T-cell Receptor Transgenic Mouse Model Research Articles

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.

Cellular dynamics of immune evasion during Leishmania major infection

Abstract Concomitant immunity against intracellular parasites is dictated by the immune response and parasite escape mechanisms that prevent clearance. Infection by Leishmania major generates a strong T cell response, yet parasite clearance is incomplete and leaves a small pool of persistently infected cells. T cell behaviours are augmented by the biochemical and spatial conditions of their immediate environments, and a gap in knowledge is the cellular and molecular mechanisms that prevent complete clearance of pathogen-infected cells in the skin. We combined MP-IVM with a novel T-cell receptor transgenic mouse model, where T cells recognize the immunodominant Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK) peptide, to provide insights into the spatiotemporal dynamics of anti-Leishmania T cell responses during active disease and persistent infection. We show that monocyte-derived macrophage:T cell interaction dynamics were transient at steady-state, but prolonged upon antigen recognition. PEPCK-specific T cells produced high levels of IFNg in response to peptide or infection. We also show that in vitro expanded Treg cells can suppress effector T cell functions, and that greater suppression was observed by PEPCK-specific Treg cells. Ongoing studies that address the cellular and molecular mechanisms of Treg-mediated response blunting will be presented. Intravital microscopy studies characterizing PEPCK-specific T cell migration dynamics and tissue localization within skin lesions directly in live mice will also be presented. The goal of our study is to identify barriers to overcome in order to achieve complete parasite clearance and to better describe the dynamic aspects of concomitant immunity generation.

Low-Density Lipoprotein-Reactive T Cells Regulate Plasma Cholesterol Levels and Development of Atherosclerosis in Humanized Hypercholesterolemic Mice

Atherosclerotic cardiovascular disease is a chronic inflammatory process initiated when cholesterol-carrying low-density lipoprotein (LDL) is retained in the arterial wall. CD4+ T cells, some of which recognize peptide components of LDL as antigen, are recruited to the forming lesion, resulting in T-cell activation. Although these T cells are thought to be proatherogenic, LDL immunization reduces disease in experimental animals. These seemingly contradictory findings have hampered the development of immune-based cardiovascular therapy. The present study was designed to clarify how activation of LDL-reactive T cells impacts on metabolism and vascular pathobiology. We have developed a T-cell receptor-transgenic mouse model to characterize the effects of immune reactions against LDL. Through adoptive cell transfers and cross-breeding to hypercholesterolemic mice expressing the antigenic human LDL protein apolipoprotein B-100, we evaluate the effects on atherosclerosis. A subpopulation of LDL-reactive T cells survived clonal selection in the thymus, developed into T follicular helper cells in lymphoid tissues on antigen recognition, and promoted B-cell activation. This led to production of anti-LDL immunoglobulin G antibodies that enhanced LDL clearance through immune complex formation. Furthermore, the cellular immune response to LDL was associated with increased cholesterol excretion in feces and with reduced vascular inflammation. These data show that anti-LDL immunoreactivity evokes 3 atheroprotective mechanisms: antibody-dependent LDL clearance, increased cholesterol excretion, and reduced vascular inflammation.

Cooperation of Th1 and Th17 cells determines transition from autoimmune myocarditis to dilated cardiomyopathy

Myocarditis is a potentially lethal inflammatory heart disease of children and young adults that frequently leads to dilated cardiomyopathy (DCM). Since diagnostic procedures and efficient therapies are lacking, it is important to characterize the critical immune effector pathways underlying the initial cardiac inflammation and the transition from myocarditis to DCM. We describe here a T-cell receptor (TCR) transgenic mouse model with spontaneously developing autoimmune myocarditis that progresses to lethal DCM. Cardiac magnetic resonance imaging revealed early inflammation-associated changes in the ventricle wall including transient thickening of the left ventricle wall. Furthermore, we found that IFN-γ was a major effector cytokine driving the initial inflammatory process and that the cooperation of IFN-γ and IL-17A was essential for the development of the progressive disease. This novel TCR transgenic mouse model permits the identification of the central pathophysiological and immunological processes involved in the transition from autoimmune myocarditis to DCM.

