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Introduction: Chimeric antigen receptor (CAR) expressing T-cells have shown great promise for the future of cancer immunotherapy with the recent clinical successes achieved in treating different hematologic cancers. Despite these early successes, several challenges remain in the field that require to be solved for the therapy to be more efficacious. One such challenge is the lack of long-term persistence of CD28 based CAR T-cells in patients. Although, CD28 based CAR T-cells elicit a robust acute anti-tumor response, they are more prone to early exhaustion, terminal differentiation and cell death due to their strong signaling patterns. Hence attenuation of signaling strength in CD28 based CARs is an accepted strategy to improve long-term CAR T-cell function and persistence in patients. Previous studies with the conventional T-cell receptor (TCR) have suggested that manipulation of CD3 immunoreceptor tyrosine-based activation motif (ITAM) sequences can alter TCR signaling strength. Based on these studies, we have designed 2 nd generation murine anti-CD19 CD28 based CARs with restricted CD3ζ ITAM sequence diversity while maintaining a multiplicity of three. They are called ζAAA, ζBBB and ζCCC based on which CD3ζ ITAM they express. The goal of the study is to understand the non-redundant signaling properties of the individual CD3ζ ITAMs and their effect on CAR T-cell function. We hypothesized that the individual CD3ζ ITAMs will exhibit unique signaling properties in the ITAM restricted CARs which may allow for optimization of CAR signaling and improve CAR T-cell persistence and function., Method: We subjected the ITAM restricted CAR T cells to various conditions of in vitro stimulation using CD19+ tumor cells or CD19-coated magnetic beads. Immunoblotting and flow cytometry based Ca2+ signaling assays were used to quantify signaling differences. Functional differences were studied using in vitro cytotoxicity, degranulation and cytokine expression assays. CAR T cell exhaustion and differentiation were studied using an in vitro exhaustion assay., Results: We observed that ζAAA CARs had stronger signaling strength compared to ζBBB and ζCCC CARs. The signaling differences were reflected in their functional activation profiles with T-cells expressing ζAAA CARs having a strong activation profile and ζCCC CARs having a weak activation profile. ζCCC CAR T cells were less prone to differentiation and exhaustion., Discussion: Since, weaker signaling ζCCC CARs favored less cell death, exhaustion and differentiation, they might be better candidates for improving long term survival and persistence of CAR T cells in patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Majumdar, Echelibe, Bettini and Bettini.)

Self-reactive T cells experience chronic antigen exposure but do not exhibit signs of exhaustion. Here, we investigated the mechanisms for sustained, functioning autoimmune CD4 + T cells despite chronic stimulation. Examination of T cell priming showed that CD4 + T cells activated in the absence of infectious signals retained TCF1 expression. At later time points and during blockade of new T cell recruitment, most islet-infiltrating autoimmune CD4 + T cells were TCF1 + , although expression was reduced on a per T cell basis. The Tcf7 locus was epigenetically modified in circulating autoimmune CD4 + T cells, suggesting a pre-programmed de novo methylation of the locus in early stages of autoimmune CD4 + T cell differentiation. This mirrored the epigenetic profile of recently recruited CD4 + CD62L + T cells in the pancreas. Collectively, these data reveal a unique environment during autoimmune CD4 + T cell priming that allows T cells to fine-tune TCF1 expression and maintain long-term survival and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Conventional immunosuppressive functions of CD4 + Foxp3 +  regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. However, whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed that upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. From this we postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin, and that both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited., (© 2023. The Author(s).)

As the prevalence of allergy and autoimmune disease in industrialized societies continues to rise, improving our understanding of the mechanistic roles behind microbiota-immune homeostasis has become critical for informing therapeutic interventions in cases of dysbiosis. Of particular importance, are alterations to intestinal microbiota occurring within the critical neonatal window, during which the immune system is highly vulnerable to environmental exposures. This review will highlight recent literature concerning mechanisms of early-life microbiota-immune homeostasis as well as discuss the potential for therapeutics in restoring dysbiosis in early life., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Reynolds and Bettini.)

