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The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly in

Central tolerance of thymocytes to self-antigen depends on the medullary thymic epithelial cell (mTEC) transcription factor autoimmune regulator (Aire), which drives tissue-restricted antigen (TRA) gene expression. Vitamin D signaling regulates Aire and TRA expression in mTECs, providing a basis for links between vitamin D deficiency and autoimmunity. We find that mice lacking Cyp27b1, which cannot produce hormonally active vitamin D, display profoundly reduced thymic cellularity, with a reduced proportion of Aire + mTECs, attenuated TRA expression, and poorly defined cortical-medullary boundaries. Markers of T cell negative selection are diminished, and organ-specific autoantibodies are present in knockout (KO) mice. Single-cell RNA sequencing revealed that loss of Cyp27b1 skews mTEC differentiation toward Ccl21 + intertypical TECs and generates a gene expression profile consistent with premature aging. KO thymi display accelerated involution and reduced expression of thymic longevity factors. Thus, loss of thymic vitamin D signaling disrupts normal mTEC differentiation and function and accelerates thymic aging.

Chronic viral infections cause thymic involution yet the potential for broader, longer-term impact on thymic composition remains unexplored. Here we show that chronic, but not acute, lymphocytic choriomeningitis virus infection promotes a unique population of immature B cells in the thymus. We show that chronic viral infection promotes signals within the thymus, including the expression of B-cell activating factor (BAFF), that favor the maturation of this population as these cells acquire expression of CD19 and immunoglobulin M. Mechanistically, type I interferon (IFN-I), predominantly IFNβ, signals to thymic hematopoietic cells, strongly delaying T-cell development at the earliest precursor stage. Furthermore, IFN-I signaling to the nonhematopoietic compartment provides a second signal essential to favor B-cell differentiation and maturation within the thymus. Importantly, chronic infection yields changes in the B-cell population for at least 50 days following infection, long after thymic atrophy has subsided. Thus, the inflammatory milieu induced by chronic viral infection has a profound, and long-lasting, effect on thymic composition leading to the generation of a novel population of thymic B cells., (© 2024 The Author(s). Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

The Canadian Society for Immunology (CSI) established a formal Equity, Diversity, and Inclusion (EDI) Committee with the goal of providing EDI advocacy and leadership within the CSI, as well as in the broader scientific community. A first task of this committee was to review the publicly available historical data on gender representation within the CSI's membership, leadership, award recipients, and conference chairs/presenters as a step in establishing a baseline reference point and monitoring the trajectory of future success in achieving true inclusion. We found that, except for overall membership and a specific subset of awards, all categories showed a historical bias toward men, particularly prior to 2010. Bias persists in various categories, evident even in recent years. However, we note an encouraging trend toward greater gender parity, particularly in the roles of President, symposium presenters, and workshop chairs, especially from 2017 onward. We present these findings as well as our recommendations to enhance inclusivity. These include a more comprehensive collection and secure storage of self-identification data, emphasis on EDI as an essential component of all annual meeting activities, and innovative measures of outreach, collaboration, and leadership with the aim of making the CSI a model for improving EDI in other professional research societies., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Adoptive T cell therapies rely on the production of T cells with an antigen receptor that directs their specificity toward tumor-specific antigens. Methods for identifying relevant T cell receptor (TCR) sequences, predominantly achieved through the enrichment of antigen-specific T cells, represent a major bottleneck in the production of TCR-engineered cell therapies. Fluctuation of intracellular calcium is a proximal readout of TCR signaling and candidate marker for antigen-specific T cell identification that does not require T cell expansion; however, calcium fluctuations downstream of TCR engagement are highly variable. We propose that machine learning algorithms may allow for T cell classification from complex datasets such as polyclonal T cell signaling events. Using deep learning tools, we demonstrate accurate prediction of TCR-transgenic CD8 + T cell activation based on calcium fluctuations and test the algorithm against T cells bearing a distinct TCR as well as polyclonal T cells. This provides the foundation for an antigen-specific TCR sequence identification pipeline for adoptive T cell therapies.

The T cell receptor (TCR) determines specificity and affinity for both foreign and self-peptides presented by the major histocompatibility complex (MHC). Although the strength of TCR interactions with self-pMHC impacts T cell function, it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naive CD4 + T cells with low versus high self-reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that identifies population-level differences between TCRβ sequence sets. This approach revealed that weakly self-reactive T cell populations were enriched for longer CDR3β regions and acidic amino acids. We tested our ML predictions of self-reactivity using retrogenic mice with fixed TCRβ sequences. Extrapolating our analyses to independent datasets, we predicted high self-reactivity for regulatory T cells and slightly reduced self-reactivity for T cells responding to chronic infections. Our analyses suggest a potential trade-off between TCR repertoire diversity and self-reactivity. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitamin D deficiency is associated with the development of autoimmunity, which arises from defects in T cell tolerance to self-antigens. Interactions of developing T cells with medullary thymic epithelial cells, which express tissue-restricted Ags, are essential for the establishment of central tolerance. However, vitamin D signaling in the thymus is poorly characterized. We find that stromal and hematopoietic cells in the mouse thymus express the vitamin D receptor (Vdr) and Cyp27b1, the enzyme that produces hormonal 1,25-dihydroxyvitamin D (1,25D). Treatment of cultured thymic slices with 1,25D enhances expression of the critical medullary thymic epithelial cell transcription factor autoimmune regulator (Aire), its colocalization with the Vdr, and enhances tissue-restricted Ag gene expression. Moreover, the Vdr interacts with Aire in a 1,25D-dependent manner and recruits Aire to DNA at vitamin D response elements, where it acts as a Vdr coactivator. These data link vitamin D signaling directly to critical transcriptional events necessary for central tolerance., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The Canadian Society for Immunology (CSI) organized an Equity, Diversity and Inclusion (EDI) training workshop during its 2022 Scientific Meeting to improve understanding of EDI and explore strategies to achieve EDI goals in the scientific environment. The workshop focused on identifying Specific, Measurable, Achievable, Realistic and Timely (SMART) goals related to EDI in academia through small group discussions and learning exercises. Attendees highlighted several equity considerations within the field of academic immunology, including financial barriers, lack of diversity in research teams and gender bias; they emphasized the importance of creating an inclusive and accessible research environment. The collection and use of data relevant to EDI goals within the CSI were also identified as challenges. Fostering a culture of active and nonjudgmental listening within the CSI community is another aspirational goal to address EDI. The workshop received positive feedback from attendees, who noted that more diverse voices and specific actions for local research environments are needed., (© 2023 the Australian and New Zealand Society for Immunology, Inc.)

