Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response.

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) ß-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8+ T cell populations specific for variants of the nonstructural protein epitope NS3133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3133-DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2+ TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second ß-chain complementarity-determining region (CDR2ß). Extensive mutagenesis studies of three distinct TRBV11-2+ TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2ß loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

Infectious pathogens may trigger specific allo-HLA reactivity via multiple mechanisms.

Transplant recipients can be sensitized against allo-HLA antigens by previous transplantation, blood transfusion, or pregnancy. While there is growing awareness that multiple components of the immune system can act as effectors of the alloresponse, the role of infectious pathogen exposure in triggering sensitization and allograft rejection has remained a matter of much debate. Here, we describe that exposure to pathogens may enhance the immune response to allogeneic HLA antigens via different pathways. The potential role of allo-HLA cross-reactivity of virus-specific memory T cells, activation of innate immunity leading to a more efficient induction of the adaptive alloimmune response by antigen-presenting cells, and bystander activation of existing memory B cell activation will be discussed in this review.

T-cell alloreactivity and transplantation outcome: a budding role for heterologous immunity?

PURPOSE OF REVIEW: Despite the association between alloreactive T cells and poor graft survival, the mechanisms behind T-cell-mediated rejection are still under investigation. In this review, we will discuss the latest insights into the impact of T-cell alloreactivity on solid organ transplantation and hematopoietic stem cell transplantation (HSCT), with special emphasis on the potential impact of heterologous immunity. RECENT FINDINGS: A large part of the memory T-cell repertoire is induced upon virus infections, and evidence for a role of T-cell receptor cross-reactivity of virus-induced memory T cells against allogeneic human leukocyte antigen (HLA) is accumulating in experimental and clinical solid organ transplantation studies. In HSCT, strong alloreactive potential of naïve T cells causes concerns for graft-versus-host disease while additional HLA-DP matching is suggested to prevent CD4 alloreactivity. Furthermore, virus-induced memory T cells hamper mixed chimerism induction, pointing once more towards a role for heterologous immunity. SUMMARY: Both memory and naïve T cells contribute to the alloimmune response after transplantation. Monitoring for T-cell phenotypes could help predict rejection episodes and/or graft-versus-host disease, allowing timely intervention. Tailoring donor lymphocyte infusions and additional HLA matching could prevent strong alloreactivity in HSCT. Furthermore, the potential role of heterologous immunity in T-cell alloreactivity and transplantation is gaining interest.

Stimulation of human EBV- and CMV-specific cytolytic effector function using allogeneic HLA molecules.

Viral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from virus-specific memory T cells is common, it is unclear whether priming with specific allogeneic cells could conversely elicit a viral peptide/self-HLA restricted cytotoxic T cell response in humans. First, we used the previously described allo-HLA-B*44:02 cross-reactivity of EBV peptide/HLA-B8 restricted T cells, to determine whether allogeneic HLA stimulation can elicit a cytolytic immune response against EBV. HLA-B8(+) HLA-B44(-) EBV-seropositive PBMCs were stimulated with either HLA-B*44:02(+) or HLA-B*44:03(+) mismatched irradiated PBMCs in a 7-10 d MLR. The allo-HLA stimulated responder cells were then evaluated for cytotoxicity using EBV peptide loaded autologous target cells and unloaded HLA-B8(+) EBV LCL target cells. PBMCs from EBV-seropositive donors gained EBV-specific cytolytic effector function following specific allo-HLA stimulation. Finally, we also elicited cytolytic CMV-specific responses using specific allogeneic cell stimulation, to confirm that this technique can be used to elicit viral peptide/self-HLA restricted responses even from nonpublic TCR responses. Allogeneic cell stimulation used as a cell therapy may be a potential tool to augment an antiviral T cell response in patients with EBV or CMV infection.

Preferential HLA-B27 Allorecognition Displayed by Multiple Cross-Reactive Antiviral CD8+ T Cell Receptors.

T cells provide essential immunosurveillance to combat and eliminate infection from pathogens, yet these cells can also induce unwanted immune responses via T cell receptor (TCR) cross-reactivity, also known as heterologous immunity. Indeed, pathogen-induced TCR cross-reactivity has shown to be a common, robust, and functionally potent mechanism that can trigger a spectrum of human immunopathologies associated with either transplant rejection, drug allergy, and autoimmunity. Here, we report that several virus-specific CD8+ T cells directed against peptides derived from chronic viruses (EBV, CMV, and HIV-1) presented by high frequency HLA-A and -B allomorphs differentially cross-react toward HLA-B27 allotypes in a highly focused and hierarchical manner. Given the commonality of cross-reactive T cells and their potential contribution to adverse outcomes in allogeneic transplants, our study demonstrates that multiple antiviral T cells recognizing the same HLA allomorph could pose an extra layer of complexity for organ matching.

Infection with a virus generates a polyclonal immune response with broad alloreactive potential.

