Cell Receptor Transgenic Mice Research Articles

An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes.

The antigen-presenting cell function of insulin-reactive B cells promotes type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by stimulating pathogenic T cells leading to destruction of insulin-producing β-cells of pancreatic islets. To target insulin-reactive B cells, AKS-107, a human IgG1 Fc molecule fused with human insulin A and B chains, was engineered to retain conformational insulin epitopes that bound mouse and human B cell receptors but prevented binding to the insulin metabolic receptor. AKS-107 Fc-mediated deletion of insulin-reactive B cells was demonstrated via ex vivo and in vivo experiments with insulin-reactive B cell receptor transgenic mouse strains, VH125Tg/NOD and Tg125(H+L)/NOD. As an additional immune tolerance feature, the Y16A mutation of the insulin B(9-23) dominant T cell epitope was engineered into AKS-107 to suppress activation of insulin-specific T cells. In mice and non-human primates, AKS-107 was well-tolerated, non-immunogenic, did not cause hypoglycemia even at high doses, and showed an expectedly protracted pharmacokinetic profile. AKS-107 reproducibly prevented spontaneous diabetes from developing in NOD and VH125Tg/NOD mice that persisted for months after cessation of treatment, demonstrating durable immune tolerance. These preclinical outcomes position AKS-107 for clinical development in T1D prevention settings.

B Cell Receptor Transgenic Mice as Tools to Study Memory B Cells.

B cell receptor (BCR) transgenic mice allow thecontrol ofthe initial target (antigen) specificity of naïve B cells and to investigate their properties following activation. Here, I describe how BCR transgenic B cells can be used in combination with adoptive cell transfer and immunization models to study memory B cell formation and reactivation.

Transgenic Mice Expressing Functional TCRs Specific to Cardiac Myhc-α 334-352 on Both CD4 and CD8 T Cells Are Resistant to the Development of Myocarditis on C57BL/6 Genetic Background.

Myocarditis is a predominant cause of congestive heart failure and sudden death in children and young adolescents that can lead to dilated cardiomyopathy. Lymphocytic myocarditis mediated by T cells can result from the recognition of cardiac antigens that may involve CD4 or CD8 T cells or both. In this report, we describe the generation of T cell receptor (TCR) transgenic mice on a C57BL/6 genetic background specific to cardiac myosin heavy chain (Myhc)-α 334-352 and make the following observations: First, we verified that Myhc-α 334-352 was immunogenic in wild-type C57BL/6 mice and induced antigen-specific CD4 T cell responses despite being a poor binder of IAb; however, the immunized animals developed only mild myocarditis. Second, TCRs specific to Myhc-α 334-352 in transgenic mice were expressed in both CD4 and CD8 T cells, suggesting that the expression of epitope-specific TCR is common to both cell types. Third, although T cells from naïve transgenic mice did not respond to Myhc-α 334-352, both CD4 and CD8 T cells from animals immunized with Myhc-α 334-352 responded to the peptide, indicating that antigen priming is necessary to break tolerance. Fourth, although the transgenic T cells could produce significant amounts of interferon-γ and interleukin-17, the immunized animals developed only mild disease, indicating that other soluble factors might be necessary for developing severe myocarditis. Alternatively, the C57BL/6 genetic background might be a major contributing factor for resistance to the development of myocarditis. Taken together, our model permits the determination of the roles of both CD4 and CD8 T cells to understand the disease-resistance mechanisms of myocarditis in a single transgenic system antigen-specifically.

Human gut commensals support development of ocular autoimmunity in retina-specific T cell receptor transgenic mice

Abstract Autoimmune uveitis is a major cause of blindness, in which intraocular inflammation is driven by T cells that target the neuroretina. In a mouse model of spontaneous autoimmune uveitis (R161H), retina-specific T cells are primed in the gut through their transgenic TCR, and trigger disease. To support the relevance to human disease, we examined the development of uveitis and its association with gut microbiota in gnotobiotic R161H mice colonized with gut flora from healthy human donors. We then performed fecal metagenomic sequencing, gut immunophenotyping by flow cytometry and disease monitoring. Our results indicate that human gut commensals support development of autoimmunity in the spontaneous uveitis model. Human flora mice compared to SPF mice harboring normal mouse flora displayed altered intestinal effector and regulatory CD4 T cell profiles, and had decreased fecal metabolites including microbe-derived short chain fatty acids. Microbiome analyses showed that human flora mice retained a distinct but simplified gut microbial community compared to their original donor sample. Mice with high disease scores appeared to harbor more diverse gut flora than those with low scores. Verrucomicrobia, Actinobacteria, and Fusobacteria were enriched in mice with high disease scores, whereas Firmicutes appeared enriched in mice with low disease scores. In-vivo association studies are underway to examine the ability of candidate microbes to modulate autoimmune uveitis. These findings suggest that some taxa of human commensals may have enhancing or suppressive effects on the development of ocular autoimmunity. These candidate microbes can be explored as targets or as probiotics for therapeutic use.

