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.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.     

Clonotype‐specific avidity influences the dynamics and hierarchy of virus‐specific regulatory and effector CD4+ T‐cell responses

A key component of immunity against viruses, CD4⁺ T cells expand and differentiate into functional subsets upon primary infection, where effector (Teff) cells facilitate infection control and regulatory (Treg) cells mitigate immunopathology. After secondary infection, Teff cells mount a robust response from the memory pool. Here, we show that Treg‐cell responses are diminished upon secondary infection, and Treg‐cell response dynamics are associated more with T‐cell receptors (TCRs) repertoire and avidity than with epitope specificity. In the murine model, the IA^bM₂₀₉ epitope of respiratory syncytial virus is recognized by both CD4⁺ Treg and Teff cells, while the IA^bM2₂₆ epitope is recognized almost exclusively by CD4⁺ Teff cells expressing high avidity TCR Vβ8.1/8.2 and dominating the CD4⁺ T‐cell response during primary and secondary infections. IA^bM₂₀₉‐Teff cells express relatively low avidity TCRs during early primary infection, but high avidity TCR Vβ7‐expressing IA^bM₂₀₉‐Teff cells emerge during the late phase, and become dominant after secondary infection. The emerging high avidity IA^bM₂₀₉‐Teff cells outcompete IA^bM₂₀₉‐Treg cells that share the same epitope, but have low avidity and are restricted to TCR Vβ2 and Vβ6 subpopulations. These data indicate that MHC‐peptide‐TCR interactions can produce different kinetic and functional profiles in CD4⁺ T‐cell populations even when responding to the same epitope.     

In vivo selection of a TCR Vbeta repertoire directed against an immunodominant influenza virus CTL epitope

Little is known about the mechanisms governing TCR repertoire selection in response to foreign antigens. Here, we evaluate the molecular features of the murine C57BL/6 (B6) TCR Vbeta repertoire directed at the NP(366-374) immunodominant epitope of the influenza virus nucleoprotein. Common or 'public' beta chains are shared among individuals following either primary or secondary infection. Importantly, repertoire diversity decreases substantially after a second viral exposure due to enrichment of TCRs sharing Vbeta CDR3 loops of identical length and highly related amino acid sequences. TCRs from these secondary T cell populations possess greater overall avidity for the NP(366-374)/D(b) complex compared to those from the primary repertoire. Thus, expansion of CD8(+) T cells expressing a favored germline Vbeta gene segment in the primary response and further selection for CDR3beta loops during the secondary response, contribute to optimization of immune recognition against certain viral epitopes.     

High‐sequence diversity and structural conservation in the human T‐cell receptor β junctional region during thymic development

The T‐cell repertoire depends on intrathymic genetic rearrangement events in the T‐cell receptor (TCR) locus, followed by positive and negative selection. The repertoire thus generated is highly diverse, but recent data indicate that the recombination of gene segments is less stochastic than previously suggested. Very little is known of the junctional complementarity determining region 3 (CDR3), which is to a large degree not germline encoded. We have analyzed the development of the human TCR β CDR3 repertoire, from the nonselected CD4⁺CD8⁺CD3^? cells up to the fully selected CD4⁺CD8^? thymocytes. In addition to spectratyping, a fraction of the CDR3 repertoire was sequenced and a structural in silico analysis of the CDR3 loop characteristics performed. Our data show that the thymic TCR repertoire is extremely diverse, and the effect of the selection events can be detected as a measurable loss of polyclonality in the CDR3 loop. However, the main physicochemical features of the CDR3 loop were found already at the nonselected repertoire and showed no progressive changes during the selection. Thus, the main structural characteristics of the CDR3 loop were already determined by the recombination process and not significantly affected by the extensive thymocyte death associated with selection in the thymus.     

CD8+ CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of the T‐cell receptor β‐chain

We identified CD8⁺ CD122⁺ regulatory T cells (CD8⁺ CD122⁺ Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8⁺ CD122⁺ Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T‐cell receptors (TCRs) expressed on CD8⁺ CD122⁺ Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8⁺ CD122⁺ Treg cells, we compared the TCR diversity of CD8⁺ CD122⁺ T cells with that of conventional, naive T cells in mice. We analysed the use of TCR‐Vβ in the interleukin 10‐producing population of CD8⁺ CD122⁺ T cells marked by high levels of CD49d expression, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the complementarity‐determining region 3 (CDR3) of the TCR β‐chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8⁺ CD122⁺ T cells. Clonal expansion in Vβ13⁺ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8⁺ CD122⁺ Treg cells.     

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE‐1 antigen was associated with a prolonged relapse‐free survival for HLA‐A2⁺ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE‐1/A2‐specific T‐cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE‐1‐specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×10⁵ CD8⁺ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Vα12.1 chain for 17/18 MELOE‐1‐specific T‐cell clones derived from healthy donors or patients, associated with diverse Vβ chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE‐1_36–44 peptide and against HLA‐A2⁺ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE‐1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.     