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. Whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. We postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin and both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum (ER) stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited., Competing Interests: Additional Information The authors declare no competing interests. Additional Declarations: No competing interests reported.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Type-1 Diabetes (T1D) is a complex polygenic autoimmune disorder involving T-cell driven beta-cell destruction leading to hyperglycemia. There is no cure for T1D and patients rely on exogenous insulin administration for disease management. T1D is associated with specific disease susceptible alleles. However, the predisposition to disease development is not solely predicted by them. This is best exemplified by the observation that a monozygotic twin has just a 35% chance of developing T1D after their twin's diagnosis. This makes a strong case for environmental triggers playing an important role in T1D incidence. Multiple studies indicate that commensal gut microbiota and environmental factors that alter their composition might exacerbate or protect against T1D onset. In this review, we discuss recent literature highlighting microbial species associated with T1D. We explore mechanistic studies which propose how some of these microbial species can modulate adaptive immune responses in T1D, with an emphasis on T-cell responses. We cover topics ranging from gut-thymus and gut-pancreas communication, microbial regulation of peripheral tolerance, to molecular mimicry of islet antigens by microbial peptides. In light of the accumulating evidence on commensal influences in neonatal thymocyte development, we also speculate on the link between molecular mimicry and thymic selection in the context of T1D pathogenesis. Finally, we explore how these observations could inform future therapeutic approaches in this disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Majumdar, Lin and Bettini.)

Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T-cell population. Hybrid peptides formed through transpeptidation within pancreatic β-cell lysosomes have been proposed as a new class of autoantigens in type 1 diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the hybrid insulin peptide (HIP) 2.5HIP toward thymic resident Langerin-positive dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti-Langerin-2.5HIP delivery can enhance T-cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti-Langerin-2.5HIP during the neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neoantigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases., (© 2022 by the American Diabetes Association.)

The contribution of low-affinity T cells to autoimmunity in the context of polyclonal T-cell responses is understudied due to the limitations in their capture by tetrameric reagents and low level of activation in response to antigenic stimulation. As a result, low-affinity T cells are often disregarded as nonantigen-specific cells irrelevant to the immune response. Our study aimed to assess how the level of self-antigen reactivity shapes T-cell lineage and effector responses in the context of spontaneous tissue-specific autoimmunity observed in NOD mice. Using multicolor flow cytometry in combination with Nur77 GFP reporter of TCR signaling, we identified a dormant population of T cells that infiltrated the pancreatic islets of prediabetic NOD mice, which exhibited reduced levels of self-tissue reactivity based on expression of CD5 and Nur77 GFP . We showed that these CD5 low T cells had a unique TCR repertoire and exhibited low activation and minimal effector function; however, induced rapid diabetes upon transfer. The CD4 + CD5 low T-cell population displayed transcriptional signature of central memory T cells, consistent with the ability to acquire effector function post-transfer. Transcriptional profile of CD5 low T cells was similar to T cells expressing a low-affinity TCR, indicating TCR affinity to be an important factor in shaping CD5 low T-cell phenotype and function at the tissue site. Overall, our study suggests that autoimmune tissue can maintain a reservoir of undifferentiated central memory-like autoreactive T cells with pathogenic effector potential that might be an important source for effector T cells during long-term chronic autoimmunity., (© 2022 Wiley-VCH GmbH.)

Accumulating evidence supports a critical role for posttranslationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen-reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115-127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes and reduced frequency of regulatory T cells. Using humanized HLA/T-cell receptor (TCR) mice, we observed that TCRs reactive to the native or deamidated GAD65115-127 led to efficient development of CD4+ effector T cells; however, regulatory T-cell development was reduced in mice expressing the PTM-reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native-specific and the deamidated-specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti-β-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T-cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T-cell development is reduced in response to the deamidated epitope, pointing to regulatory T-cell development as a key mechanism for loss of tolerance to PTM antigenic targets., (© 2022 by the American Diabetes Association.)

Critical insights into the etiology of type 1 diabetes (T1D) came from genome-wide association studies that unequivocally connected genetic susceptibility to immune cell function. At the top of the susceptibility are genes involved in regulatory T-cell (Treg) function and development. The advances in epigenetic and transcriptional analyses have provided increasing evidence for Treg dysfunction in T1D. These are well supported by functional studies in mouse models and analysis of peripheral blood during T1D. For these reasons, Treg-based therapies are at the forefront of research and development and have a tangible probability to deliver a long-sought-after successful immune-targeted treatment for T1D. The current challenge in the field is whether we can directly assess Treg function at the tissue site or make informative interpretations based on peripheral data. Future studies focused on Treg function in pancreatic lymph nodes and pancreas could provide key insight into the ultimate mechanisms underlying Treg failure in T1D. In this Perspective we will provide an overview of current literature regarding Treg development and function in T1D and how this knowledge has been applied to Treg therapies., (© 2021 by the American Diabetes Association.)