Positive selecting cell type determines the phenotype of MHC class Ib-restricted CD8 + T cells

Several studies have demonstrated an apparent link between positive selection on hematopoietic cells (HCs) and an "innate" T-cell phenotype. Whereas conventional CD8(+) T cells are primarily selected on thymic epithelial cells (TECs) and certain innate T cells are exclusively selected on HCs, MHC class Ib-restricted CD8(+) T cells appear to be selected on both TECs and HCs. However, whether TEC- and HC-selected T cells represent distinct lineages or whether the same T-cell precursors have the capacity to be selected on either cell type is unknown. Using an M3-restricted T-cell receptor transgenic mouse model, we demonstrate that not only are MHC class Ib-restricted CD8(+) T cells capable of being selected on either cell type but that selecting cell type directly affects the phenotype of the resulting CD8(+) T cells. M3-restricted CD8(+) T cells selected on HCs acquire a more activated phenotype and possess more potent effector functions than those selected on TECs. Additionally, these two developmental pathways are active in the generation of the natural pool of M3-restricted CD8(+) T cells. Our results suggest that these two distinct populations may allow MHC class Ib-restricted CD8(+) T cells to occupy different immunological niches playing unique roles in immune responses to infection.

Development of a transgenic mouse with high affinity TCR reactive to human tyrosinase epitope (41.39)

Abstract Most tumor-associated antigen epitopes recognized on human melanomas by autologous T lymphocytes are derived from nonmutated self-proteins. Given the belief that T cells bearing high affinity T cell receptor (TCR) would be more effective at targeting self antigens expressed by tumors, understanding tolerance with reference to T cells expressing high affinity TCRs against self-tumor antigens is important for optimizing the anti-tumor T cell response. Thus, using a high affinity TCR isolated from a class-I restricted CD4+ T cells from tumor infiltrating lymphocytes of a patient with metastatic melanoma and reactive to human tyrosinase-derived peptide YMDGTMSQV we developed a novel TCR transgenic mice. Our results show that HLA-A2 restricted TCR passed thymic selection in C57BL/6J (H2-Kb) wild type mice and could be tracked in the periphery. Around 25% of the peripheral CD4+ T cells and more than 75% of the peripheral CD8+ T cells from these TCR transgenic mice expressed functional human tyrosinase TCR. Since tyrosinase is a self-antigen and C57BL/6J mice lack HLA-A2/tyrosinase complexes on cells in their thymus -we developed HLA-A2+TCR+ double transgenic mice by breeding our TCR transgenic mouse and HLA-A2 transgenic mouse strains. Surprisingly, more than 90% of TCR transgenic T cells in the HLA-A2+TCR+ double transgenic mice lacked expression of CD4 or CD8 co-receptors and had CD3+CD4-CD8- phenotype. These double transgenic strains also developed spontaneous autoimmune vitiligo. Thus, our novel TCR transgenic mouse model would allow us to undertake various developmental and translational studies.

Reduced Fas ligand-expressing splenic CD5+ B lymphocytes in severe collagen-induced arthritis

IntroductionThe objective was to study immune regulation in a mouse model of rheumatoid arthritis that exhibits considerable heterogeneity of disease activity.MethodsT-cell receptor transgenic mice, in which nearly all CD4+ T cells recognize a single peptide of type II collagen, were immunized with collagen and observed for development of arthritis for 4 weeks. At 28 days post-immunization, splenocytes were analyzed by flow cytometry and in vitro assays for markers of immune activation and regulation.ResultsDisease severities ranging from 0 to 12 (on a 12-point scale) were observed. Among splenic lymphocyte populations, only the CD5+ B-cell subset displayed a decrease in relative numbers as arthritis severity increased. Splenic CD5+ B cells expressed higher levels of Fas ligand (FasL) than did CD4+ T cells or CD5- B cells in all mice, and antigen-dependent T-cell death correlated with higher levels of CD5+ B cells in cocultures. Ratios of interleukin (IL)-17 to interferon-gamma production were higher in antigen-driven cultures of splenocytes from severely arthritic mice compared to mildly or nonarthritic mice. A correlation was established between the reduced production of IL-17 in antigen-driven T-cell/B-cell cocultures and FasL, but not IL-10. Confirmation of the direct killing effect of B cells on T cells was demonstrated using an antigen-specific T hybridoma cell line.ConclusionsReduced numbers of splenic FasL+ CD5+ B cells correlated with increasing arthritis severity and decreased T-cell death in a T-cell receptor transgenic mouse model of collagen-induced arthritis. These 'killer' B cells may provide a novel mechanism for inducing T-cell death as a treatment for arthritis.

