Dual TCR Research Articles

ScRNA+TCR-seq reveals the pivotal role of dual receptor T lymphocytes in the pathogenesis of Kawasaki disease and during IVIG treatment.

Kawasaki disease (KD), a common cause of acquired heart disease in children in developed countries, is primarily treated with intravenous immunoglobulin (IVIG), but some children demonstrate IVIG resistance with increased coronary artery injury risk. T cells have been demonstrated to be involved in the pathogenesis of KD and its treatment with IVIG. However, the role and mechanism of dual TCR T lymphocytes in the occurrence of KD and IVIG therapy remain unclear. This study, based on scRNA-seq combined with TCR-seq technology, clustered the peripheral blood mononuclear cells of 3 healthy controls and 6 KD patients before and after IVIG treatment. Comparative analysis was conducted to investigate the differences in the proportion of single/dual receptor T cells, the characteristics of CDR3 repertoires, cell types, and the expression of transcription factors among the three groups. The study aimed to explore the correlation between dual TCR T cells and KD as well as IVIG treatment. In our experimental results, we observed the presence of dual TCR T cells in all three groups. However, compared to the healthy control group and the IVIG-treated group, the KD patients before IVIG treatment exhibited a lower proportion of dual TCR T cells, with variability between samples, ranging from 4% to 15%. Notably, after IVIG treatment, the proportion of dual TCR T cells significantly increased, stabilizing above 12%, and these T cells also exhibited clonal expansion and a preference for V gene usage. In addition we found differences in dual TCR T cell subsets among the three groups, for example, IVIG treatment increases the proportion of dual TCR Treg cells, but it still remains below that of healthy control groups, significantly higher proportions of both dual TCR CD8 central and effector memory T cells in IVIG-treated KD patients, and differences in the expression of transcription factors between single and dual TCR T cells. These results suggest dual TCR T cells correlate with KD and IVIG treatment. Dual TCR T lymphocytes, especially dual TCR CD8 T cells and Treg cells, play crucial roles in the pathogenesis of KD and during IVIG treatment, providing strong support for further elucidating KD pathogenesis and optimizing treatment strategies.

Dual TCR T Cells: Identity Crisis or Multitaskers?

Dual TCR T cells are a common and natural product of TCR gene rearrangement and thymocyte development. As much as one third of the T cell population may have the capability to express two different TCR specificities on the cell surface. This discovery provoked a reconsideration of the classic model of thymic selection. Many potential roles for dual TCR T cells have since been hypothesized, including posing an autoimmune hazard, dominating alloreactive T cell responses, inducing allergy, and expanding the TCR repertoire to improve protective immunity. Yet, since the initial wave of publications following the discovery of dual TCR T cells, research in the area has slowed. In this study, we aim to provide a brief but comprehensive history of dual TCR T cell research, re-evaluate past observations in the context of current knowledge of the immune system, and identify key issues for future study.

Endogenous co-expression of two T cell receptors promotes lymphopenia-induced proliferation via increased affinity for self-antigen.

Approximately 10% of peripheral T cells express 2 functional TCR αβheterodimers. Receptor co-expression changes the repertoire of TCRs produced during thymic development, enabling generation of T cells bearing TCRs not capable of mediating positive selection or that would normally be negatively selected. The effect of receptor co-expression on the composition and functionality of the peripheral TCR repertoire is not well defined, though evidence demonstrates dual TCR cells pose an increased risk for unwanted immune responses such as autoimmunity and alloreactivity. Based on our previous finding that dual TCR expression promotes positive selection, we hypothesized that dual TCR expression may enhance T cell homeostasis via increased reactivity against self-peptide:MHC (pMHC) ligands. To examine the effect of dual TCR expression on T cell homeostasis, we performed cotransfer experiments comparing T cells genetically deficient for dual TCR expression (TCRα+/- ) with wild-type T cells in models of acute and chronic lymphopenia-induced proliferation (LIP). Lack of dual TCR expression resulted in reduced LIP. The effect of dual TCR expression on LIP was most pronounced in acute lymphopenia, which is driven by recognition of low-affinity self-pMHC ligands. Differences in homeostatic proliferation were not attributable to differences in total TCR expression or signaling, but were dependent on interaction with MHC and associated with increased affinity for positively selecting self-pMHC as evidenced by higher expression of CD5 by dual TCR cells from wild-type mice. These results represent an unappreciated novel mechanism driving homeostasis and shaping the T cell repertoire, potentially promoting autoreactive or heterologous immune responses.