Virus-specific T cells have been shown to cross-react with allogeneic HLA (allo-HLA) at a clonal level. However, the impact of a single virus on the allorepertoire has never been investigated at the polyclonal level. We made an inventory of the incidence and specificity of allo-HLA-cross-reactive-virus-specific CD8+ T cells in 24 healthy individuals. T cells were stained for 25 virus-specific tetramers, and mixed-lymphocyte reactions were performed against a panel of HLA-typed allostimulators. Allospecificity was confirmed by IFNγ-ELISA using T-cell clones against a panel of HLA-typed cell-lines. The polyclonal immune repertoire directed against CMV alone was associated with a memory response against six allo-HLA molecules. Besides, a single allostimulator activated memory T-cell responses with multiple viral specificities. Concluding, a single virus can substantially broaden the allo-HLA memory T-cell repertoire. This study only looked at CMV- and EBV-specific T cells, whereas the immune repertoire consists of T cells directed against many different viruses. Hence, transplant patients receiving an HLA-mismatched graft may already express a polyclonal repertoire of anti-donor-memory T cells before transplantation.

Cross-Reactivity of Virus-Specific CD8+ T Cells Against Allogeneic HLA-C: Possible Implications for Pregnancy Outcome.

Heterologous immunity of virus-specific T cells poses a potential barrier to transplantation tolerance. Cross-reactivity to HLA-A and -B molecules has broadly been described, whereas responses to allo-HLA-C have remained ill defined. In contrast to the transplant setting, HLA-C is the only polymorphic HLA molecule expressed by extravillous trophoblasts at the maternal-fetal interface during pregnancy. Uncontrolled placental viral infections, accompanied by a pro-inflammatory milieu, can alter the activation status and stability of effector T cells. Potential cross-reactivity of maternal decidual virus-specific T cells to fetal allo-HLA-C may thereby have detrimental consequences for the success of pregnancy. To explore the presence of cross-reactivity to HLA-C and the other non-classical HLA antigens expressed by trophoblasts, HLA-A and -B-restricted CD8+ T cells specific for Epstein-Barr virus, Cytomegalovirus, Varicella-Zoster virus, and Influenza virus were tested against target cells expressing HLA-C, -E, and -G molecules. An HLA-B*08:01-restricted EBV-specific T cell clone displayed cross-reactivity against HLA-C*01:02. Furthermore, cross-reactivity of HLA-C-restricted virus-specific CD8+ T cells was observed for HCMV HLA-C*06:02/TRA CD8+ T cell lines and clones against HLA-C*03:02. Collectively, these results demonstrate that cross-reactivity against HLA-C can occur and thereby may affect pregnancy outcome.

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8+ T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8+ T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions.

Detection of Virus-Specific CD8+ T Cells With Cross-Reactivity Against Alloantigens: Potency and Flaws of Present Experimental Methods.

UNLABELLED: Virus-specific T cells have the intrinsic capacity to cross-react against allogeneic HLA antigens, a phenomenon known as heterologous immunity. In transplantation, these cells may contribute to the alloimmune response and negatively impact graft outcome. This study describes the various techniques that can be used to detect heterologous immune responses of virus-specific CD8(+) T cells against allogeneic HLA antigens. The strengths and weaknesses of the different approaches are discussed and illustrated by experimental data. METHODS: Mixed-lymphocyte reactions (MLRs) were performed to detect allo-HLA cross-reactivity of virus-specific CD8(+) T cells in total peripheral blood mononuclear cells. T-cell lines and clones were generated to confirm allo-HLA cross-reactivity by IFNγ production and cytotoxicity. In addition, the conventional MLR protocol was adjusted by introducing a 3-day resting phase and subsequent short restimulation with alloantigen or viral peptide, whereupon the expression of IFNγ, IL-2, CD107a, and CD137 was determined. RESULTS: The accuracy of conventional MLR is challenged by potential bystander activation. T-cell lines and clones can circumvent this issue, yet their generation is laborious and time-consuming. Using the adjusted MLR and restimulation protocol, we found that only truly cross-reactive T cells responded to re-encounter of alloantigen and viral peptide, whereas bystander-activated cells did not. CONCLUSIONS: The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8(+) T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard.

The interplay between antiviral immunity and allo-immune reactivity after renal transplantation: consortium between the Centres Amsterdam, Leiden and Nijmegen (ALLOVIR).

In the consortium "ALLOVIR" we aim to characterize the effect of CMV and BKV infections on the innate immune responses to viral and alloantigens. Furthermore, we want to characterize the interplay between adaptive immune responses to viral and alloantigens with emphasis on the role of heterologous immunity. Thirdly, we will characterize the manifestations of these immune responses in the allograft, as reflected in tissue and urine, and their correlation with graft function. Finally, we will assess how immunosuppressive drugs interfere with these cross-reactive immune responses.