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection 1 . Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses 1,2 . Antigen-specific recognition of intestinal microorganisms by T cells has previously been described 3,4 . Although the local environment shapes the differentiation of effector cells 3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αβ T cell numbers. MPC1-deficient T cells have defective thymic development at the β-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αβ T cell development and that its deficiency results in reduced but activated peripheral T cell populations., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Type 1 diabetes is an autoimmune-mediated disease that culminates in the targeted destruction of insulin-producing β-cells. CD4 responses in NOD mice are dominated by insulin epitope B:9-23 (InsB 9-23 ) specificity, and mutation of the key T-cell receptor (TCR) contact residue within the epitope prevents diabetes development. However, it is not clear how insulin self-antigen controls the selection of autoimmune and regulatory T cells (Tregs). Here we demonstrate that mutation of insulin epitope results in escape of highly pathogenic T cells. We observe an increase in antigen reactivity, clonality, and pathogenicity of insulin-specific T cells that develop in the absence of cognate antigen. Using a single TCR system, we demonstrate that Treg development is greatly diminished in mice with the Y16A mutant epitope. Collectively, these results suggest that the tyrosine residue at position 16 is necessary to constrain TCR reactivity for InsB 9-23 by both limiting the development of pathogenic T cells and supporting the selection of Tregs., (© 2019 by the American Diabetes Association.)

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of infants that is characterized by interrupted lung development. Postnatal sepsis causes BPD, yet the contributory mechanisms are unclear. To address this gap, studies have used lipopolysaccharide (LPS) during the alveolar phase of lung development. However, the lungs of infants who develop BPD are still in the saccular phase of development, and the effects of LPS during this phase are poorly characterized. We hypothesized that chronic LPS exposure during the saccular phase disrupts lung development by mechanisms that promote inflammation and prevent optimal lung development and repair. Wild-type C57BL6J mice were intraperitoneally administered 3, 6, or 10 mg/kg of LPS or a vehicle once daily on postnatal days (PNDs) 3-5. The lungs were collected for proteomic and genomic analyses and flow cytometric detection on PND6. The impact of LPS on lung development, cell proliferation, and apoptosis was determined on PND7. Finally, we determined differences in the LPS effects between the saccular and alveolar lungs. LPS decreased the survival and growth rate and lung development in a dose-dependent manner. These effects were associated with a decreased expression of proteins regulating cell proliferation and differentiation and increased expression of those mediating inflammation. While the lung macrophage population of LPS-treated mice increased, the T-regulatory cell population decreased. Furthermore, LPS-induced inflammatory and apoptotic response and interruption of cell proliferation and alveolarization was greater in alveolar than in saccular lungs. Collectively, the data support our hypothesis and reveal several potential therapeutic targets for sepsis-mediated BPD in infants.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX 3 CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX 3 CR1 + APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Regulatory T cells (Tregs) use a distinct TCR repertoire and are more self-reactive compared with conventional T cells. However, the extent to which TCR affinity regulates the function of self-reactive Tregs is largely unknown. In this study, we used a two-TCR model to assess the role of TCR affinity in Treg function during autoimmunity. We observed that high- and low-affinity Tregs were recruited to the pancreas and contributed to protection from autoimmune diabetes. Interestingly, high-affinity cells preferentially upregulated the TCR-dependent Treg functional mediators IL-10, TIGIT, GITR, and CTLA4, whereas low-affinity cells displayed increased transcripts for Areg and Ebi3 , suggesting distinct functional profiles. The results of this study suggest mechanistically distinct and potentially nonredundant roles for high- and low-affinity Tregs in controlling autoimmunity., (Copyright © 2018 by The American Association of Immunologists, Inc.)