Dual T cell receptor expression promotes agonist selection of regulatory T cells

Abstract Generation of CD4+FoxP3+ regulatory T cells (Treg) in the thymus is essential for maintenance of self-tolerance. Treg lineage commitment occurs via agonist selection, via TCR high-affinity interaction with self-peptide-MHC (pMHC) ligand. We have previously demonstrated that natural co-expression of dual TCRs, occurring in ~10% of T cells, results in increased ability to recognize self-pMHC ligands and promote positive selection. We hypothesized that dual TCR expression could similarly promote agonist Treg selection. Examination of Treg development in B6.TCRa+/− mice, heterozygous for disruptive mutation of TCRAC and unable to express dual TCRs, demonstrated decreased frequency of Tregs in the thymus (2.76+0.08% of CD4 single-positive thymocytes) compared to age-matched wild-type B6 mice (3.26+0.09%, P < 0.001). To more precisely compare Treg agonist selection, we co-transferred 1:1 ratios of T cell-depleted B6.TCRa+/−.Thy1.1 and B6.Ly5.1 bone marrow to lethally-irradiated B6 recipients and monitored thymic selection. TCRa+/− cells were deficient in Treg selection compared to co-transferred wild-type cells; TCRa+/− cells produced 37.57+4.16% of donor-derived thymic Tregs (P = 0.003). This defect was specific for Treg selection, as TCRa+/− cells comprised 44.68+6.33% of double-positive thymocytes and 45.50+5.77% of CD4 single-positive thymocytes. Tregs generated from TCRa+/− or wild-type cells were phenotypically similar, with no differences in expression of FoxP3 or CD5. We propose that the difference in agonist selection of dual TCR and single TCR Tregs could have important consequences in the TCR repertoire and antigenic specificity of peripheral Tregs, impacting immunologic tolerance.

Cutting Edge: Dual TCRα Expression Poses an Autoimmune Hazard by Limiting Regulatory T Cell Generation.

Despite accounting for 10-30% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an autoimmune hazard by allowing self-reactive TCRs to escape thymic selection. We revisited this hypothesis using the NOD murine model of type 1 diabetes. We bred NOD mice hemizygous at both TCRα and β (TCRα+/- β+/-) loci, rendering them incapable of producing dual TCR T cells. We found that the lack of dual TCRα expression skewed the insulin-specific thymocyte population toward greater regulatory T (Treg) cell commitment, resulting in a more tolerogenic Treg to conventional T cell ratio and protection from diabetes. These data support a novel hypothesis by which dual TCR expression can promote autoimmunity by limiting agonist selection of self-reactive thymocytes into the Treg cell lineage.

Dual TCRα expression alters agonist selection, redirecting Treg-biased insulin-specific thymocytes into the conventional T cell lineage

Abstract Despite accounting for ~10% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an “autoimmune hazard” by allowing self-reactive TCRs to escape negative selection. We revisited this hypothesis using the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). We bred NOD mice hemizygous at both TCRα and β (TCRα+/− β+/−) loci, rendering them incapable of producing dual TCR T cells. We found that NOD mice lacking dual TCRα expression were resistant to developing diabetes resulting from an increased insulin-specific Treg:Tconv ratio. Furthermore, early depletion of CD25-expressing cells was sufficient to induce diabetes in single TCR T cell NOD mice demonstrating that these mice are capable of developing diabetes and that Treg cells are responsible for their observed resistance to diabetes. Further investigation uncovered a previously-unrecognized impact of dual TCRα expression on agonist selection. Specifically, we found in single TCR T cell mice that single positive (SP) thymocytes had a higher Treg:CD4 ratio indicating increased Treg commitment, that insulin-specific thymocytes exhibited higher CD5 expression suggesting increased TCR signaling, and that deletion of SP thymocytes was reduced. Altogether these data demonstrate that dual TCRα expression perturbs agonist selection of the insulin-specific thymocyte subset, resulting in skewing of this population toward the diabetogenic conventional CD4+ T cell lineage and away from the regulatory T cell lineage. Thus, dual TCR expression can promote autoimmunity by reducing the ratio of protective Treg cells to pathogenic T cells.