Induction of CD8 T cell-mediated EAE by viral infections in a CD8 TCR transgenic mouse model (130.31)

Abstract Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Besides CD4 T cells, CD8 T cells have recently been shown to mediate the pathogenesis of EAE. The cause of MS is not clear, but infections have been suggested. In this study, we investigated the roles of viral infections in the development of EAE on a CD8 T cell receptor (TCR) transgenic mouse model. Infection with wildtype vaccinia virus broke tolerance and triggered atypical EAE in a TCR transgenic mouse specific for myelin basic protein 79–87 (MBP79-87). CD8 T cells up-regulated activation markers and trafficked to the CNS in virus-infected mice. Interestingly, CD8 T cells from TCR-transgenic MBP deficient mice also displayed activation markers, and had cytolytic effector function similar to that from TCR transgenic MBP sufficient mice. T cell activation was not due to MBP TCR cross-reactivity against vaccinia virus. Furthermore, preliminary data showed that disease induction by vaccinia virus required endogenous TCRs. Ongoing experiments are extending these observations into another viral infection. In conclusion, the results suggest that viral infection may induce EAE by activation through dual TCR.

Arthritis induced by systemic autoimmunity against glucose-6-phosphate isomerase in normal mice

The antigens that trigger the pathogenic immune response in rheumatoid arthritis remain unknown. Until recently it was assumed that joint-specific antigens were the targets of arthritogenic T lymphocytes and B lymphocytes in rheumatoid arthritis. Consequently, murine models of arthritis are induced by immunization with either joint-specific antigens such as type II collagen or microbial products such as streptococcal cell wall. In the K/BxN T-cell receptor transgenic mouse model, arthritis is caused by a systemic autoimmune response to the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI). More recently it was shown that G6PI immunization induces severe symmetrical peripheral polyarthritis in genetically unaltered DBA/I or SJL mice [1,2]. T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4 cells. In contrast, antibodies and FcγR effector cells are necessary but not sufficient for G6PI-induced arthritis in genetically unaltered mice [1]. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.

The role and clinical implications of G6PI in experimental models of rheumatoid arthritis

The antigens that trigger the pathogenic immune response in rheumatoid arthritis (RA) remain unknown. Until recently it was assumed that either viral or microbial antigens, or joint-specific antigens were the target of arthritogenic T and B lymphocytes in RA. Consequently, murine models of arthritis are induced by immunization with either joint-specific antigens such as type II collagen or microbial products such as streptococcal cell wall. In the K/B×N T-cell receptor transgenic mouse model arthritis is caused by a systemic autoimmune response to the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI). The autoreactive transgenic T cells recognize G6PI and provide help for the production of arthritogenic IgG antibodies against G6PI. More recently it was shown that G6PI immunization induces severe symmetrical peripheral polyarthritis in genetically unaltered DBA/I mice. In that model CD4+ T cells are necessary not only for the induction but also for the effector phase of arthritis. Here we review the pathomechanisms that lead from systemic autoreactivity to arthritis in these models, consider the relevance of anti-G6PI immune reactivity for RA, and discuss the insights into the pathogenesis of RA and possibly other autoimmune conditions that can be gained from these models.

Therapeutic effect of idiotype-specific CD4+ T cells against B-cell lymphoma in the absence of anti-idiotypic antibodies

AbstractImmunoglobulin (Ig) variable (V) region idiotypes (Id's) are highly tumor-specific antigens produced by B-lymphoma cells and are promising targets for immunotherapy. Id vaccination has proven effective in experimental mouse models and may possibly prevent recurrence of B lymphomas in humans. It has previously been shown that anti-Id antibodies protect against B-cell lymphoma in the absence of T cells. We here demonstrate in a T-cell–receptor transgenic mouse model that the contrary is also true: Id-specific CD4+ T cells can protect against Id+ B-lymphoma cells in the absence of B cells, antibodies, and CD8+ T cells. Moreover, Id-specific CD4+ T cells have a curative potential since they could be transferred as late as 17 days after subcutaneous tumor cell injection of severe combined immunodeficiency (SCID) mice and still abrogate tumor development in about 50% of mice. Such mice undergo an acute inflammatory swelling with infiltration of neutrophils at the site of tumor injection, which subsides over weeks, with some mice cured and delayed emergence of lymphomas in other mice. Adoptively transferred CD4+ T cells accumulated in the tumor and were activated (CD69+). In vitro experiments demonstrated that memory, but not naive, Id-specific CD4+ T cells kill Id+ B-lymphoma cells. The results show that Id-specific CD4+ T cells, in the absence of antibodies home to subcutaneous Id+ B lymphoma, become activated, induce inflammation, and prevent tumor development.

