+), however little is known about their function in the tumor microenvironment in glioblastoma or their expression of PD-1. METHODS: We characterized functional differences between T effector cells derived from blood and tumors of patients with glioblastoma (n = 20), with the hypothesis that PD-1 would mark a dysfunctional T effector population. T effector cell proliferative capacity was assessed by stimulation with anti-CD3/anti-CD28/anti-CD2 in thymidine incorporation assays; cytokine production profiles were assessed ex vivo by stimulation with PMA and ionomycin; transcriptional profiling was determined by RNA sequencing. We also performed transcriptional profiling on PD-1+ and PD-1− T effector subsets isolated from the peripheral blood and tumors of patients with glioblastoma and from the peripheral blood of healthy donors. RESULTS: The frequency of PD-1+TIM-3+ T effectors in the peripheral blood of patients with glioblastoma was significantly increased compared to healthy controls. Tumor-derived PD-1+ T effectors displayed significantly less proliferation but retained the ability to produce inflammatory cytokines, including IFNγ. PD-1+ T effectors derived from healthy donors displayed impaired proliferation, which could not be rescued by PD-1/PD-L1 blockade or the addition of IL-2. PD-1+ T effector cells were enriched in several gene sets, indicating that PD-1+ T effector cells may be clonally exhausted. We also identified several genes that differentiated tumor-derived and peripheral blood-derived PD-1+ T effectors in glioblastoma patients. CONCLUSIONS: These data indicate that PD-1 expression marks a dysfunctional population of clonally exhausted T effector cells that may be important in the context of glioblastoma.

Association studies have greatly refined the understanding of how variation within the human leukocyte antigen (HLA) genes influences risk of multiple sclerosis. However, the extent to which major effects are modulated by interactions is poorly characterized. We analyzed high-density SNP data on 17,465 cases and 30,385 controls from 11 cohorts of European ancestry, in combination with imputation of classical HLA alleles, to build a high-resolution map of HLA genetic risk and assess the evidence for interactions involving classical HLA alleles. Among new and previously identified class II risk alleles (HLA-DRB1*15:01, HLA-DRB1*13:03, HLA-DRB1*03:01, HLA-DRB1*08:01 and HLA-DQB1*03:02) and class I protective alleles (HLA-A*02:01, HLA-B*44:02, HLA-B*38:01 and HLA-B*55:01), we find evidence for two interactions involving pairs of class II alleles: HLA-DQA1*01:01-HLA-DRB1*15:01 and HLA-DQB1*03:01-HLA-DQB1*03:02. We find no evidence for interactions between classical HLA alleles and non-HLA risk-associated variants and estimate a minimal effect of polygenic epistasis in modulating major risk alleles.

High salt is positively associated with the risk of many diseases. However, little is known about the mechanisms. Here we showed that high salt increased proinflammatory molecules, while decreased anti-inflammatory and proendocytic molecules in both human and mouse macrophages. High salt also potentiated lipopolysaccharide-induced macrophage activation and suppressed interleukin 4-induced macrophage activation. High salt induced the proinflammatory aspects by activating p38/cFos and/or Erk1/2/cFos pathways, while inhibited the anti-inflammatory and proendocytic aspects by Erk1/2/signal transducer and activator of transcription 6 pathway. Consistent with the in vitro results, high-salt diet increased proinflammatory gene expression of mouse alveolar macrophages. In mouse models of acute lung injury, high-salt diet aggravated lipopolysaccharide-induced pulmonary macrophage activation and inflammation in lungs. These results identify a novel macrophage activation state, M(Na), and high salt as a potential environmental risk factor for lung inflammation through the induction of M(Na).

The transcription factor nuclear factor κB (NFκB) is a central regulator of inflammation, and genome-wide association studies in subjects with autoimmune disease have identified a number of variants within the NFκB signaling cascade. In addition, causal variant fine-mapping has demonstrated that autoimmune disease susceptibility variants for multiple sclerosis (MS) and ulcerative colitis are strongly enriched within binding sites for NFκB. We report that MS-associated variants proximal to NF κ B1 and in an intron of TNFRSF1A ( TNFR1 ) are associated with increased NFκB signaling after tumor necrosis factor–α (TNFα) stimulation. Both variants result in increased degradation of inhibitor of NFκB α (IκBα), a negative regulator of NFκB, and nuclear translocation of p65 NFκB. The variant proximal to NF κ B1 controls signaling responses by altering the expression of NFκB itself, with the GG risk genotype expressing 20-fold more p50 NFκB and diminished expression of the negative regulators of the NFκB pathway: TNFα-induced protein 3 (TNFAIP3), B cell leukemia 3 (BCL3), and cellular inhibitor of apoptosis 1 (CIAP1). Finally, naive CD4 T cells from patients with MS express enhanced activation of p65 NFκB. These results demonstrate that genetic variants associated with risk of developing MS alter NFκB signaling pathways, resulting in enhanced NFκB activation and greater responsiveness to inflammatory stimuli. As such, this suggests that rapid genetic screening for variants associated with NFκB signaling may identify individuals amenable to NFκB or cytokine blockade.

