Disease etiology and diagnosis by TCR repertoire analysis goes viral

The importance of T‐cell receptor (TCR) repertoire diversity is highlighted in murine models of immunodeficiency and in many human pathologies. However, the true extent of TCR diversity and how this diversity varies in health and disease is poorly understood. In a previous issue of the European Journal of Immunology, Lossius et al. [Eur. J. Immunol. 2014. 44: 3439–3452] dissected the composition of the TCR repertoire in the context of multiple sclerosis (MS) using high‐throughput sequencing of TCR‐β chains in cerebrospinal fluid samples and blood. The authors demonstrated that the TCR repertoire of the CSF was largely distinct from the blood and enriched in EBV‐reactive CD8⁺ T cells in MS patients. Studies of this kind have long been hindered by technical limitations and remain scarce in the literature. However, TCR sequencing methodologies are progressing apace and will undoubtedly shed light on the genetic basis of T‐cell responses and the ontogeny of T‐cell‐mediated diseases, such as MS.     

CD8+ MAIT cells infiltrate into the CNS and alterations in their blood frequencies correlate with IL‐18 serum levels in multiple sclerosis

Recent findings indicate a pathogenic involvement of IL‐17‐producing CD8⁺ T cells in multiple sclerosis (MS). IL‐17 production has been attributed to a subset of CD8⁺ T cells that belong to the mucosal‐associated invariant T (MAIT) cell population. Here, we report a reduction of CD8⁺ MAIT cells in the blood of MS patients compared with healthy individuals, which significantly correlated with IL‐18 serum levels in MS patients. In vitro stimulation of peripheral blood mononuclear cells from healthy individuals and MS patients with IL‐18 specifically activated CD8⁺ MAIT cells. Moreover, IL‐18 together with T‐cell receptor stimulation induced, specifically on CD8⁺ MAIT cells, an upregulation of the integrin very late antigen‐4 that is essential for the infiltration of CD8⁺ T cells into the CNS. Notably, we were able to identify CD8⁺ MAIT cells in MS brain lesions by immunohistochemistry while they were almost absent in the cerebrospinal fluid (CSF). In summary, our findings indicate that an IL‐18–driven activation of CD8⁺ MAIT cells contributes to their CNS infiltration in MS, in turn leading to reduced CD8⁺ MAIT‐cell frequencies in the blood. Therefore, CD8⁺ MAIT cells seem to play a role in the innate arm of immunopathology in MS.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

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.     

Intrathecal CD4+ and CD8+ T‐cell responses to endogenously synthesized candidate disease‐associated human autoantigens in multiple sclerosis patients

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T‐cell platform was developed to determine intrathecal CD4⁺ and CD8⁺ T‐cell responses to candidate MS‐associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein–Barr virus (EBV)‐based vector to express cMSAg at high levels in EBV‐transformed B‐cells (BLCLs). Human cMSAg cloned were myelin‐associated and ‐oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP‐dependent potassium channel ATP‐dependent inwards rectifying potassium channel 4.1, S100 calcium‐binding protein B, contactin‐2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T‐cell assays to determine cMSAg‐specific T‐cell frequencies in cerebrospinal fluid derived T‐cell lines (CSF‐TCLs) by intracellular IFN‐γ flow cytometry. Whereas all CSF‐TCL responded strongly to mitogenic stimulation, no substantial T‐cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein‐specific CD4⁺ and CD8⁺ T‐cell clones, used as control of the APC/T‐cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T‐cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T‐cell target antigens in CIS and MS patients early after onset of disease.     

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.     

Stimulation through very late antigen‐4 and ‐5 improves the multifunctionality and memory formation of CD8+ T cells

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T‐cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH‐296, we demonstrated that stimulation via very late Ag (VLA)‐4 and VLA‐5 in human and BALB/c mouse CD8⁺ T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR‐transgenic mouse‐derived CD8⁺ T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma‐derived tumor Ag, we showed that stimulation by CH‐296 improved the ability of tumor‐specific CD8⁺ T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3⁺CD4⁺ Treg cells in tumors. These results suggest that stimulation via VLA‐4 and VLA‐5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.     

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.     

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.     

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.     

Age‐associated Epstein–Barr virus‐specific T cell responses in seropositive healthy adults

Epstein–Barr virus (EBV) is present in 95% of the world's adult population. The immune response participates in immune vigilance and persistent infection control, and this condition is maintained by both a good quality (functionality) and quantity of specific T cells throughout life. In the present study, we evaluated EBV‐specific CD4⁺ and CD8⁺ T lymphocyte responses in seropositive healthy individuals younger and older than 50 years of age. The assessment comprised the frequency, phenotype, functionality and clonotypic distribution of T lymphocytes. We found that in both age groups a similar EBV‐specific T cell response was found, with overlapping numbers of tumour necrosis factor (TNF)‐α⁺ T lymphocytes (CD4⁺ and CD8⁺) within the memory and effector cell compartments, in addition to monofunctional and multi‐functional T cells producing interleukin (IL)‐2 and/or interferon (IFN)‐γ. However, individuals aged more than 50 years showed significantly higher frequencies of IL‐2‐producing CD4⁺ T lymphocytes in association with greater production of soluble IFN‐γ, TNF‐α and IL‐6 than subjects younger than 50 years. A polyclonal T cell receptor (TCR)‐variable beta region (Vβ) repertoire exists in both age groups under basal conditions and in response to EBV; the major TCR families found in TNF‐α⁺/CD4⁺ T lymphocytes were Vβ1, Vβ2, Vβ17 and Vβ22 in both age groups, and the major TCR family in TNF‐α⁺/CD8⁺ T cells was Vβ13·1 for individuals younger than 50 years and Vβ9 for individuals aged more than 50 years. Our findings suggest that the EBV‐specific T cell response (using a polyclonal stimulation model) is distributed throughout several T cell differentiation compartments in an age‐independent manner and includes both monofunctional and multi‐functional T lymphocytes.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