RSV-specific T cell epitope presentation in the respiratory tract differs between adults and neonates as revealed by a novel T cell proliferation assay

Abstract Respiratory syncytial virus (RSV) invades the respiratory epithelium and provokes severe symptoms in young children leading to infant mortality. CD8 tissue resident memory (TRM) cells are associated with protection in adults against RSV, but are poorly established in neonatal mice consequent to primary infection. We hypothesized that antigen presentation contributes to age-dependent TRM establishment, thus we sought to define antigen kinetics in RSV-infected adult and neonatal mice. CB6F1/J adult and neonatal mice were infected with RSV and lung tissue were isolated over the course of acute and convalescent infection. Single cell lung suspension was co-cultured in vitrowith RSV K dM2 82–90(M2) or D bM 187–195(M) epitope-specific T cells isolated from CD8 T cell receptor (TCR) transgenic mice. T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) to quantify proliferative response 48 hours post-incubation at 37°C via flow cytometry. From lung tissue isolated at 7 days post infection (dpi), M and M2-specific CD8 T cells proliferated in response to co-culture; however, higher rates were induced by neonatal lung tissue primarily by M-specific T cells. By 8 and 9 dpi, proliferation induced by isolated neonatal lung continued while proliferation induced by adult lung tissue was not quantifiable by our assay. Our findings suggest antigen availability in neonates exceeds that of adults and is age and epitope-dependent. These discoveries indicate that antigen accessibility does not independentlycorrespond with the magnitude of the T cell response in early life. Exploring the inflammatory contributions to RSV-specific T cell defenses is required as this may aid in vaccine design and understanding disease pathogenesis. Supported by grant from NIH R01-AI154619.

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.

Dissecting the cardiac cell landscape in myocarditis

Abstract Introduction Myocarditis is the prototypical cardiac inflammatory disease, with an incidence of 20–30 per 100000 individuals. Acute myocarditis potentially develops into lethal inflammatory cardiomyopathy in a substantial fraction of patients, rendering myocarditis and its sequelae the underlying reason for many heart transplants and 10–20% of cardiovascular sudden deaths in young adults. Different pathological routes had been described to lead to myocardial inflammation (eg. viral, bacterial or parasitic infection, autoimmune processes, toxins). Moreover, genetic predisposition and environmental factors such as the microbiome influence the progression of the disease. Therefore, it is plausible that the inherent complexity of the disease hampers the elucidation of effective therapeutic interventions because the molecular mechanisms underlying this severe heart condition are – to a large extent – incompletely understood. Therefore, it is important to elucidate the molecular pathways governing the interplay between immune cells and stromal cells in the cardiac microenvironment in myocarditis. Methods To dissect the processes involved in myocarditis progression, we took advantage of the T cell receptor transgenic mouse model of spontaneous autoimmune myocarditis (TCRM) were all mice develop acute myocarditis around 4 weeks of age that progresses to lethal dilated cardiomyopathy in 50% of the mice. To analyse the mechanisms involved in cardiac inflammation in mouse and human patients, we used single cell and single nucleus transcriptomic approaches in combination with high dimensional flow cytometry, confocal microscopy and echocardiographic analysis of the cardiac function. To examine the cardiac landscape in humans we applied the same analysis methodologies to right ventricle endomyocardial biopsies from myocarditis or heart transplantation patients (control). Results Our analysis shows that dynamic changes occur in the cardiac microenvironment during the course of myocarditis; these changes involve not only the recruitment of immune cells expressing pro-inflammatory signatures to the myocardium (T cells, inflammatory monocytes, neutrophils) but also the alteration of transcriptional programs in non-immune cells including fibroblast, endothelial cells and cardiomyocytes. The analysis of endomyocardial biopsies from myocarditis patients revealed similar changes in the cardiac cell population's transcriptome. Conclusion Our study reveal that the transcriptional landscape of cardiac cells is strongly influenced by the inflammatory microenvironment. Notably, during the acute phase of myocarditis pathways supporting inflammation and fibrosis overrule circuits involved in cardiac homeostasis. Moreover, we show that the analysis of endomyocardial biopsies by single-nucleus RNAseq represents a powerful tool to unveil the processes involved in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC personal grant to Burkhard Ludewig