T cell receptor (TCR) affinity is a critical factor of Treg lineage commitment, but whether self-reactivity is a determining factor in peripheral Treg function remains unknown. Here, we report that a high degree of self-reactivity is crucial for tissue-specific Treg function in autoimmunity. Based on high expression of CD5, we identified a subset of self-reactive Tregs expressing elevated levels of T-bet, GITR, CTLA-4, and ICOS, which imparted significant protection from autoimmune diabetes. We observed that T-bet expression in Tregs, necessary for control of Th1 autoimmunity, could be induced in an IFNγ-independent fashion and, unlike in conventional T cells (Tconv), was strongly correlated with the strength of TCR signaling. The level of CD5 similarly identified human Tregs with an increased functional profile, suggesting that CD5hi Tregs may constitute an efficacious subpopulation appropriate for use in adoptive Treg therapies for treatment of inflammatory conditions. Overall, this work establishes an instrumental role of high TCR self-reactivity in driving Treg function.

The strongest susceptibility allele for Type 1 Diabetes (T1D) is human leukocyte antigen (HLA), which supports a central role for T cells as the drivers of autoimmunity. However, the precise mechanisms that allow thymic escape and peripheral activation of beta cell antigen-specific T cells are still largely unknown. Studies performed with the non-obese diabetic (NOD) mouse have challenged several immunological dogmas, and have made the NOD mouse a key experimental system to study the steps of immunodysregulation that lead to autoimmune diabetes. The structural similarities between the NOD I-A g7 and HLA-DQ8 have revealed the stability of the T cell receptor (TCR)/HLA/peptide tri-molecular complex as an important parameter in the development of autoimmune T cells, as well as afforded insights into the key antigens targeted in T1D. In this review, we will provide a summary of the current understanding with regard to autoimmune T cell development, the significance of the antigens targeted in T1D, and the relationship between TCR affinity and immune regulation.

Type 1 diabetes is a T cell-mediated autoimmune disease that is characterized by Ag-specific targeting and destruction of insulin-producing β cells. Although multiple studies have characterized the pathogenic potential of β cell-specific T cells, we have limited mechanistic insight into self-reactive autoimmune T cell development and their escape from negative selection in the thymus. In this study, we demonstrate that ectopic expression of insulin epitope B:9-23 (InsB 9-23 ) by thymic APCs is insufficient to induce deletion of high- or low-affinity InsB 9-23 -reactive CD4 + T cells; however, we observe an increase in the proportion and number of thymic and peripheral Foxp3 + regulatory T cells. In contrast, the MHC stable insulin mimetope (InsB 9-23 R22E) efficiently deletes insulin-specific T cells and prevents escape of high-affinity thymocytes. Collectively, these results suggest that Ag dose and peptide-MHC complex stability can lead to multiple fates of insulin-reactive CD4 + T cell development and autoimmune disease outcome., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

For the αβ or γδTCR chains to integrate extracellular stimuli into the appropriate intracellular cellular response, they must use the 10 ITAMs found within the CD3 subunits (CD3γε, CD3δε, and ζζ) of the TCR signaling complex. However, it remains unclear whether each specific ITAM sequence of the individual subunit (γεδζ) is required for thymocyte development or whether any particular CD3 ITAM motif is sufficient. In this article, we show that mice utilizing a single ITAM sequence (γ, ε, δ, ζa, ζb, or ζc) at each of the 10 ITAM locations exhibit a substantial reduction in thymic cellularity and limited CD4 - CD8 - (double-negative) to CD4 + CD8 + (double-positive) maturation because of low TCR expression and signaling. Together, the data suggest that ITAM sequence diversity is required for optimal TCR signal transduction and subsequent T cell maturation., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Single-cell paired TCR identification is a powerful tool, but has been limited in its previous incompatibility with further functional analysis. The current protocol describes a method to clone and functionally evaluate in vivo TCRs derived from single antigen-responsive human T cells and monoclonal T cell lines. We have improved upon current PCR-based TCR sequencing protocols by developing primers that allow amplification of human TCRα and TCRβ variable regions, while incorporating specific restriction cut sites for direct subcloning into the template retroviral vector. This streamlined approach for generating human:mouse chimeric TCR vectors allows for rapid TCR expression in humanized-retrogenic (hu-Rg) mice through retroviral mediated stem cell gene transfer. Using widely available techniques and equipment, this method is easily adaptable by most laboratories. This is the first TCR identification protocol that is efficiently combined with subsequent in vivo TCR expression., (Copyright © 2016 Elsevier B.V. All rights reserved.)