Absence of dual TCR T cells protects NOD mice from diabetes due to an increased insulin-specific Treg:Teff ratio

Abstract Due to allelic exclusion, most αβ T cells express only a single αβ TCR specificity. However, around 10% of human and mouse T cells express two different α chains with the potential for dual specificities. Dual β-expressing T cells also exist and account for ~1% of all T cells. Expression of two different TCR specificities by a single T cell is one hypothesized mechanism by which self-reactive TCRs escape central tolerance to initiate autoimmunity. While peripheral tolerance mechanisms typically control any self-reactive T cells in healthy animals, these mechanisms are hypo-functional in NOD mice. We hypothesized that eliminating dual TCR T cells would protect NOD mice from diabetes. To test this hypothesis we generated NOD mice hemizygous at both the TCRα and β loci (TCRα+/−β+/−). Indeed, we found that NOD mice incapable of generating dual TCR T cells were protected from diabetes with both lower diabetes incidence than wildtype (WT) NOD mice (0% versus 70% at 30 weeks of age) and also lower insulitis scores at 10 weeks of age. Examination of the insulin-specific T cell population in the pancreas-draining lymph node revealed a lower Treg:Teff ratio in WT NOD mice compared to single TCR T cell NOD mice. Anti-PD-1 treatment resulted in the rapid development of diabetes in 100% of WT NOD mice but in only 30% of single TCR T cell mice, suggesting that the diabetes resistance phenotype of single TCR T cell mice is not primarily due to increased anergy. These data support the hypothesis that the presence of dual TCR T cells increases the risk of diabetes in NOD mice by altering the insulin-specific Treg:Teff ratio.

Dual T cell receptor-expressing CD8 T cells potentiate autoreactivity

Abstract Central tolerance serves to eliminate newly developing T cells that express strongly autoreactive T cell receptors. Although central tolerance is efficient in deleting high avidity autoreactive T cells, some lower avidity autoreactive T cells escape negative selection to cause autoimmune diseases. Although tight allelic exclusion limits thymocytes to expressing a single TCRbeta chain, rearrangement of the TCRalpha chain continues unabated until halted by positive selection, enabling thymocytes to express up to two TCRalpha chains and thus two TCRs. Moreover, it has been postulated that pathogenic low avidity autoreactive CD8 T cells may escape central tolerance through expression of a secondary benign TCR that mediates positive selection. To determine the role of dual TCR expressing CD8 T cells in autoreactivity and autoimmunity, we have compared CD8 T cell autoreactivity against the model autoantigen ovalbumin between T cells capable of expressing two TCRs (TCRalpha+/+) versus T cells capable of expressing a single TCR (TCRalpha+/−). TCRalpha+/− CD8 T cells exhibited reduced proliferative capacity upon OVA stimulation relative to TCRalpha+/+ CD8 T cells. In addition, a lower frequency of TCRalpha+/− CD8 T cell effectors produced IFN-gamma upon activation with OVA compared to TCRalpha+/+ CD8 T cell effectors. Taken together, we shown that dual TCR expression by CD8 T cells reduces the efficiency of T cell tolerance and may potentiate T cell autoreactivity. We are investigating whether dual TCR-expressing T cells are key to the pathogenesis of autoimmune diabetes in our mouse model system. Our results will provide valuable insight into the escape mechanisms exploited by pathogenic autoreactive CD8 T cells.

Bi-Allelic TCRα or β Recombination Enhances T Cell Development but Is Dispensable for Antigen Responses and Experimental Autoimmune Encephalomyelitis.

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα+/-β+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions.

