T-cell Receptor Transgenic Mice Research Articles

Psychological stress to ovalbumin peptide-specific T-cell receptor transgenic mice impairs the suppressive ability of type 1 regulatory T cell.

Numerous diseases of the immune system can be traced back to the malfunctioning of the regulatory T cells. The aetiology is unclear. Psychological stress can cause disruption to the immune regulation. The synergistic effects of psychological stress and immune response on immune regulation have yet to be fully understood. The intention of this study is to analyse the interaction between psychological stress and immune responses and how it affects the functional status of type 1 regulatory T (Tr1) cells. In this study, ovalbumin peptide T-cell receptor transgenic mice were utilised. Mice were subjected to restraint stress to induce psychological stress. An airway allergy murine model was established, in which a mouse strain with RING finger protein 20 (Rnf20)-deficient CD4+ T cells were used. The results showed that concomitant exposure to restraint stress and immune response could exacerbate endoplasmic reticulum stress in Tr1 cells. Corticosterone was responsible for the elevated expression of X-box protein-1 (XBP1) in mouse Tr1 cells after exposure to both restraint stress and immune response. XBP1 mediated the effects of corticosterone on inducing Rnf20 in Tr1 cells. The reduction of the interleukin-10 expression in Tr1 cells was facilitated by Rnf20. Inhibition of Rnf20 alleviated experimental airway allergy by restoring the immune regulatory ability of Tr1 cells. In conclusion, the functions of Tr1 cells are negatively impacted by simultaneous exposure to psychological stress and immune response. Tr1 cells' immune suppressive functions can be restored by inhibiting Rnf20, which has the translational potential for the treatment of diseases of the immune system.

Investigation into Cardiac Myhc-α 334-352-Specific TCR Transgenic Mice Reveals a Role for Cytotoxic CD4 T Cells in the Development of Cardiac Autoimmunity.

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4+ and CD8+ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334-352 and found that Myhc-α-specific TCRs were expressed in both CD4+ and CD8+ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At the fourth generation of backcrossing, we observed that Tg T cells from naïve mice responded to Myhc-α 334-352, as evaluated by proliferation assay and carboxyfluorescein succinimidyl ester staining. The T cell responses included significant production of mainly pro-inflammatory cytokines, namely interferon (IFN)-γ, interleukin-17, and granulocyte macrophage-colony stimulating factor. While the naïve Tg mice had isolated myocardial lesions, immunization with Myhc-α 334-352 led to mild myocarditis, suggesting that further backcrossing to increase the percentage of A/J genome close to 99.99% might show a more severe disease phenotype. Further investigations led us to note that CD4+ T cells displayed the phenotype of cytotoxic T cells (CTLs) akin to those of conventional CD8+ CTLs, as determined by the expression of CD107a, IFN-γ, granzyme B natural killer cell receptor (NKG)2A, NKG2D, cytotoxic and regulatory T cell molecules, and eomesodermin. Taken together, the transgenic system described in this report may be a helpful tool to distinguish the roles of cytotoxic cardiac antigen-specific CD4+ T cells vs. those of CD8+ T cells in the pathogenesis of myocarditis.

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.

Noninvasive PET Detection of CD69-Positive Immune Cells Before Signs of Clinical Disease in Inflammatory Arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [68Ga]Ga-DOTA-ZCAM241, for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T-cell receptor transgenic B6 mice into T-cell-deficient I-Ag7 major histocompatibility complex class II-expressing recipient mice. The mice were examined longitudinally by [68Ga]Ga-DOTA-ZCAM241 PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the paws was analyzed and expressed as SUVmean Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size-matched Affibody molecule as the control. Results: Clinical symptoms appeared 5-7 d after induction of arthritis. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [68Ga]Ga-DOTA-ZCAM241 correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [68Ga]Ga-DOTA-ZCAM241 Conclusion: The uptake of [68Ga]Ga-DOTA-ZCAM241 in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [68Ga]Ga-DOTA-ZCAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset.

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

TCR-engineered adoptive cell therapy effectively treats intracranial murine glioblastoma

BackgroundAdoptive cellular therapies with chimeric antigen receptor T cells have revolutionized the treatment of some malignancies but have shown limited efficacy in solid tumors such as glioblastoma and face a...