Autoimmune diseases affect up to approximately 10% of the population. While rare Mendelian autoimmunity syndromes can result from monogenic mutations disrupting essential mechanisms of central and peripheral tolerance, more common human autoimmune diseases are complex disorders that arise from the interaction between polygenic risk factors and environmental factors. Although the risk attributable to most individual nucleotide variants is modest, genome-wide association studies (GWAS) have the potential to provide an unbiased view of biological pathways that drive human autoimmune diseases. Interpretation of GWAS requires integration of multiple genomic datasets including dense genotyping, cis-regulatory maps of primary immune cells, and genotyped studies of gene expression in relevant cell types and cellular conditions. Improved understanding of the genetic basis of autoimmunity may lead to a more sophisticated understanding of underlying cellular phenotypes and, eventually, novel diagnostics and targeted therapies.

Myelin-reactive T cells have been identified in patients with multiple sclerosis (MS) and healthy subjects with comparable frequencies, but the contribution of these autoreactive T cells to disease pathology remains unknown. A total of 13,324 T cell libraries generated from blood of 23 patients and 22 healthy controls were interrogated for reactivity to myelin antigens. Libraries derived from CCR6(+) myelin-reactive T cells from patients with MS exhibited significantly enhanced production of interferon-γ (IFN-γ), interleukin-17 (IL-17), and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to healthy controls. Single-cell clones isolated by major histocompatibility complex/peptide tetramers from CCR6(+) T cell libraries also secreted more proinflammatory cytokines, whereas clones isolated from controls secreted more IL-10. The transcriptomes of myelin-specific CCR6(+) T cells from patients with MS were distinct from those derived from healthy controls and, notably, were enriched in T helper cell 17 (TH17)-induced experimental autoimmune encephalitis gene signatures, and gene signatures derived from TH17 cells isolated other human autoimmune diseases. These data, although not causal, imply that functional differences between antigen-specific T cells from MS and healthy controls are fundamental to disease development and support the notion that IL-10 production from myelin-reactive T cells may act to limit disease progression or even pathogenesis.

Genome-wide association studies have identified loci underlying human diseases, but the causal nucleotide changes and mechanisms remain largely unknown. Here we developed a fine-mapping algorithm to identify candidate causal variants for 21 autoimmune diseases from genotyping data. We integrated these predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4(+) T-cell subsets, regulatory T cells, CD8(+) T cells, B cells, and monocytes. We find that ∼90% of causal variants are non-coding, with ∼60% mapping to immune-cell enhancers, many of which gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation. Causal variants tend to occur near binding sites for master regulators of immune differentiation and stimulus-dependent gene activation, but only 10-20% directly alter recognizable transcription factor binding motifs. Rather, most non-coding risk variants, including those that alter gene expression, affect non-canonical sequence determinants not well-explained by current gene regulatory models.

Naive T cells get activated upon encounter with their cognate antigen and differentiate into a specific subset of effector cells. These T cells are themselves plastic and are able to re-differentiate into another subset, changing both phenotype and function. Differentiation into a specific subset depends on the nature of the antigen and of the environmental milieu. Notably, certain nutrients, such as vitamins A and D, sodium chloride, have been shown to modulate T cell responses and influence T cell differentiation. Parasite infection can also skew Th differentiation. Similarly, the gut microbiota regulates the development of immune responses. Lastly, the key role of metabolism on T cells has also been demonstrated. This series of articles highlights some of the multiple links existing between environmental factors and T cell responses.

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.