The Ability To Rearrange Dual TCRs Enhances Positive Selection, Leading to Increased Allo- and Autoreactive T Cell Repertoires

Thymic selection is designed to ensure TCR reactivity to foreign Ags presented by self-MHC while minimizing reactivity to self-Ags. We hypothesized that the repertoire of T cells with unwanted specificities such as alloreactivity or autoreactivity are a consequence of simultaneous rearrangement of both TCRα loci. We hypothesized that this process helps maximize production of thymocytes capable of successfully completing thymic selection, but results in secondary TCRs that escape stringent selection. In T cells expressing two TCRs, one TCR can mediate positive selection and mask secondary TCR from negative selection. Examination of mice heterozygous for TRAC (TCRα(+/-)), capable of only one functional TCRα rearrangement, demonstrated a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of TCRα repertoires of dual TCR T cells and TCRα(+/-) T cells demonstrated unique TCRs in the presence of secondary rearrangements. The functional impact of secondary TCRs on the naive peripheral repertoire was evidenced by reduced frequencies of T cells responding to autoantigen and alloantigen peptide-MHC tetramers in TCRα(+/-) mice. T cell populations with secondary TCRs had significantly increased ability to respond to altered peptide ligands related to their allogeneic ligand as compared with TCRα(+/-) cells, suggesting increased breadth in peptide recognition may be a mechanism for their reactivity. Our results imply that the role of secondary TCRs in forming the T cell repertoire is perhaps more significant than what has been assumed.

TCR‐engineered T cells: A model of inducible TCR expression to dissect the interrelationship between two TCRs

TCR gene modified T cells for adoptive therapy simultaneously express the Tg TCR and the endogenous TCR, which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-Tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a Tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization).

Dual Receptor T Cells Mediate Pathologic Alloreactivity in Patients with Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGVHD) results from a robust response of donor T cells transferred during hematopoietic stem cell transplantation (HSCT) to allogeneic peptide-major histocompatibility complex antigens. Previous investigations have not identified T cell subsets that selectively mediate either protective immunity or pathogenic alloreactivity. We demonstrate that the small subset of peripheral T cells that naturally express two T cell receptors (TCRs) on the cell surface contributes disproportionately to aGVHD in patients after allogeneic HSCT. Dual TCR T cells from patients with aGVHD demonstrate an activated phenotype and produce pathogenic cytokines ex vivo. Dual receptor clones from a patient with symptomatic aGVHD responded specifically to mismatched recipient human leukocyte antigens (HLAs), demonstrating pathologic alloreactivity. Human dual TCR T cells are strongly activated and expanded by allogeneic stimulation in vitro, and disproportionately contribute to the repertoire of T cells recognizing both major (HLA) and minor histocompatibility antigens, providing a mechanism for their observed activity in vivo in patients with aGVHD. These results identify dual TCR T cells as a target for focused analysis of a T cell subset mediating GVHD and as a potential prognostic indicator.

Key role of Free Heavy Chain of HLA class I molecules in HCMV and tumor stress sensing by gamma-delta TCR