Thymus‐resident memory CD8+ T cells mediate local immunity

The thymus is a primary lymphoid organ responsible for production and selection of T cells. Nonetheless, mature T cells and in particular activated T cells can reenter the thymus. Here, we identified memory CD8⁺ T cells specific for lymphocytic choriomeningitis virus or vaccinia virus in the thymus of mice long‐time after the infection. CD8⁺ T cells were mainly located in the thymic medulla, but also in the cortical areas. Interestingly, virus‐specific memory CD8⁺ T cells in the thymus expressed the cell surface markers CD69 and CD103 that are characteristic of tissue‐resident memory T cells in a time‐dependent manner. Kinetic analyses and selective depletion of peripheral CD8⁺ T cells by antibodies further revealed that thymic virus‐specific memory CD8⁺ T cells did not belong to the circulating pool of lymphocytes. Finally, we demonstrate that these thymus‐resident virus‐specific memory CD8⁺ T cells efficiently mounted a secondary proliferative response, exhibited immediate effector functions and were able to protect the thymus from lymphocytic choriomeningitis virus reinfection. In conclusion, the present study not only describes for the first time virus‐specific memory CD8⁺ T cells with characteristics of tissue‐resident memory T (T_RM) cells in a primary lymphoid organ but also extends our knowledge about local T‐cell immunity in the thymus.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

Patient‐shared TCRβ‐CDR3 clonotypes correlate with favorable prognosis in chronic hepatitis B

The presence of shared T‐cell clonotypes was found in several different diseases, but its relationship with the progression of disease remains unclear. By sequencing the complementary determining region 3 of T‐cell receptor (TCR) β chains from the purified antigen‐experienced CD8⁺ T cells, we characterized the T‐cell repertoire in a prospective cohort study among 75 patients with chronic hepatitis B in China, as well as a healthy control and a validation cohort. We found that most T‐cell clones from patients harbored the “patient‐specific” TCR sequences. However, “patient‐shared” TCR clonotypes were also widely found, which correlated with the favorable turnover of disease. Interestingly, the frequency of the “patient‐shared” clonotypes can serve as a biomarker for favorable prognosis. Based on the clonotypes in those patients with favorable outcomes, we created a database including several clusters of protective anti‐HBV CD8⁺ T‐cell clonotypes that might be a reasonable target for therapeutic vaccine development or adoptive cell transfer therapy. These findings were validated in an additional independent cohort of patients. These results suggest that the “patient‐shared” TCR clonotypes may serve as a valuable prognostic tool in the treatment of chronic hepatitis B and possibly other chronic viral diseases.     

Naturally processed HLA‐DR3‐restricted HHV‐6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T‐cell responses

Human herpes virus 6B (HHV‐6B) is a widespread virus that infects most people early in infancy and establishes a chronic life‐long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV‐6B, but antigenic targets and functional characteristics of the CD4 T‐cell response are poorly understood. We identified 25 naturally processed MHC‐II peptides, derived from six different HHV‐6B proteins, and showed that they were recognized by CD4 T‐cell responses in HLA‐matched donors. The peptides were identified by mass spectrometry after elution from HLA‐DR molecules isolated from HHV‐6B‐infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T‐cell responses in vitro. T‐cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3⁺CD4⁺, produced IFN‐γ, TNF‐α, and low levels of IL‐2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide‐pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long‐term control of HHV‐6B infection.     

Cell contact interaction between adipose‐derived stromal cells and allo‐activated T lymphocytes

Mesenchymal stromal cells regulate immune cell function via the secretion of soluble factors. Cell membrane interactions between these cell types may play an additional role. Here, we demonstrate that subpopulations of allo‐activated T cells are capable of binding to human adipose‐derived stromal cells (ASC). The bound T‐cell population contained CD8⁺ T cells and was enriched for CD4^?CD8^? T cells, whereas the proportion of CD4⁺ T cells was decreased compared with the non‐bound T‐cell population. Bound CD4⁺ T cells had high proliferative activity and increased CD25 and FoxP3 expression. However, they also expressed CD127, excluding regulatory T‐cell function. In CD8⁺ T cells, IL‐2 sensitivity, as determined by the analysis of phosphorylated STAT5, was lower in the presence of ASC and even lower in bound cells. In contrast, IL‐2‐induced phosphorylated STAT5 levels were higher in bound CD4⁺ T cells than in non‐bound CD4⁺ T cells. Additionally, pro‐proliferative TGF‐β signalling via endoglin and SMAD1/5/8 phosphorylation was detected in bound CD4⁺ T cells. Even after prolonged co‐culture with ASC, the activated phenotype of bound CD4⁺ T cells persisted. In conclusion, these results demonstrate that the binding of lymphocytes to ASC represents an immunomodulatory mechanism in which CD8⁺ T cells are inhibited in their responsiveness to pro‐inflammatory stimuli and reactive CD4⁺ T cells are depleted from the immune response.