The proteins encoded by the classical HLA class I and class II genes in the major histocompatibility complex (MHC) are highly polymorphic and play an essential role in self/non-self immune recognition. HLA variation is a crucial determinant of transplant rejection and susceptibility to a large number of infectious and autoimmune disease1. Yet identification of causal variants is problematic due to linkage disequilibrium (LD) that extends across multiple HLA and non-HLA genes in the MHC2,3. We therefore set out to characterize the LD patterns between the highly polymorphic HLA genes and background variation by typing the classical HLA genes and >7,500 common single nucleotide polymorphisms (SNPs) and deletion/insertion polymorphisms (DIPs) across four population samples. The analysis provides informative tag SNPs that capture some of the variation in the MHC region and that could be used in initial disease association studies, and provides new insight into the evolutionary dynamics and ancestral origins of the HLA loci and their haplotypes.

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.

To date, three loci have been validated to confer susceptibility to inflammatory bowel disease (IBD): the CARD15/NOD2 gene, the discs large homolog 5 gene (DLG5), and the IBD5 locus on 5q31 (IBD5). We have explored the possibility that these loci may also be associated with susceptibility to two other chronic inflammatory diseases, multiple sclerosis (MS) and systemic lupus erythematosus (SLE). As the CARD15 risk alleles had previously been assessed in our collection of 496 MS trios, we focused our efforts on the DLG5 risk allele and the IBD5(risk) haplotype (IBD5(risk)) for MS. While there is no evidence of association within our MS sample with either of these polymorphisms, screening of 1027 subjects with SLE suggests that IBD5(risk) may have a modest contribution to disease risk in the subset of SLE subjects without lupus nephritis. In addition, a pooled analysis of existing published and unpublished data in 1305 cases of SLE genotyped for the CARD15 risk alleles suggests that only the CARD15(908R) IBD risk allele may have a strong effect on risk of SLE. Our data, therefore, suggest that both the CARD15 gene and the IBD5 locus may have a role as general susceptibility loci for certain common, genetically complex inflammatory 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.

In autoimmune type 1 diabetes, pathogenic T lymphocytes are associated with the specific destruction of insulin-producing beta-islet cells. Identification of the autoantigens involved in triggering this process is a central question. Here we examined T cells from pancreatic draining lymph nodes, the site of islet-cell-specific self-antigen presentation. We cloned single T cells in a non-biased manner from pancreatic draining lymph nodes of subjects with type 1 diabetes and from non-diabetic controls. A high degree of T-cell clonal expansion was observed in pancreatic lymph nodes from long-term diabetic patients but not from control subjects. The oligoclonally expanded T cells from diabetic subjects with DR4, a susceptibility allele for type 1 diabetes, recognized the insulin A 1-15 epitope restricted by DR4. These results identify insulin-reactive, clonally expanded T cells from the site of autoinflammatory drainage in long-term type 1 diabetics, indicating that insulin may indeed be the target antigen causing autoimmune diabetes.

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.

The sequencing of the human genome and the intense study of its variation in different human populations have improved our understanding of the genome's architecture. It is now becoming clear that segments of the genome that are unbroken by reshuffling or recombination during meiosis create a mosaic of DNA 'haplotype blocks'. Here, we discuss the advantages and limitations of this block structure. Haplotype blocks hold the promise of reducing the complexity of analysing the human genome for association with disease. But can they deliver on this promise? First generation maps of these block patterns, such as the admixture and haplotype maps, are now emerging and, it is to be hoped, will accelerate the discovery of alleles that contribute to susceptibility to human inflammatory diseases.

Admixture mapping (also known as "mapping by admixture linkage disequilibrium," or MALD) provides a way of localizing genes that cause disease, in admixed ethnic groups such as African Americans, with approximately 100 times fewer markers than are required for whole-genome haplotype scans. However, it has not been possible to perform powerful scans with admixture mapping because the method requires a dense map of validated markers known to have large frequency differences between Europeans and Africans. To create such a map, we screened through databases containing approximately 450000 single-nucleotide polymorphisms (SNPs) for which frequencies had been estimated in African and European population samples. We experimentally confirmed the frequencies of the most promising SNPs in a multiethnic panel of unrelated samples and identified 3011 as a MALD map (1.2 cM average spacing). We estimate that this map is approximately 70% informative in differentiating African versus European origins of chromosomal segments. This map provides a practical and powerful tool, which is freely available without restriction, for screening for disease genes in African American patient cohorts. The map is especially appropriate for those diseases that differ in incidence between the parental African and European populations.

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.