Event Abstract Back to Event Key role of Free Heavy Chain of HLA class I molecules in HCMV and tumor stress sensing by gamma-delta TCR Romain Marlin1, Sonia Netzer1, Vincent Pitard1, Sophie Daburon1, Christelle Harly1, Thomas Bachelet1, Jean-François Moreau1, 2, Jean-Luc Taupin1, 2 and Julie Dechanet1* 1 UMR 5164 CIRID, Univ. Bordeaux Segalen - CNRS, France 2 Immunology and Immunogenetic laboratory, France Non-Vδ2 γδ T cell clones isolated from Cytomegalovirus (HCMV)-infected individuals show dual TCR dependent reactivity against tumor cell lines and HCMV-infected cells suggesting recognition of stress-associated self antigens by non-Vδ2 TCRs. In order to identify these uncharacterized ligands, we immunize mice with tumor cells targeted by HCMV-reactive non-Vδ2 TCRs and select mAbs able to specifically abrogate the reactivity of γδ TCR transductants against these targets. In this study, we focused on a Vγ9Vδ3 TCR, which confers to transductants a restricted reactivity against three tumor B cell lines only. By contrast, several blocking mAbs (LSM) selected to recognize Vγ9Vδ3 antigen, ubiquitously targeted HLA class I (HLA-I) molecules expressed by a wide panel of cells. These results suggested the Vγ9Vδ3 TCR recognition of HLA-I molecules expressed in a particular shape or environment in the three B cell targets. Interestingly, B cell targets and HCMV-infected cells expressed β-2-microglobulin-free HLA-I heavy chains (FHC). Silencing β-2-microglobulin in B cell targets abrogated both HLA-I FHC cell surface expression and Vγ9Vδ3 TCR reactivity, suggesting the recognition of HLA-I FHC by the Vγ9Vδ3 TCR. Moreover, the original Vγ9Vδ3 T clone expressed CD85j and CD158b inhibitory receptors specific for HLA-I, that do not bind FHC, suggesting a regulation of TCR-mediated HLA-I recognition in physiological context. Blocking these receptors markedly increased the Vγ9Vδ3 T clone reactivity against B cell targets and HCMV-infected cells. Altogether, our results identify HLA-I FHC as a potential stress-associated antigen common to HCMV-infection or cellular transformation that mediate stress surveillance by γδ T cells. Keywords: gamma-delta T cells, HLA Antigens, ligand-receptor interaction, CMV, B cells Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Marlin R, Netzer S, Pitard V, Daburon S, Harly C, Bachelet T, Moreau J, Taupin J and Dechanet J (2013). Key role of Free Heavy Chain of HLA class I molecules in HCMV and tumor stress sensing by gamma-delta TCR . Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00960 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Julie Dechanet, UMR 5164 CIRID, Univ. Bordeaux Segalen - CNRS, Bordeaux, France, jdechanet@cirid.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Romain Marlin Sonia Netzer Vincent Pitard Sophie Daburon Christelle Harly Thomas Bachelet Jean-François Moreau Jean-Luc Taupin Julie Dechanet Google Romain Marlin Sonia Netzer Vincent Pitard Sophie Daburon Christelle Harly Thomas Bachelet Jean-François Moreau Jean-Luc Taupin Julie Dechanet Google Scholar Romain Marlin Sonia Netzer Vincent Pitard Sophie Daburon Christelle Harly Thomas Bachelet Jean-François Moreau Jean-Luc Taupin Julie Dechanet PubMed Romain Marlin Sonia Netzer Vincent Pitard Sophie Daburon Christelle Harly Thomas Bachelet Jean-François Moreau Jean-Luc Taupin Julie Dechanet Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Dual TCR Expression Biases Lung Inflammation in DO11.10 Transgenic Mice and Promotes Neutrophilia via Microbiota-Induced Th17 Differentiation

A commonly used mouse model of asthma is based on i.p. sensitization to OVA together with aluminum hydroxide (alum). In wild-type BALB/c mice, subsequent aerosol challenge using this protein generates an eosinophilic inflammation associated with Th2 cytokine expression. By constrast, in DO11.10 mice, which are transgenic for an OVA-specific TCR, the same treatment fails to induce eosinophilia, but instead promotes lung neutrophilia. In this study, we show that this neutrophilic infiltration results from increased IL-17A and IL-17F production, whereas the eosinophilic response could be restored upon blockade of IFN-γ, independently of the Th17 response. In addition, we identified a CD4(+) cell population specifically present in DO11.10 mice that mediates the same inflammatory response upon transfer into RAG2(-/-) mice. This population contained a significant proportion of cells expressing an additional endogenous TCR α-chain and was not present in RAG2(-/-) DO11.10 mice, suggesting dual antigenic specificities. This particular cell population expressed markers of memory cells, secreted high levels of IL-17A, and other cytokines after short-term restimulation in vitro, and triggered a neutrophilic response in vivo upon OVA aerosol challenge. The relative numbers of these dual TCR lymphocytes increased with the age of the animals, and IL-17 production was abolished if mice were treated with large-spectrum antibiotics, suggesting that their differentiation depends on foreign Ags provided by gut microflora. Taken together, our data indicate that dual TCR expression biases the OVA-specific response in DO11.10 mice by inhibiting eosinophilic responses via IFN-γ and promoting a neutrophilic inflammation via microbiota-induced Th17 differentiation.