Autoreactive B cells recruited to lungs by silica exposure contribute to local autoantibody production in autoimmune-prone BXSB and B cell receptor transgenic mice.

Occupational exposure to inhaled crystalline silica dust (cSiO2) is linked to systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and anti-neutrophil cytoplasmic autoantibody vasculitis. Each disease has a characteristic autoantibody profile used in diagnosis and implicated in pathogenesis. A role for cSiO2 in modulating humoral autoimmunity in vivo is supported by findings in mice, where respirable cSiO2 induces ectopic lymphoid structures as well as inflammation in exposed lungs across genetically diverse backgrounds. In lupus-prone mice cSiO2 exposure also leads to early onset autoantibody production and accelerated disease. Elevated autoantibody levels in bronchoalveolar lavage fluid (BALF) and lung transcriptome analysis suggest that the lung is a hub of cSiO2-evoked autoimmune activity. However, mechanisms by which cSiO2 and lung microenvironments interact to promote autoantibody production remain unclear. We previously demonstrated elevated anti-DNA Ig in BALF but not in lung cell cultures from cSiO2-exposed C57BL/6 mice, suggesting that BALF autoantibodies did not arise locally in this non-autoimmune strain. Autoantibodies were also elevated in BALF of cSiO2-exposed lupus-prone BXSB mice. In this report we test the hypothesis that dysregulated autoreactive B cells recruited to cSiO2-exposed lungs in the context of autoimmune predisposition contribute to local autoantibody production. We found that anti-DNA and anti-myeloperoxidase (MPO) Ig were significantly elevated in cultures of TLR ligand-stimulated lung cells from cSiO2-exposed BXSB mice. To further explore the impact of strain genetic susceptibility versus B cell intrinsic dysfunction on cSiO2-recruited B cell fate, we used an anti-basement membrane autoantibody transgenic (autoAb Tg) mouse line termed M7. In M7 mice, autoAb Tg B cells are aberrantly regulated and escape from tolerance on the C57BL/6 background. Exposure to cSiO2 elicited prominent pulmonary B cell and T cell aggregates and autoAb Tg Ig were readily detected in lung cell culture supernatants. Taken together, diverse disease-relevant autoreactive B cells, including cells specific for DNA, MPO, and basement membrane, are recruited to lung ectopic lymphoid aggregates in response to cSiO2 instillation. B cells that escape tolerance can contribute to local autoantibody production. Our demonstration of significantly enhanced autoantibody induction by TLR ligands further suggests that a coordinated environmental co-exposure can magnify autoimmune vulnerability.

Abstract 586: Orthogonal IL-2/IL-2RB signaling in adoptively transferred T cells controls tumor growth without the need for lymphodepletion in a B16 tumor model