Germline T cell receptor exchange results in physiological T cell development and function

T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin (Msln), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac-targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. Together, our results support that the TRex methodology is an advanced tool to study physiological antigen-specific T cell behavior.

Abstract 5507: Generating optimal-affinity T cell receptors targeting the shared neoantigen KRAS G12V using the humanized TCR transgenic mouse platform HuTCR

Abstract Background T cell receptor (TCR)-engineered T cell therapy (TCR-T) is a promising therapeutic modality to address key limitations of targeting solid tumors. Neoantigens are excellent therapeutic targets for TCR-T cell therapy, because they are highly specific and often homogeneously expressed in the tumor. While most neoantigens are patient-specific, there are rare exceptions of widely shared neoantigens, such as the driver oncogene KRAS containing mutations at position 12. The KRAS mutations G12C, G12D, and G12V are among the most common mutations in solid tumors, including indications with high unmet medical need, such as pancreatic, colorectal, and non-small cell lung cancer. Previous TCR-T approaches targeting KRAS G12V have been primarily focused on HLA-A*02:01, which appeared to be unable to present a mutant KRAS epitope. Here, we aim to generate TCRs of optimal affinity and high specificity to a KRAS G12V epitope in an HLA-agnostic manner using our HuTCR mouse platform. Experimental procedures HuTCR mice represent a fully humanized TCR-MHC system and bear a diverse TCR repertoire, being transgenic for the entire human TCR alpha and beta gene loci and multiple human HLA class I molecules, while lacking murine TCRs and murine MHC class I molecules. The multi-HLA HuTCR mice were immunized with KRAS G12V peptides or the neoantigen-encoding adenovirus, leading to robust immune responses. Results Single-cell sequencing of responding T cells revealed HLA-A*11:01-restricted immune responses, indicating that the epitope presented on HLA-A*11:01 was more immunogenic than epitopes presented on other HLA alleles. HLA-A*11:01 is expressed in approximately 11% of the United States and the European Union population and in around 30% of Asian populations. We were able to generate 30 KRAS G12V-specific TCRs, which we characterized for efficacy and safety. We selected a lead candidate, which demonstrated potent anti-tumor efficacy and no detectable off-target toxicity, to be pursued further into the clinic. Conclusion Using the HuTCR mouse platform, we identified TCRs of optimal affinity and high specificity for the shared oncogenic KRAS G12V mutation. Importantly, the HuTCR platform was a powerful tool enabling us to determine the HLA allele responsible for generating the strongest immune response, and consequently, to define the patient population that may benefit most from targeting KRAS G12V. Citation Format: Alexei Leliavski, Lorenz Knackstedt, Jennifer Oduro, Claudia Selck, Arunraj Dhamodaran, Thomas Blankenstein, Elisa Kieback, Lucia Poncette. Generating optimal-affinity T cell receptors targeting the shared neoantigen KRAS G12V using the humanized TCR transgenic mouse platform HuTCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5507.

Differential location of NKT and MAIT cells within lymphoid tissue

Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αβ T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.

Generation and characterization of HLA-A2 transgenic mice expressing the human TCR 1G4 specific for the HLA-A2 restricted NY-ESO-1157-165 tumor-specific peptide

BackgroundNY-ESO-1 is a tumor-specific, highly immunogenic, human germ cell antigen of the MAGE-1 family that is a promising vaccine and cell therapy candidate in clinical trial development. The mouse genome...

CD49d marks Th1 and Tfh-like antigen-specific CD4+ T cells during Plasmodium chabaudi infection.