Primary mediastinal (thymic) large B cell lymphoma with aberrant expression of CD3: a case report with review of the literature

We report the first case of primary mediastinal large B cell lymphoma (PMBL) with aberrant expression of CD3. PMBL is a subtype of diffuse large B cell lymphoma (DLBCL) and usually presents with bulky mediastinal lesions. Lineage ambiguity/infidelity is uncommon in DLBCL but has been described in sporadic case reports/series. A literature search identifies 13 additional cases of DLBCL expressing CD3, with the majority displaying cytoplasmic expression. Of the 14 total cases, 6 are pyothorax-associated lymphoma, 4 are conventional DLBCL, 2 are plasmablastic lymphoma, one is primary effusion lymphoma and one is PMBL. Two cases show genotypic ambiguity/infidelity with dual clonal IG and TCR gene rearrangements in addition to ambiguous immunophenotypes. Of the 13 cases tested for EBV status, 11 are positive, suggesting an important role of EBV in promoting lineage ambiguity/infidelity. A low threshold for testing EBV status is advocated in DLBCL with phenotypic ambiguity along with panels of immunohistochemical and molecular studies.

Donor CD4+ T Cells That Possess Both Allo- and Autoreactivity in Transplants Mediate Chronic Graft Versus Host Disease.

Abstract 3555Poster Board III-492Chronic graft versus host disease (GVHD) is considered an autoimmune-like disease mediated by donor CD4+ T cells, but the role and origin of the autoreactive T cells remain controversial. Here, we report that, in a chronic GVHD model of MHC-matched DBA/2 (H-2d) donor to BALB/c (H-2d) host, donor spleen cells induced autoimmune-like chronic GVHD in thymectomized allogeneic BALB/c but not in syngeneic DBA/2 recipients. The spleen cells from the former but not the latter recipients induced autoimmune-like disease in the secondary DBA/2 recipients, indicating that autoreactive donor CD4+ T cells from transplants are expanded and contribute to chronic GVHD pathogenesis. In addition, we found that both auto- and alloreactive donor CD4+ T cells generated from primary chronic GVHD recipients via serial in vivo and in vitro expansion proliferated to donor and host DC stimulation and both induced autoimmune-like disease in syngeneic and allogeneic recipients. Furthermore, the clonal expansion and TCR spreading of the autoreactive T cells in chronic GVHD recipients were following the alloreactive T cells, as revealed by TCR-spectrum typing and skewing of TCR-CDR3 length; No dual TCR was expressed by the donor-type T cells with both allo- and autoreactivity; and the autoreactive hybridoma T clones proliferated to stimulation by both syngeneic and allogeneic DCs. Taken together, these results demonstrate that donor CD4+ T cells that possess both allo- and autoreactivity in transplants are expanded in recipients and contribute to chronic GVHD pathogenesis, and the allo- and autoreactivity of the donor T cells can be mediated by a single TCR. Disclosures:No relevant conflicts of interest to declare.

Cutting Edge: Highly Alloreactive Dual TCR T Cells Play a Dominant Role in Graft-versus-Host Disease

Alloreactivity is the response of T cells to MHC molecules not encountered during thymic development. A small population (1-8%) of peripheral T cells in mice and humans express two TCRs due to incomplete allelic exclusion of TCRalpha, and we hypothesized they are highly alloreactive. FACS analysis of mouse T cell MLR revealed increased dual TCR T cells among alloreactive cells. Quantitative assessment of the alloreactive repertoire demonstrated a nearly 50% reduction in alloreactive T cell frequency among T cells incapable of expressing a secondary TCR. We directly demonstrated expansion of the alloreactive T cell repertoire at the single cell level by identifying a dual TCR T cell with distinct alloreactivities for each TCR. The importance of dual TCR T cells is clearly demonstrated in a parent-into-F(1) model of graft-vs-host disease, where dual TCR T cells comprised up to 60% of peripheral activated T cells, demonstrating a disproportionate contribution to disease.

