Reduced TCR‐dependent activation through citrullination of a T‐cell epitope enhances Th17 development by disruption of the STAT3/5 balance

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4⁺ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.     

T‐bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death‐1‐deficient mice

Programmed cell death‐1 (PD‐1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD‐1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs). PD‐1‐deficient T cell‐specific T‐bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T‐bet over‐expression, increased interferon (IFN)‐γ production by CD4⁺ T cells and significantly low FoxP3⁺ T_reg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3⁺ T_reg count. The study identified a unique, previously undescribed role for PD‐1 in Th1 and T_reg differentiation, with potential implication in the development of Th1 cell‐targeted therapy.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.     

Distinct IL‐7 signaling in recent thymic emigrants versus mature naïve T cells controls T‐cell homeostasis

IL‐7 is essential for T‐cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL‐7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T‐cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL‐7. Moreover, RTEs express low levels of IL‐7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL‐7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL‐7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL‐7‐induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL‐7 signaling in RTEs preferentially upregulated expression of Bcl‐2, which is anti‐apoptotic but also anti‐proliferative. In contrast, naïve T cells showed diminished Bcl‐2 induction but greater proliferative response to IL‐7. Collectively, these data indicate that IL‐7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

Thymocyte‐derived BDNF influences T‐cell maturation at the DN3/DN4 transition stage

Brain‐derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, and plasticity. Emerging evidence also indicates an essential role for BDNF outside the nervous system, for instance in immune cells. We therefore investigated the impact of BDNF on T cells using BDNF knockout (KO) mice and conditional KO mice lacking BDNF specifically in this lymphoid subset. In both settings, we observed diminished T‐cell cellularity in peripheral lymphoid organs and an increase in CD4⁺CD44⁺ memory T cells. Analysis of thymocyte development revealed diminished total thymocyte numbers, accompanied by a significant increase in CD4/CD8 double‐negative (DN) thymocytes due to a partial block in the transition from the DN3 to the DN4 stage. This was neither due to increased thymocyte apoptosis nor defects in the expression of the TCR‐β chain or the pre‐TCR. In contrast, pERK but not pAKT levels were diminished in DN3 BDNF‐deficient thymocytes. BDNF deficiency in T cells did not result in gross deficits in peripheral acute immune responses nor in changes of the homeostatic proliferation of peripheral T cells. Taken together, our data reveal a critical autocrine and/or paracrine role of T‐cell‐derived BDNF in thymocyte maturation involving ERK‐mediated TCR signaling pathways.     

Absence of both Sos‐1 and Sos‐2 in peripheral CD4+ T cells leads to PI3K pathway activation and defects in migration

Sos‐1 and Sos‐2 are ubiquitously expressed Ras‐guanine exchange factors involved in Erk‐MAP kinase pathway activation. Using mice lacking genes encoding Sos‐1 and Sos‐2, we evaluated the role of these proteins in peripheral T‐cell signaling and function. Our results confirmed that TCR‐mediated Erk activation in peripheral CD4⁺ T cells does not depend on Sos‐1 and Sos‐2, although IL‐2‐mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos‐1/2dKO CD4⁺ T cells upon TCR and IL‐2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL‐2 stimulation in Sos‐1/2dKO CD4⁺ T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos‐1/2dKO T cells and a subsequent impairment in T‐cell migration.     

OX40 controls effector CD4+ T‐cell expansion,not follicular T helper cell generation in acute Listeria infection

To investigate the importance of OX40 signals for physiological CD4⁺ T‐cell responses, an endogenous antigen‐specific population of CD4⁺ T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S‐specific CD4⁺ T cells, but only on a subset that co‐expressed effector cell markers. This population was specifically expanded by Ab‐ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S‐specific effector cells. OX40 was not expressed by 2W1S‐specific memory cells, although it was rapidly up‐regulated upon challenge whereupon Ab‐ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4⁺ T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.     

Antigen presentation by B cells guides programing of memory CD4+ T‐cell responses to a TLR4‐agonist containing vaccine in mice

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T‐helper 1 (T_H1) CD4⁺ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B‐cell deficient mice (μMT^?/?), tetramer‐positive CD4⁺ T cells, markers of memory “precursor” effector cells (MPECs), and T‐cell adoptive transfers and demonstrated that the early antigen‐specific cytokine‐producing T_H1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T_H1 response in μMT^?/? mice. We further show that antigen‐presentation by B cells is necessary for their role in MPEC generation using B‐cell adoptive transfers from wt or MHC class II knock‐out mice into μMT^?/? mice. Our study challenges the view that B‐cell deficiency exclusively alters the T_H1 response at memory time‐points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T_H1 cell‐mediated immunity.     