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCRα and TCRβ chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The use of retrogenic mice offers a rapid and flexible approach to T cell receptor (TCR)-transgenic mice. By transducing bone marrow progenitor cells with a retrovirus that encodes a given TCR-α/β subunit, TCR-retrogenic mice can be generated in as few as 4-6 weeks, whereas conventional TCR transgenics can take 6 months or longer. In this updated protocol, we have increased the efficiency of the bone marrow transduction and bone marrow reconstitution compared with our previously published protocol. The main departure from the previous protocol is the implementation of spin transduction with the viral supernatant instead of coculture with the viral producer cell line. The changes in this protocol improve bone marrow viability, increase consistency of the bone marrow transduction and bone marrow engraftment, and they reduce the ratio of bone marrow donor mice to bone marrow recipients.

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a-Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

What does it take to make a regulatory T (Treg) cell? In this issue of Immunity, Ohkura et al. show that Treg-cell-specific CpG hypomethylation and Foxp3 expression are independent events required for Treg cell development, stability, and full suppressive activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The T-cell receptor (TCR) is unique in its complexity. It determines not only positive (life) and negative (death) selection in the thymus, but also mediates proliferation, anergy, differentiation, cytotoxicity and cytokine production in the periphery. Through its association with six CD3 signalling chains (εγ, δε and ζζ), the TCR is capable of recognizing an extensive variety of antigenic peptides, from both pathogens and self-antigens, and translating these interactions into multiple signalling pathways that mediate diverse T-cell developmental and functional responses. The analysis of TCR biology has been revolutionized by the development of TCR transgenic mice, which express a single clonotypic T-cell population, with diverse specificities and genetic backgrounds. However, they are time consuming to generate and characterize, limiting the analysis of large numbers of TCR over a short period of time in multiple genetic backgrounds. The recent development of TCR retrogenic technology resolves these limitations and could in time have a similarly important impact on our understanding of T-cell development and function. In this review, we will discuss the advantages and limitations of retrogenic technology compared with the generation and use of TCR transgenic mice for studying all aspects of T-cell biology., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

Regulatory T (Treg) cells, driven by the Foxp3 transcription factor, are responsible for limiting autoimmunity and chronic inflammation. We showed that a well-characterized Foxp3(gfp) reporter mouse, which expresses an N-terminal GFP-Foxp3 fusion protein, is a hypomorph that causes profoundly accelerated autoimmune diabetes on a NOD background. Although natural Treg cell development and in vitro function are not markedly altered in Foxp3(gfp) NOD and C57BL/6 mice, Treg cell function in inflammatory environments was perturbed and TGF-β-induced Treg cell development was reduced. Foxp3(gfp) was unable to interact with the histone acetyltransferase Tip60, the histone deacetylase HDAC7, and the Ikaros family zinc finger 4, Eos, which led to reduced Foxp3 acetylation and enhanced K48-linked polyubiquitylation. Collectively this results in an altered transcriptional landscape and reduced Foxp3-mediated gene repression, notably at the hallmark IL-2 promoter. Loss of controlled Foxp3-driven epigenetic modification leads to Treg cell insufficiency that enables autoimmunity in susceptible environments., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

Natural regulatory T cells (nTregs) are defined by their inherent ability to establish and maintain peripheral self-tolerance. In recent years, the development of nTregs has come under close examination with the advent of Forkhead Box P3 protein (FOXP3)-green fluorescent protein reporter mice that pinpointed the initiation of FOXP3 expression within the thymus. The mechanism and pathway of nTreg development has only recently been studied in detail and to a large degree remains unclear. In this review, we will discuss our current understanding of nTreg lineage choice and development from a cellular and intracellular standpoint.

Phosphorylation of MAP kinases is important for proper translation of T cell antigen receptor (TCR) signals into thymocyte cell fates, but the role of MAP kinase phosphatase (MKP) activity in thymocyte development has not been characterized. To explore the role of MKP in thymocytes, we constructed a double mutant MKP-3 (DM-MKP3) that acts as a dominant-negative inhibitor of ERK- and JNK-specific MKP. Thymocytes developing in the presence of DM-MKP3 have enhanced frequencies of both CD4 and CD8 mature, single-positive cells and no increase in apoptosis. Expression of DM-MKP3 also results in an increased proportion of thymocytes with high levels of both CD69 and TCRbeta, suggesting that the increased proportion of mature thymocytes is the result of an increased probability that CD4(+)CD8(+) cells will be positively selected. Thus, MKP activity controls thymocyte cell fate by regulating the threshold of TCR signaling that is able to induce positive selection.