Abstract Multiple adoptive T-cell therapy modalities (ACT) have delivered promising clinical responses in cancer patients. However, challenges including poor T cell effector function, lack of proliferation, and limited persistence have prevented ACTs from reaching their full curative potential. In addition, ACTs typically require lymphodepletion to aid cell engraftment. Lymphodepletion has been shown to improve persistence and efficacy of ACTs by elevating T-cell common gamma-chain cytokines like IL-7 and IL-15. However, lymphodepletion regimens have been identified as a risk factor for cytokine release syndrome (CRS) and infectious complications from opportunistic pathogens. IL-2, another common gamma-chain cytokine, is a potent stimulator of T cells, making it an attractive cytokine to support ACT and potentially bypass the need for lymphodepletion. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of immune cells. To facilitate selective delivery of an IL-2 signal to engineered T cells and avoid signaling in bystander T cells and NK cells, we developed a mouse orthogonal receptor/ligand system consisting of a mutated IL-2 Receptor Beta (moRβ) and a pegylated, IL-2 mutein (moIL-2) that does not significantly activate the wild type IL-2β receptor but does activate moRβ. T cells from pmel-1 T cell receptor-transgenic mice, recognizing gp100 on B16 melanoma cells were transduced with moRβ (orthoPmel). A highly active moIL-2 was continuously dosed for four weeks in mice. Thy1.1+ orthoPmel T cells were tracked by FACS and IHC systemically and in the tumor. During orthoPmel manufacturing, moIL-2 specifically enriched orthoPmel compared to mouse WT IL-2. OrthoPmel in combination with moIL-2 controlled tumor growth in lymphoreplete mice bearing established B16 tumors while neither component alone inhibited tumor growth. moIL-2 significantly expanded orthoPmel systemically and intratumorally, with orthoPmel ultimately accounting for greater than 80% or 40% of all peripheral and intratumoral T cells, respectively. Systemic orthoPmel maintained a consistent central memory and effector memory mix throughout the four-week moIL-2 treatment course. moIL-2 also induced the expression of activation markers, CD25 and Granzyme B, in intratumoral orthoPmel. These findings validate than an orthogonal IL-2/IL-2Rβ platform can enhance efficacy of ACTs without peripheral expansion or activation of NK cells or non-tumor specific T cells and the toxicities typically associated with high dose IL-2 therapy. Importantly, these results demonstrate the potential of this platform overcome the requirement of lymphodepletion in adoptive cell therapies. Citation Format: Christina Kochel, Meng Sun, Navneet Ratti, Sandro Vivona, Mahalaksmi Ramadass, Marie Semana, Michele Bauer, Mohammed Ali, Jan Emmerich, Rob Kastelein, Patrick J. Lupardus, Paul-Joseph Aspuria, Martin Oft. Orthogonal IL-2/IL-2RB signaling in adoptively transferred T cells controls tumor growth without the need for lymphodepletion in a B16 tumor model [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 586.

Measuring the Manipulation of T Helper Immune Responses by Schistosoma mansoni.

Schistosoma mansoni uses different mechanisms to escape its host’s immunity. Understanding the ability of memory T cells to withstand this pathogen’s manipulation is important for the development of effective vaccines against this immunomodulatory pathogen. In this study, ovalbumin (OVA) transgenic S. mansoni is used as a tool to investigate whether fully differentiated Th1, Th2 and Th17 cells are able to withstand pathogen manipulation. Naïve T cells from OT-II T cell receptor transgenic mice with a specificity for OVA were differentiated into Th1, Th2, and Th17 polarised memory cells in vitro. These cells were adoptively transferred into recipient mice to investigate whether these polarised immune memory T cells are resilient in the face of pathogen-mediated manipulation. After transferring memory cells, mice were challenged with OVA-transduced S. mansoni eggs as well as wild-type controls. The in vitro differentiated Th1, Th2 and Th17 memory cells continued to produce the same cytokines when challenged by OVA-expressing S. mansoni eggs as to these they produced when transferred in vivo, suggesting that the Th phenotypes of the memory T cells remains unaltered in the face of stimulation by S. mansoni. The ability of memory T cells to remain resilient to manipulation by the parasite suggests that vaccines might be able to produce immune memory responses able to withstand S. mansoni immune manipulation and hence protect the host from infection.

Tolerization of CD4+ Recent Thymic Emigrants Is Required to Prevent RBC-Specific Autoimmunity