Rapamycin inhibits the proliferation of many mammalian cell types, including lymphocytes, making the compound useful as an immunosuppressant. Rapamycin has also been a useful tool for studying signaling mechanisms regulating cellular proliferation. However, the effects of rapamycin remain poorly understood, and the precise mechanisms of clinical action remain elusive. Previously, we found that, depending on the strength of the signal delivered to the T cell via both the T cell receptor and the costimulatory molecule CD28, CD8+ T cells are capable of rapamycin-resistant proliferation. Here, we have further elucidated the mechanism of rapamycin-resistant proliferation of human CD8+ T cells. Under conditions where rapamycin inhibited proliferation, p27kip1 down-regulation was prevented, whereas under conditions resulting in rapamycin-resistant proliferation, p27kip1 was down-regulated. Further, T cell receptor/CD28-dependent induction of bcl-xL expression was not inhibited by rapamycin, which correlated with both rapamycin-resistant proliferation and increased cell survival. Moreover, an inhibitor of phosphoinositide 3-kinase activity was able to eliminate rapamycin-resistant proliferation of freshly isolated CD8+ human cells, strongly suggesting that phosphoinositide 3-kinase activity was required for the rapamycin-resistant proliferation of CD8+ T cells. The selective immunosuppressive effect of rapamycin in human CD8+ T cell populations could be predictive of a selective effect allowing cytotoxic responses during microbial infections where there are strong strengths of signals associated with high affinity T cell receptors and strong costimulatory second signals. In contrast, the weaker autoimmune and perhaps allogeneic responses can be selectively inhibited by the actions of rapamycin.

An enzyme-linked immunosorbent assay of antibodies in the serum of 86 patients with multiple sclerosis and that of 51 healthy controls that used KIR4.1 as the target protein showed no significant between-group difference.

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.

CD8+T cells can become anergic following activation, though the cellular mechanism, as compared to CD4+ T cells, remains poorly understood. Here, we examined the effects of different antigen-dose, peptide ligands, and engagement of costimulatory molecules on the induction of CD8+ T cell anergy. We observed that increasing strengths of signals delivered to CD8+ T cells by varying the antigen-dose and the nature of peptide ligands induced increasing degrees of non-responsiveness to secondary stimulation. Furthermore, higher levels of LFA-3 engagement of CD2 rendered CD8+ T cells unresponsive to secondary antigenic re-challenge. This pattern of secondary responsiveness lasted up to 2 weeks following primary stimulation and was not correlated with prior cell division history. These results indicate that the strength of prior stimuli, which is determined by the sum of signals from both TCR and costimulatory molecules, determines the activation threshold and magnitude of CD8+ T cell responses.

CD4(+)CD25(high)CD127(low/-) forkhead box p3 (Foxp3)(+) regulatory T cells (T(reg) cells) possess functional plasticity. Here we describe a higher frequency of T helper type 1 (T(H)1)-like, interferon-γ (IFN-γ)-secreting Foxp3(+) T cells in untreated subjects with relapsing remitting multiple sclerosis (RRMS) as compared to healthy control individuals. In subjects treated with IFN-β, the frequency of IFN-γ(+)Foxp3(+) T cells is similar to that in healthy control subjects. In vitro, human T(reg) cells from healthy subjects acquire a T(H)1-like phenotype when cultured in the presence of interleukin-12 (IL-12). T(H)1-like T(reg) cells show reduced suppressive activity in vitro, which can partially be reversed by IFN-γ-specific antibodies or by removal of IL-12.

The recent descriptions of widespread random monoallelic expression (RMAE) of genes distributed throughout the autosomal genome indicate that there are more genes subject to RMAE on autosomes than the number of genes on the X chromosome where X-inactivation dictates RMAE of X-linked genes. Several of the autosomal genes that undergo RMAE have independently been implicated in human Mendelian disorders. Thus, parsing the relationship between allele-specific expression of these genes and disease is of interest. Mutations in the human forkhead box P2 gene, FOXP2 , cause developmental verbal dyspraxia with profound speech and language deficits. Here, we show that the human FOXP2 gene undergoes RMAE. Studying an individual with developmental verbal dyspraxia, we identify a deletion 3 Mb away from the FOXP2 gene, which impacts FOXP2 gene expression in cis. Together these data suggest the intriguing possibility that RMAE impacts the haploinsufficiency phenotypes observed for FOXP2 mutations.