Diversity and clonotypic composition of influenza‐specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Strong and sustained effector function of memory‐ versus naïve‐derived T cells upon T‐cell receptor RNA transfer: Implications for cellular therapy

Current protocols used to select CMV‐specific T cells for adoptive immunotherapy focus on virus‐specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem‐cell transplantation (HSCT) from CMV‐seronegative donors. Here, we redirected T cells of CMV‐seronegative donors with a human genetically engineered TCR recognizing an HLA‐A*0201‐binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non‐viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve‐derived T‐cell subsets were both efficiently transfected by TCR‐RNA, memory‐derived T cells showed much stronger levels of HLA‐A*0201‐restricted cytolytic activity to CMV‐infected fibroblasts and maintained acquired function for 5–10 days. In addition to redirection of CD8⁺ cytotoxic T cells, TCR‐RNA transfection was capable of redirecting CD4⁺ T cells into potent Ag‐specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve‐derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus‐specific CD4⁺ and CD8⁺ T‐cell function. CMV TCR‐RNA may represent a suitable therapeutic ‘off‐the‐shelf’ reagent to be used in severe CMV infections of HSCT patients when endogenous CMV‐specific T‐cell immunity is insufficient.     

Prevention and treatment of experimental autoimmune encephalomyelitis with clonotypic CDR3 peptides: CD4+ FoxP3+ T‐regulatory cells suppress interleukin‐2‐dependent expansion of myelin basic protein‐specific T cells

T‐cell receptor (TCR)‐derived peptides are recognized by the immune system and are capable of modulating autoimmune responses. Using the myelin basic protein (MBP) TCR 1501 transgenic mouse model, we demonstrated that TCR CDR3 peptides from the transgenic TCR can provide a protective effect when therapy is initiated before the induction of experimental autoimmune encephalomyelitis (EAE). More importantly, TCR CDR3 peptide therapy can ameliorate the disease when administered after EAE onset. Concurrent with the therapeutic effects, we observed reduced T‐cell proliferation and reduced interleukin‐2 (IL‐2) levels in response to stimulation with MBP‐85‐99 peptide in splenocyte cultures from mice receiving TCR CDR3 peptides compared with that of control mice. Moreover, we found that Foxp3⁺ CD4 T cells from mice protected with TCR CDR3 peptide are preferentially expanded in the presence of IL‐2. This is supportive of a proposed mechanism where Foxp3⁺ T‐regulatory cells induced by therapy with MBP‐85‐99 TCR CDR3 peptides limit expansion and the encephalitogenic activity of MBP‐85‐99‐specific T cells by regulating the levels of secreted IL‐2.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Gut commensal microbes do not represent a dominant antigenic source for continuous CD4+ T‐cell activation during HIV‐1 infection

Chronic immune activation is a hallmark of HIV‐1 infection; specifically, the activation of T cells has predictive value for progression to AIDS. The majority of hyperactivated T cells are not HIV‐specific and their antigenic specificities remain poorly understood. Translocation of gut luminal microbial products to systemic sites contributes to chronic immune activation during HIV‐1 infection, but how it affects (TCR‐dependent) immune activation remains elusive. We hypothesized that gut luminal antigens foster activation of CD4⁺ T cells with specificities for commensal bacterial antigens, thereby contributing to the pool of activated CD4⁺ T cells in the circulation of HIV‐1 infected individuals. To test this hypothesis, we quantified the frequencies of gut microbe‐specific CD4⁺ T cells by cytokine production upon restimulation with selected gut commensal microbial antigens. Contrary to our hypothesis, we did not observe increased but rather decreased frequencies of gut microbe‐specific CD4⁺ T cells in HIV‐1 infected individuals compared to healthy controls. We conclude that the increased activation status of circulating CD4⁺ T cells in HIV‐1 infected individuals is not driven by CD4⁺ T cells with specificities for commensal bacterial antigens.     

Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8+ T cells in an epitope‐specific manner

Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus‐harboring hepatocytes influences de novo priming of CD8⁺ T cells. We showed that surface antigen‐expressing transfectants exclusively display a K^b/S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K^b/S208 epitope to CD8⁺ T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K^b/S190‐ and K^b/S208‐specific T cells in C57BL/6 mice by endogenous/DNA‐ or exogenous/protein‐based vaccines, respectively. Silencing the K^b/S190 epitope (K^b/S190_V194F) in antigen‐expressing vectors rescued the presentation of the K^b/S208 epitope in stable transfectants and significantly enhanced priming of K^b/S208‐specific T cells in C57BL/6 mice. A K^b/S190‐mediated immunodominance operating in surface antigen‐expressing cells, but not in rSP‐pulsed cells, led to an efficient suppression in the presentation of the K^b/S208 epitope and a consequent decrease in the priming of K^b/S208‐specific T cells. This K^b/S190‐mediated immunodominance also operated in 1.4HBV‐S^mut transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K^b/S208‐ but not K^b/S190‐specific T cells in 1.4HBV‐S^mut tg mice. However, IFN‐γ⁺ K^b/S208‐specific T cells could not inhibit HBV replication in the liver of 1.4HBV‐S^mut tg mice. These results have practical implications for the design of T‐cell‐stimulating therapeutic vaccines.     

Immunological characteristics and T‐cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T‐cell‐depleted autologous stem cell transplantation

Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T‐cell‐depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T‐cell receptor (TCR) diversity by analysis of the β variable region (TCRVb) complementarity determining region‐3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11·5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8⁺ TCRVb repertoire was highly oligoclonal early in immune reconstitution and re‐emergence of pre‐transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re‐emergence of pre‐ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T‐cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis.     

T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis

Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161^hiTRAV1-2⁺ T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161^hiTRAV1-2⁺ TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells.     

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.