The adaptor protein SAP regulates type II NKT‐cell development,cytokine production,and cytotoxicity against lymphoma

CD1d‐restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT‐cell TCR transgenic mouse model, we demonstrated that CD1d‐expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT‐cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP‐deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine‐producing capacity and diminished cytotoxicity to CD1d‐expressing lymphoma cells. The impaired IL‐4 production by SAP‐deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA‐3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X‐linked lymphoproliferative disease caused by mutations in SAP.     

T cell receptor alpha variable 12‐2 bias in the immunodominant response to Yellow fever virus

The repertoire of human αβ T‐cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen‐specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA‐A*0201‐restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8⁺ T cell response to the highly effective YF‐17D vaccine. We discover that these A2/LLW‐specific CD8⁺ T cells are highly biased for the TCR α chain TRAV12‐2. This bias is already present in A2/LLW‐specific naïve T cells before vaccination with YF‐17D. Using CD8⁺ T cell clones, we show that TRAV12‐2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline‐encoded complementarity determining region (CDR) 1α loop of TRAV12‐2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T‐cell responses specific for the A2/LLW epitope.     

Critical role of IL‐33 receptor ST2 in experimental cerebral malaria development

Cerebral malaria, a severe complication of Plasmodium falciparum infection, can be modeled in murine Plasmodium berghei ANKA (PbA) infection. PbA‐induced experimental cerebral malaria (ECM) is CD8⁺ T‐cell mediated, and influenced by T_H1/T_H2 balance. Here, we show that IL‐33 expression is increased in brain undergoing ECM and we address the role of the IL‐33/ST2 pathway in ECM development. ST2‐deficient mice were resistant to PbA‐induced neuropathology. They survived >20 days with no ECM neurological sign and a preserved cerebral microcirculation, while WT mice succumbed within 10 days with ECM, brain vascular leakage, distinct microvascular pathology obstruction, and hemorrhages. Parasitemia and brain parasite load were similar in ST2‐deficient and WT mice. Protection was accompanied by reduced brain sequestration of activated CD4⁺ T cells and perforin⁺ CD8⁺ T cells. While IFN‐γ and T‐cell‐attracting chemokines CXCL9 and CXCL10 were not affected in the absence of functional ST2 pathway, the local expression of ICAM‐1, CXCR3, and LT‐α, crucial for ECM development, was strongly reduced, and this may explain the diminished pathogenic T‐cell recruitment and resistance to ECM. Therefore, IL‐33 is induced in PbA sporozoite infection, and the pathogenic T‐cell responses with local microvascular pathology are dependent on IL‐33/ST2 signaling, identifying IL‐33 as a new actor in ECM development.     

Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA‐E during active tuberculosis and express type 2 cytokines

CD8 T cells contribute to protective immunity against Mycobacterium tuberculosis. In humans, M. tuberculosis reactive CD8 T cells typically recognize peptides associated to classical MHC class Ia molecules, but little information is available on CD8 T cells recognizing M. tuberculosis Ags presented by nonclassical MHC class Ib molecules. We show here that CD8 T cells from tuberculosis (TB) patients recognize HLA‐E‐binding M. tuberculosis peptides in a CD3/TCR αβ mediated and CD8‐dependent manner, and represent an additional type of effector cells playing a role in immune response to M. tuberculosis during active infection. HLA‐E‐restricted recognition of M. tuberculosis peptides is detectable by a significant enhanced ex vivo frequency of tetramer‐specific circulating CD8 T cells during active TB. These CD8 T cells produce type 2 cytokines upon antigenic in vitro stimulation, help B cells for Ab production, and mediate limited TRAIL‐dependent cytolytic and microbicidal activity toward M. tuberculosis infected target cells. Our results, together with the finding that HLA‐E/M. tuberculosis peptide specific CD8 T cells are detected in TB patients with or without HIV coinfection, suggest that this is a new human T‐cell population that participates in immune response in TB.     

High‐throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV‐reactive CD8+ T cells

Epstein‐Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high‐throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV‐reactivity of the T‐cell receptor (TCR) repertoires in MS. TCR‐β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T‐cell clones, represented by TCR‐β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR‐β libraries generated from peripheral blood T cells reactive against autologous EBV‐transformed B cells were highly enriched for public EBV‐specific sequences and were used to quantify EBV‐reactive TCR‐β sequences in CSF. TCR‐β sequences of EBV‐reactive CD8⁺ T cells, including several public EBV‐specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV‐reactive CD4⁺ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T‐cell response in MS. The presented strategy links TCR sequence to intrathecal T‐cell specificity, demonstrating enrichment of EBV‐reactive CD8⁺ T cells in MS.     