Upon activation, specific CD4+ T cells up-regulate the expression of CD11a and CD49d, surrogate markers of pathogen-specific CD4+ T cells. However, using T-cell receptor transgenic mice specific for a Plasmodium antigen, termed PbT-II, we found that activated CD4+ T cells develop not only to CD11ahiCD49dhi cells, but also to CD11ahiCD49dlo cells during acute Plasmodium infection. CD49dhi PbT-II cells, localized in the red pulp of spleens, expressed transcription factor T-bet and produced IFN-γ, indicating that they were type 1 helper T (Th1)-type cells. In contrast, CD49dlo PbT-II cells resided in the white pulp/marginal zones and were a heterogeneous population, with approximately half of them expressing CXCR5 and a third expressing Bcl-6, a master regulator of follicular helper T (Tfh) cells. In adoptive transfer experiments, both CD49dhi and CD49dlo PbT-II cells differentiated into CD49dhi Th1-type cells after stimulation with antigen-pulsed dendritic cells, while CD49dhi and CD49dlo phenotypes were generally maintained in mice infected with Plasmodium chabaudi. These results suggest that CD49d is expressed on Th1-type Plasmodium-specific CD4+ T cells, which are localized in the red pulp of the spleen, and can be used as a marker of antigen-specific Th1 CD4+ T cells, rather than that of all pathogen-specific CD4+ T cells.

CD5 levels reveal distinct basal T-cell receptor signals in T cells from non-obese diabetic mice.

Type 1 diabetes in non-obese diabetic (NOD) mice occurs when autoreactive T cells eliminate insulin producing pancreatic β cells. While extensively studied in T-cell receptor (TCR) transgenic mice, the contribution of alterations in thymic selection to the polyclonal T-cell pool in NOD mice is not yet resolved. The magnitude of signals downstream of TCR engagement with self-peptide directs the development of a functional T-cell pool, in part by ensuring tolerance to self. TCR interactions with self-peptide are also necessary for T-cell homeostasis in the peripheral lymphoid organs. To identify differences in TCR signal strength that accompany thymic selection and peripheral T-cell maintenance, we compared CD5 levels, a marker of basal TCR signal strength, on immature and mature T cells from autoimmune diabetes-prone NOD and -resistant B6 mice. The data suggest that there is no preferential selection of NOD thymocytes that perceive stronger TCR signals from self-peptide engagement. Instead, NOD mice have an MHC-dependent increase in CD4+ thymocytes and mature T cells that express lower levels of CD5. In contrast, T cell-intrinsic mechanisms lead to higher levels of CD5 on peripheral CD8+ T cells from NOD relative to B6 mice, suggesting that peripheral CD8+ T cells with higher basal TCR signals may have survival advantages in NOD mice. These differences in the T-cell pool in NOD mice may contribute to the development or progression of autoimmune diabetes.

Isolevuglandin-Modified Cardiac Proteins Drive CD4+ T-Cell Activation in the Heart and Promote Cardiac Dysfunction.

Despite the well-established association between T-cell-mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)-modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation-deficient MhcII-/- mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII-/- mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction-induced cardiac dysfunction despite the presence of left ventricle-infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species-dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. Our study demonstrates an important role of reactive oxygen species-induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

TCR Transgenic Mice: A Valuable Tool for Studying Viral Immunopathogenesis Mechanisms.

Viral infectious diseases are a significant burden on public health and the global economy, and new viral threats emerge continuously. Since CD4+ and CD8+ T cell responses are essential to eliminating viruses, it is important to understand the underlying mechanisms of anti-viral T cell-mediated immunopathogenesis during viral infections. Remarkable progress in transgenic (Tg) techniques has enabled scientists to more readily understand the mechanisms of viral pathogenesis. T cell receptor (TCR) Tg mice are extremely useful in studying T cell-mediated immune responses because the majority of T cells in these mice express specific TCRs for partner antigens. In this review, we discuss the important studies utilizing TCR Tg mice to unveil underlying mechanisms of T cell-mediated immunopathogenesis during viral infections.