Mucosal immune responses to fed soluble protein antigen in t cell receptor (TCR) transgenic mice

Background: Oral tolerance defines a state of systemic hyporesponsiveness following oral antigen administration, however, the mechanisms underlying the induction of suppressive T cells are not well understood. We investigated kinetics and dose-dependent features of mucosal and peripheral immune responses following oral antigen application in a TCR transgenic mouse model. Methods: Ovalbumin (OVA)-TCR transgenic mice which have a <80% expression of the clonotype receptor on their peripheral T cells were fed OVA in different doses (5-250 rag) and various frequencies (1-7 times, or continuous feeding in the drinking water). Subsequently, lymphoid tissues were isolated and proliferative and cytokine resonses were determined. Results: Low and medium dose (10, 20, 100 mg) OVA feeding resulted in priming of immune responses, i.e., antigen-specific proliferation as well as interieukin (IL)-2, IL-4, IL-10, and interferon-? (IFN-7) secretion upon in vitro restimulation, both in the mucosal immune system (Peyer's patches [PP]) and in the periphery (spleen and peripheral lymph nodes) as compared to unfed control mice. Proliferative responses in the mesenteric lymph nodes (MLN) were supressed with every feeding regimen. One time high dose feeding (250 mg of OVA) suppressed antigen-specific immune responses in the periphery but not in the PP. Only the highest OVA feeding doses (7 x 250 mg OVA) and continuous feeding (5 mg daily over a 10 week period) diminished the predominant IFN-7 response in the PP and resulted in an occurence of transforming growth factor 13 (TGFI3) production in the PP and the spleen. Conclusions: Our data characterize the differential cytokine production in response to fed soluble protein antigen in the periphery versus the gastrointestinal mucosa. In addition, the occurence of a TGFfl response is based on the counterregulation of Thl cytokines and TGF[I and seems to originate from the PP. These results provide thus further insight into mechanisms involved in regulating suppressive immune functions following antigen feeding. This work was supported by a research grant from the Deutsche Forschungsgemeinschaft to T. M. (DFG Ma 1711/2-1 to 2-3).

Mechanisms of recovery from experimental autoimmune encephalomyelitis: T cell deletion and immune deviation in myelin basic protein T cell receptor transgenic mice

Experimental autoimmune encephalomyelitis (EAE) is a Th1-type cell-mediated autoimmune disease directed against central nervous system (CNS) myelin antigens such as myelin basic protein (MBP). EAE is usually characterized by spontaneous remission of clinical disease and immune pathology despite the persistence of self myelin antigens in the central nervous system. Following induction of an acute episode of EAE, spontaneous remission also occurs in MBP T cell receptor (TCR) transgenic mice even though most T cells express a TCR specific for MBP. To investigate the mechanisms of recovery associated with EAE, we examined the behavior of MBP-specific T cells in the MBP TCR transgenic mouse model during disease progression and recovery. We found that recovery from EAE was associated with three major immunologic changes: (1) deletion of encephalitogenic T cells in the brain; (2) deviation of MBP-specific transgenic (Tg +) T cells both in the periphery and in the central nervous system from IFN- γ secreting Th1 type cells to cells that secrete IL-4, IL-10, and TGF- β; and (3) deletion of Tg + T cells in the thymus through apoptosis. Thus spontaneous recovery from a classic Th1 type organ specific autoimmune disease is associated with two mechanisms of immune tolerance, deletion of autoreactive cells and immune deviation of autoreactive cells to a non-pathogenic phenotype.

From naive to effector ‐ alterations with aging

We have used a T-cell receptor transgenic mouse model to study the role of antigen in the changes that occur as T cells age. We find that the characteristic shift in the CD4 population to a predominance of memory phenotype T cells which accompanies aging in non-transgenic mice does not occur, suggesting that this shift is a result of antigenic stimulation. Thus at least one component of aging must be antigen dependent. When responses of naive CD4 T cells from aged and young mice are directly compared in vitro, the former are relatively deficient in their ability to produce IL-2 and IL-3, they express altered levels of P-glycoprotein and they proliferate less well in the absence of exogenous cytokines. When the ability of both naive populations to generate effectors is compared, the number of effectors generated from aged naive cells is much reduced and the effectors generated express lower levels of IL-2R alpha and produce reduced levels of cytokines. Importantly, addition of IL-2 restores proliferation of aged naive T cells, restores efficient effector generation and results in effectors seemingly indistinguishable from those derived from young CD4 cells. Similar phenotypic and functional changes seen with aging are also found in T-cell populations from IL-2 and IL-2R alpha knockout mice. Thus the loss of optimal IL-2 production may participate in the aging process and may represent the main antigen-independent defect in the CD4 T-cell population.

T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.