A20 deletion in T cells modulates acute graft‐versus‐host disease in mice

The NF‐κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo‐HSCT) with A20‐deficient CD4⁺ and CD8⁺ donor T cells in mice. Severity and mortality of graft‐versus‐host disease (GVHD) after allo‐HSCT was drastically reduced in recipients transplanted with conventional doses of A20‐deficient T cells. Consistently, we found that the A20‐deficient donor T‐cell compartment was strongly diminished at various timepoints after allo‐HSCT. However, proportionally more A20‐deficient donor T cells produced IFN‐γ and systemic inflammation was elevated early after allo‐HSCT. Consequently, increasing the dose of transplanted A20‐deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20^+/+ T cells. Still, A20‐deficient T cells, activated either through T cell receptor‐dependent or ‐independent mechanisms, were less viable than control A20^+/+ T cells, highlighting that A20 balances both, T‐cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T‐cell‐mediated inflammatory diseases like GVHD.     

Emerging role of interleukin‐31 and interleukin‐31 receptor in pruritus in atopic dermatitis

Atopic dermatitis (AD) is a chronic or chronically relapsing, eczematous, severely pruritic skin disorder associated with skin barrier dysfunction. The lesional skin of AD exhibits T helper 2 (T_H2)‐deviated immune reactions. Interleukin‐31 (IL‐31), preferentially produced from T_H2 cells, is a potent pruritogenic cytokine, and its systemic and local administration induces scratching behavior in rodents, dogs and monkeys. Recent clinical trials have revealed that administration of an anti‐IL‐31 receptor antibody significantly alleviates pruritus in patients with AD. In this review, we summarize recent topics related to IL‐31 and its receptor with special references to atopic itch.     

Induction of T helper cell subsets

Conclusions Long-term murine CD4⁺ T cell clones can be classified into two distinct subsets which produce either IL-2 and IFN- (T_h1) or IL-4 and IL-5 (T_h2). This functional separation of CD4⁺ T cells has subsequently been shown to be of great biological relevance, since these T helper cell subsets can be associated with protective or pathogenic immune responses. While a large number of in vitro and in vivo experiments underlined the biological relevance of T_h1 and T_h2 phenotypes, the ontogeny of T_h1 or T_h2 cells remained unknown until recently. Experiments published during the last 3 years have shown that cytokines such as IL-2, IL-4 and IFN- can influence the LK response pattern of CD4⁺ T cells. However, further investigations demonstrated that these cytokines alone had no effect on the modulation of the LK response pattern.Prolonged TCR binding by mitogenic mAb delivered a signal required for the induction of the T_h2 phenotype. Whereas this signal cannot be replaced by soluble factors, ligation of the TCR together with LK leads to the development of T_h2. The fact that TCR-mediated induction of T_h2 in vitro takes the same time as the antigen-mediated commitment of T_h1- or T_h2-like cells in vivo underlines the role of the TCR in T helper cell subset differentiation. Since the two subsets can be derived from a common postthymic CD4⁺ precursor, the T_h1 or T_h2 phenotype of a T cell is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations which are predetermined for the production of a specific LK pattern.The structure of the natural ligands of the TCR, i. e., antigens and MHC class II-encoded molecules, have been shown to influence the development of T helper cells with distinct LK profiles in vivo. This suggests that the TCR-ligand interaction is decisive for the commitment of a peripheral CD4⁺ T cell towards a T_h1- or a T_h2-like response pattern.     

Abundance of follicular helper T cells in Peyer's patches is modulated by CD155

The secondary humoral immune response is characterized by plasma B cells secreting isotype‐switched and affinity‐matured antibodies. The efficient generation of plasma B cells in the GC depends on the presence of follicular helper T (T_FH) cells, a cell type thought to arise from naive CD4‐positive T cells by a hitherto unresolved differentiation pathway. Mice deficient for CD155, an adhesion receptor of the immunoglobulin superfamily, are impaired to mount a secondary humoral immune response upon oral administration of antigen, while the primary IgM response is unaffected. Here, we show that mice lacking CD155 harbor significantly reduced numbers of T_FH cells in their Peyer's patches. This was paralleled by a decreased frequency of T_FH cells in the GC. Moreover, the CD155 ligand CD226, which is involved in T‐cell activation, is down‐regulated during T_FH cell differentiation, resulting in a complete absence of CD226 on those T_FH cells residing in the GC. Concurrently, the expression of TIGIT/WUCAM, a newly discovered CD155 ligand, is induced in T_FH cells. Thus, these cells replace an activating by a putative inhibitory CD155‐binding partner during their differentiation.