BackgroundLoss of humoral tolerance to red blood cell (RBC)-specific antigens may lead to the development of pathogenic autoantibodies and result in autoimmune hemolytic anemia, a severe and sometimes fatal disease. Recently, it has been demonstrated that although RBC-specific autoreactive B cells escape central tolerance (and can secrete autoantibodies), stringent T cell tolerance is a stopgap to autoimmunity; however, T cell tolerance mechanisms utilized against RBC antigens is largely unknown.Thymic central tolerance is a process by which newly-formed autoreactive T cells undergo negative selection by deletion, anergy, or diversion into a regulatory T cell (Treg). This process ensures that newly-minted T cells are self-tolerant and functional. Elimination of >95% of thymocytes occurs during central tolerance. However, while rigorous, central tolerance sometimes fails and autoreactive T cells escape to the periphery. The youngest peripheral T cells, recent thymic emigrants (RTEs), are functionally distinct from mature T cells and are more susceptible to peripheral tolerance mechanisms in response to autoantigens. Herein, we assess central and peripheral T tolerance to RBC-specific autoantigens.MethodsHOD transgenic mice express an RBC-specific fusion protein consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and human blood group, Duffy (D). To assess tolerance, HOD mice were bred with OTII mice, CD4+ T cell receptor (TCR) transgenic mice with specificity for OVA (contained within HOD). HODxOTII F1s were immunized with 100ug of OVA/CFA. To assess central tolerance, thymi from 8-12 week old HODxOTII F1 mice were harvested and stained with antibodies to identify OTIIs and Tregs. For RTE analysis, HOD mice were bred with OTII.Rag2p-GFP (referred to as HODxRTE mice), OTII T cells that express GFP during TCR rearrangement. GFP+ OTII RTEs were stained with antibodies against CD73, FR4, and PD1. To assess function, GFP+ OTII RTEs were sorted and 3x105 RTEs were infused into naïve B6 mice. Recipient mice received a 100uL packed HOD RBC transfusion and RTE proliferation was assessed at 3 days by Ki67 staining.ResultsHOD+OTII+ mice had profound tolerance to the HOD RBC autoantigen; immunization with OVA/CFA elicited strong antibody responses in HOD-OTII+ littermates, but not autoreactive HOD+OTII+ mice. To determine whether OTIIs underwent central tolerance, thymi were analyzed. No significant differences in total numbers of thymocytes or CD4+ OTIIs was observed in autoreactive HOD+OTII+ mice, compared to control HOD-OTII+. Additional phenotyping for co-expression of CD25 and FoxP3 (a phenotype consistent with Tregs) revealed no difference, suggesting autoreactive OTII T cells were not diverted into a regulatory program. Thus, negative selection of RBC autoreactive CD4+ T cells does not occur in the thymus.Given that RTEs are subject to tolerization in response to peripherally expressed autoantigens, HODxRTE F1s were used to assess whether RBC-specific autoreactive OTIIs are tolerized as RTEs. RTEs were divided into GFPhi (< 1 week in the periphery), GFPint (2-3 weeks in the periphery), and GFPneg (mature T cells). In HOD+RTE+ mice, GFPhi RTEs had significantly higher levels of PD1, a marker of exhaustion, compared to controls. As RTEs matured, PD1 expression slightly decreased, but remained high compared to controls. Additionally, as autoreactive OTII RTEs matured, a significant number OTIIs expressed CD73hiFR4hi, a phenotype consistent with anergy. On average, 5% of GFPneg OTIIs from HOD-RTE+ mice expressed CD73hiFR4hi, compared to over 60% in autoreactive HOD+RTE+ mice. To assess function, sorted RTEs from HODxRTE mice were adoptively transferred into naïve mice and assessed for proliferation post HOD RBC transfusion. All RTEs sorted from HOD-RTE+ mice proliferated (>95% of OTIIs were Ki67+). However, RTEs derived from HOD+RTE+ mice had diminished proliferative capacity upon maturation (Ki67+ RTEs: GFPhi 65%, GFPint 35%, GFPneg 15%).ConclusionsThese data demonstrate that RBC-specific autoreactive CD4+ T cells are neither deleted nor diverted into a Tregs during thymic central tolerance, rather peripheral tolerance mechanisms occurring during RTE maturation is required. Together, these data provide insight into RBC-specific T cell tolerance mechanisms, pinpoint when they occur, and demonstrate that if RTE maturation does not occur, RBC autoimmunity may ensue. DisclosuresNo relevant conflicts of interest to declare.