Rare dual TCR T cells contribute substantially to alloreactivity (141.2)

Abstract T cell recognition of allogeneic MHC has long been recognized, though the nature of the alloreactive TCR repertoire is unknown. We hypothesize that the 1-3% of T cells expressing dual TCR due to incomplete allelic exclusion of TCRα are highly alloreactive due to non-thymically selected secondary TCR. Using the 4 available antibodies against TCRVα we observe an increased frequency of dual TCR T cells among alloreactive T cells in vitro. We quantitatively assessed the contribution of secondary TCR to the alloreactive repertoire by ELISPOT analysis; TCRA-C mice, incapable of expressing secondary TCRα, have a 50% decrease in alloreactive precursor frequency indicating secondary TCR contribute disproportionately to the alloreactive repertoire. We directly demonstrate expansion of the alloreactive repertoire by identification of a secondary non-thymically selected TCR with specific alloreactivity distinct from the primary TCR. The importance of dual TCR T cells is underscored by the dramatic increase of dual TCR among T cells mediating parent to irradiated F1 graft-vs.-host disease (GvHD); dual TCR comprised as much as 40% of peripheral T cells in mice with GvHD, compared to 3% observed in control mice. Together these studies convincingly demonstrate the importance of dual TCR T cells in alloreactivity. NIH AI061173 (PMA) and W.M. Keck Foundation Fellowship in Molecular Medicine (GPM).

Mechanism of MDSC induced T cell tolerance in cancer (88.26)

Abstract Tumor antigen-specific T-cell tolerance is a critical element of tumor escape. It is known that MDSC can induce antigen-specific CD8+ T cell tolerance in an experimental system with adoptive transfer of transgenic T cells. In this study we addressed the critical question whether a similar phenomenon can be observed in a "natural" non-transgenic experimental model. Balb/C mice were subcutaneously inoculated with two different tumor cell lines, a derivative A2L2 cell line, which expresses high levels of HER2/neu and the parental cell line 66.3. HER2/neu specific CD8+T cells were generated by immunization of mice with peptide. MDSC were able to tolerize antigen-specific T cells in vivo if they were collected from A2L2 but not from 66.3 tumor-bearing mice indicating the tumor-specific nature of T cell tolerance induced by MDSC. Next we investigated the type of signaling caused by MDSC in CD8+T cells that could lead to tolerance. MDSC dramatically reduced the ability of CD8+ T cells to respond to the specific peptide. However, study of various signaling molecules using different experimental systems and tests found that MDSC did not induce negative signaling in T cells. This conclusion was confirmed in adoptive transfer experiments with CD8+T cells expressing dual TCR: LCMV glycoprotein peptide gp33-41 and OVA-derived H-2Kb-restricted peptide ova257-264. We found that MDSC loaded with peptide specific for one TCR induced tolerance only against that specific epitope but CD8+ T cells retained the ability to respond to the peptide specific for the other TCR expressed on the same cell.

Rapid and Selective Expansion of Nonclonotypic T Cells in Regulatory T Cell-Deficient, Foreign Antigen-Specific TCR-Transgenic Scurfy Mice: Antigen-Dependent Expansion and TCR Analysis

Foreign Ag-specific TCR-transgenic (Tg) mice contain a small fraction of T cells bearing the endogenous Vbeta and Valpha chains as well as a population expressing an intermediate level of Tg TCR. Importantly, these minor nonclonotypic populations contain > or = 99% of the CD4(+)Foxp3(+) regulatory T cells (Treg) and, despite low overall Treg expression, peripheral tolerance is maintained. In the OT-II TCR (OVA-specific, Vbeta5(high)Valpha2(high)) Tg scurfy (Sf) mice (OT-II Sf) that lack Treg, nonclonotypic T cells markedly expanded in the periphery but not in the thymus. Expanded T cells expressed memory/effector phenotype and were enriched in blood and inflamed lungs. In contrast, Vbeta5(high)Valpha2(high) clonotypic T cells were not expanded, displayed the naive phenotype, and found mainly in the lymph nodes. Importantly, Vbeta5(neg) T cells were able to transfer multiorgan inflammation in Rag1(-/-) recipients. T cells bearing dual TCR (dual Vbeta or dual Valpha) were demonstrated frequently in the Vbeta5(int) and Valpha2(int) populations. Our study demonstrated that in the absence of Treg, the lack of peripheral expansion of clonotypic T cells is due to the absence of its high-affinity Ag OVA. Thus, the rapid expansion of nonclonotypic T cells in OT-II Sf mice must require Ag (self and foreign) with sufficient affinity. Our study has implications with respect to the roles of Ag and dual TCR in the selection and regulation of Treg and Treg-controlled Ag-dependent T cell expansion in TCR Tg and TCR Tg Sf mice, respectively.