IL-21 and IL-5 coordinately induce surface IgA+ cells

Intestinal IgA is induced by microbes and food antigens. Peyer’s patches (PPs) are known as one of the inductive sites for intestinal IgA production. However, the precise mechanism of IgA induction is as yet unknown. IgA secretion was induced from IgD+ B cells in vitro by stimulus with lipopolysaccharide in the presence of only retinoic acid (RA) and low doses of TGF-β1. Surface IgA+ cells were effectively induced from IgD+ B cells in vitro by the mixture of RA and the cytokines TGF-β1, APRIL, IL-5 and IL-21. rIL-21 upregulated surface IgA+ but impaired the proliferation of stimulated B cells in the presence of rTGF-β1, RA and rAPRIL, in vitro. The addition of rIL-5 restored the impaired proliferation by rIL-21, resulting in the expansion of IgA+ cells. rIL-21 induced the expression of Aicda and Prdm1, and impaired Rel in IgD+ B cells. Blockade of IL-21R signaling by a neutralizing mAb in vivo led to lower frequencies of IgA+ and IgG2b+ cells and lower germinal center B cells in PPs in a homeostatic condition. Although amounts of small intestinal IgA and titers of anti-dsDNA, the major target of intestinal IgA, in these mice were not altered, anti-OVA IgA titers induced by OVA drinking in OVA-specific T-cell receptor (TCR) transgenic mice were decreased. PP-deficient TCR transgenic mice showed diminished anti-OVA IgA induction. Blockade of IL-5R signaling in vivo led to similar results with relatively weaker effects than that of IL-21R mAb administration. These results suggest that IL-21 and IL-5 play cooperative roles in surface expression of IgA in PPs.

Cardiac myosin-specific TCR transgenic mouse models for studying dilated cardiomyopathy

Abstract We previously reported that the cardiac myosin heavy chain-alpha (Myhc) 334–352 encompasses epitopes for both CD4 and CD8 T cells that can induce autoimmune myocarditis/dilated cardiomyopathy (DCM) in A/J mice expressing the IAk and IEk molecules. More recently, we made an unexpected observation that, Myhc 334–352 although by being a poor binder of IAb molecule in C57Bl/6 mice, could induce strong T cell responses, but myocarditogenicity was severely dented. In our efforts to delineate the roles of Myhc-specific, CD4 and CD8 T cells in the DCM pathogenesis, we have now created the T cell receptor (TCR) transgenic (tg) mice on C57Bl/6 background for Myhc 334–352, and demonstrate that the Myhc 334–352-specific TCR was found common to both CD4 and CD8 T cells. While, the naïve tg T cells do not respond, antigen-primed CD4 and CD8 T cells from immunized mice respond to Myhc 334–352 that produce predominantly, interferon-γ. Furthermore, although mild, the immunized tg mice developed heart lesions containing the mononuclear cells, and such a phenotype was more apparent in females than males. As the tg mice become available on both resistant (C57Bl/6) and susceptible (A/J) backgrounds, our TCR tg system can be used as a helpful tool to dissect the complexities of antigen-specific CD4 and CD8 T cells in the causation of DCM in various experimental settings.

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.

Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus

During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9–23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8+ HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases.

NLRX1 inhibits the early stages of CNS inflammation and prevents the onset of spontaneous autoimmunity.

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1−/− mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity.

Mechanism of recombinant Mycobacterium smegmatis in alleviating airway inflammation in mice with non-eosinophilic asthma

Objective To observe the effects of intranasal inoculation with recombinant Mycobacterium smegmatis (rMS) on the airway inflammation of non-eosinophilic asthmatic mouse and further investigate the relative mechanism. Methods DO11.10 T-cell receptor transgenic mice were divided randomly into three groups.Non-eosinophilic asthmatic model was established via OVA challenge, rMS were administrated into mice before challenge.Anti-IL-17A autoantibody in sera, IL-6 and IL-17A in BALF were measured by ELISA, the proportion of neutrophil in BALF was measured by FCM, MPO activity in lung tissue was detected by colorimetry, and the mRNA expression of CXCL2 and CXCL5 was measured by real-time PCR. Results Compared to control group, the number of neutrophils, IL-6 and IL-17A levels in BALF from asthmatic mice was significantly increased, meanwhile MPO activity and the expression of CXCL2, CXCL5 in lung tissue were both significantly upregulated.The results showed that high titer of autoantibody of IL-17A in sera of mice vaccined with recombinant Mycobacterium smegmatis was detected.Compared to asthma group, neutrophils and IL-6, IL-17A in BALF were significantly decreased, meanwhile MPO activity and CXCL2, CXL5 mRNA expression were significantly down-regulated. Conclusion Recombinant Mycobacterium smegmatis exhibits anti-inflammatory activity in murine neutrophilic asthma model and it may have protective effects on asthma. Key words: Bronchial asthma; IL-17A; Autoantibody; Airway inflammation