Critical role of the CD44lowCD62Llow CD8+ T cell subset in restoring antitumor immunity in aged mice

CD8+ T cells play a central role in antitumor immune responses that kill cancer cells directly. In aged individuals, CD8+ T cell immunity is strongly suppressed, which is associated with cancer and other age-related diseases. The mechanism underlying this age-related decrease in immune function remains largely unknown. This study investigated the role of T cell function in age-related unresponsiveness to PD-1 blockade cancer therapy. We found inefficient generation of CD44lowCD62Llow CD8+ T cell subset (P4) in draining lymph nodes of tumor-bearing aged mice. In vitro stimulation of naive CD8+ T cells first generated P4 cells, followed by effector/memory T cells. The P4 cells contained a unique set of genes related to enzymes involved in one-carbon (1C) metabolism, which is critical to antigen-specific T cell activation and mitochondrial function. Consistent with this finding, 1C-metabolism-related gene expression and mitochondrial respiration were down-regulated in aged CD8+ T cells compared with young CD8+ T cells. In aged OVA-specific T cell receptor (TCR) transgenic mice, ZAP-70 was not activated, even after inoculation with OVA-expressing tumor cells. The attenuation of TCR signaling appeared to be due to elevated expression of CD45RB phosphatase in aged CD8+ T cells. Surprisingly, strong stimulation by nonself cell injection into aged PD-1-deficient mice restored normal levels of CD45RB and ameliorated the emergence of P4 cells and 1C metabolic enzyme expression in CD8+ T cells, and antitumor activity. These findings indicate that impaired induction of the P4 subset may be responsible for the age-related resistance to PD-1 blockade, which can be rescued by strong TCR stimulation.

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...

Priming of myelin-specific T cells in the absence of dendritic cells results in accelerated development of Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis (MS). Inflammatory CD4+ T cell responses directed against CNS antigens, including myelin proteolipid protein (PLP), are key mediators of EAE. Dendritic cells (DCs) are critical for the induction of T cell responses against infectious agents. However, the importance of DCs in priming self-reactive CD4+ T cells in autoimmune disease such as MS has been unclear. To determine the requirement of DCs in PLP-specific CD4+ T cell responses and EAE, we genetically deleted CD11c+ DCs in PLP T cell receptor (TCR) transgenic SJL mice constitutively. DC deficiency did not impair the development, selection or the pathogenic function of PLP-specific CD4+ T cells in these mice, and resulted in accelerated spontaneous EAE compared to DC sufficient controls. In addition, using a genetic approach to ablate DCs conditionally in SJL mice, we show that CD11c+ DCs were dispensable for presenting exogenous or endogenous myelin antigen to PLP-specific T cells and for promoting pro-inflammatory T cell responses and severe EAE. Our findings demonstrate that constitutive or conditional ablation of CD11c+ DCs diminished self-tolerance to PLP autoantigen. They further show that in the absence of DCs, non-DCs can efficiently present CNS myelin antigens such as PLP to self-reactive T cells, resulting in accelerated onset of spontaneous or induced EAE.

A LAT-Based Signaling Complex in the Immunological Synapse as Determined with Live Cell Imaging Is Less Stable in T Cells with Regulatory Capability.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

Gut Helicobacter presentation by multiple dendritic cell subsets enables context-specific regulatory T cell generation.

Generation of tolerogenic peripheral regulatory T (pTreg) cells is commonly thought to involve CD103+ gut dendritic cells (DCs), yet their role in commensal-reactive pTreg development is unclear. Using two Helicobacter-specific T cell receptor (TCR) transgenic mouse lines, we found that both CD103+ and CD103- migratory, but not resident, DCs from the colon-draining mesenteric lymph node presented Helicobacter antigens to T cells ex vivo. Loss of most CD103+ migratory DCs in vivo using murine genetic models did not affect the frequency of Helicobacter-specific pTreg cell generation or induce compensatory tolerogenic changes in the remaining CD103- DCs. By contrast, activation in a Th1-promoting niche in vivo blocked Helicobacter-specific pTreg generation. Thus, these data suggest a model where DC-mediated effector T cell differentiation is 'dominant', necessitating that all DC subsets presenting antigen are permissive for pTreg cell induction to maintain gut tolerance.

Insulin-Reactive T Cells Convert Diabetogenic Insulin-Reactive VH125 B Cells Into Tolerogenic Cells by Reducing Germinal Center T:B Cell Interactions in NOD Mice.

Insulin is a key autoantigen in Type 1 Diabetes (T1D), targeted by both T and B cells. Therefore, understanding insulin-specific T:B cell interactions is important. We have previously reported an insulin-reactive CD4+ T cell, (designated 2H6). Unlike other insulin-reactive T cells, 2H6 cells protect non-obese diabetic (NOD) mice from T1D development, mediated by TGFβ. To investigate insulin-specific T:B cell interactions, we bred 2H6αβ T cell receptor transgenic NOD mice (2H6) with the insulin-reactive B cell receptor transgenic NOD mice (VH125), generating 2H6VH125 NOD mice. Similar to 2H6 mice, 2H6VH125 mice are protected from T1D development. Interestingly, VH125 B cells did not alter the phenotype of 2H6 T cells; however, 2H6 T cells significantly altered the VH125 B cells by reducing the insulin-reactive non-germinal center (GC) and GC B cells, as well as MHC and costimulatory molecule expression on the B cells. Furthermore, the B cells in 2H6VH125 NOD mice exhibited increased non-insulin-specific and a class switched IgG isotype, which can be recapitulated in vivo in Rag-deficient NOD mice by adoptive transfer. In vitro, VH125 B cells from 2H6VH125 mice suppressed the proliferation of 2H6 T cells to insulin antigen but enhanced TGFβ secretion by 2H6 T cells from 2H6VH125 mice compared to 2H6 mice. In summary, our data showed that 2H6 CD4+ T cells alter the phenotype and function of insulin-reactive B cells from pathogenic to tolerogenic cells. In turn, VH125 B cells also modulate the function of the 2H6 T cells. Thus, promoting the interactions between antigen-specific regulatory T cells and B cells may lead to protection from T1D.

CD4+ T cells promote delayed B cell responses in the ischemic brain after experimental stroke

CD4+ T lymphocytes are key mediators of tissue damage after ischemic stroke. However, their infiltration kinetics and interactions with other immune cells in the delayed phase of ischemia remain elusive. We hypothesized that CD4+ T cells facilitate delayed autoreactive B cell responses in the brain, which have been previously linked to post-stroke cognitive impairment (PSCI). Therefore, we treated myelin oligodendrocyte glycoprotein T cell receptor transgenic 2D2 mice of both sexes with anti-CD4 antibody following 60-minute middle cerebral artery occlusion and assessed lymphocyte infiltration for up to 72 days. Anti-CD4-treatment eliminated CD4+ T cells from the circulation and ischemic brain for 28 days and inhibited B cell infiltration into the brain, particularly in animals with large infarcts. Absence of CD4+ T cells did not influence infarct maturation or survival. Once the CD4+ population recovered in the periphery, both CD4+ T and B lymphocytes entered the infarct site forming follicle-like structures. Additionally, we provide further evidence for PSCI that could be attenuated by CD4 depletion. Our findings demonstrate that CD4+ T cells are essential in delayed B cell infiltration into the ischemic brain after stroke. Importantly, lymphocyte infiltration after stroke is a long-lasting process. As CD4 depletion improved cognitive functions in an experimental set-up, these findings set the stage to elaborate more specific immune modulating therapies in treating PSCI.

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.

Th1 guided evolution of protective de novo Th17 response in severe influenza

Abstract The 6.5 CD4+ T cell receptor (TCR) transgenic mice have a monotonous CD4+ TCR which responds to the hemagglutinin (HA) antigen of PR8 influenza virus. Naive 6.5 CD4+ T cells adoptively transferred into wild type mice were activated by PR8 influenza virus with Th1 response. With PR8 influenza virus infection, all CD4+ T cells in 6.5 mice were activated and they were predominantly Th1 in phenotype at the early phase of infection and Th17 at the late phase. Th1 effector responses made infected mice sick and even succumb to death. The 6.5 mice were sicker at the early phase but recovered with better survival at last. IL-17KO 6.5 mice cannot display Th17 responses and lost the survival benefit as well. IL-17 supplement at the late phase of infection enhanced survival of wild type mice with PR8 influenza virus infection. With consecutive two adoptive transfers of naive 6.5 CD4+ T cells, 6.5 CD4+ T cells of the first transfer evolved into Th1 cells but 6.5 CD4+ T cells of the second transfer demonstrated a de novoTh17 response. Thymectomy or aging in 6.5 mice limited the thymic output of naive 6.5 CD4+ T cells and diminished the Th17 response. Viral neuraminidase activated TGF-β in the Th1 6.5 CD4+ T cells at the early phase. T-bet dominance was altered with excessive ROR-γt activation in the following responding 6.5 CD4+ T cells. They have substantial IL-17 with restrained IFN-γ production. We demonstrated a Th1 guided evolution of protective Th17 response in severe influenza.