Pathological neovascularization in age-related macular degeneration (nvAMD) drives the principal cause of blindness in the elderly. While there is a robust genetic association between genes of innate immunity and AMD, genome-to-phenome relationships are low, suggesting a critical contribution of environmental triggers of disease. Possible insight comes from the observation that a past history of infection with pathogens such as Chlamydia pneumoniae, or other systemic inflammation, can predispose to nvAMD in later life. Using a mouse model of nvAMD with prior C. pneumoniae infection, endotoxin exposure, and genetic ablation of distinct immune cell populations, we demonstrated that peripheral infections elicited epigenetic reprogramming that led to a persistent memory state in retinal CX3CR1+ mononuclear phagocytes (MNPs). The immune imprinting persisted long after the initial inflammation had subsided and ultimately exacerbated choroidal neovascularization in a model of nvAMD. Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) identified activating transcription factor 3 (ATF3) as a central mediator of retina-resident MNP reprogramming following peripheral inflammation. ATF3 polarized MNPs toward a reparative phenotype biased toward production of proangiogenic factors in response to subsequent injury. Therefore, a past history of bacterial endotoxin-induced inflammation can lead to immunological reprograming within CNS-resident MNPs and aggravate pathological angiogenesis in the aging retina.

Age-related macular degeneration is a prevalent neuroinflammatory condition and a major cause of blindness driven by genetic and environmental factors such as obesity. In diseases of aging, modifiable factors can be compounded over the life span. We report that diet-induced obesity earlier in life triggers persistent reprogramming of the innate immune system, lasting long after normalization of metabolic abnormalities. Stearic acid, acting through Toll-like receptor 4 (TLR4), is sufficient to remodel chromatin landscapes and selectively enhance accessibility at binding sites for activator protein-1 (AP-1). Myeloid cells show less oxidative phosphorylation and shift to glycolysis, ultimately leading to proinflammatory cytokine transcription, aggravation of pathological retinal angiogenesis, and neuronal degeneration associated with loss of visual function. Thus, a past history of obesity reprograms mononuclear phagocytes and predisposes to neuroinflammation.

There is a long-standing assumption that naive CD4 + and CD8 + T cells are largely homogeneous populations despite the extraordinary diversity of their T cell receptors (TCR). The self-immunopeptidome plays a key role in the selection of the naive T cell repertoire in the thymus, and self-peptides are also an important driver of differences between individual naive T cells with regard to their subsequent functional contributions to an immune response. Accumulating evidence suggests that as early as the β-selection stage of T cell development, when only one of the recombined chains of the mature TCR is expressed, signaling thresholds may be established for positive selection of immature thymocytes. Stochastic encounters subsequently made with self-ligands during positive selection in the thymus imprint functional biases that a T cell will carry with it throughout its lifetime, although ongoing interactions with self in the periphery ensure a level of plasticity in the gene expression wiring of naive T cells. Identifying the sources of heterogeneity in the naive T cell population and which functional attributes of T cells can be modulated through post-thymic interventions versus those that are fixed during T cell development, could enable us to better select or generate T cells with particular traits to improve the efficacy of T cell therapies., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Elimination of self-reactive T cells in the thymus is critical to establish T-cell tolerance. A growing body of evidence suggests a role for thymic B cells in the elimination of self-reactive thymocytes. To specifically address the role of thymic B cells in central tolerance, we investigated the phenotype of thymic B cells in various mouse strains, including non-obese diabetic (NOD) mice, a model of autoimmune diabetes. We noted that isotype switching of NOD thymic B cells is reduced as compared to other, autoimmune-resistant, mouse strains. To determine the impact of B cell isotype switching on thymocyte selection and tolerance, we generated NOD.AID -/- mice. Diabetes incidence was enhanced in these mice. Moreover, we observed reduced clonal deletion and a resulting increase in self-reactive CD4 + T cells in NOD.AID -/- mice relative to NOD controls. Together, this study reveals that AID expression in thymic B cells contributes to T-cell tolerance., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

T cells comprise a functionally heterogeneous cell population that has important roles in the immune system. While T cells are broadly considered to be a component of the antigen-specific adaptive immune response, certain T-cell subsets display innate-like effector characteristics whereas others perform immunosuppressive functions. These functionally diverse T-cell populations preferentially arise at different stages of ontogeny and are tailored to the immunological priorities of the organism over time. Many differences in early life versus adult T-cell phenotypes can be attributed to the cell-intrinsic properties of the distinct progenitors that seed the thymus throughout development. It is becoming clear that Lin28, an evolutionarily conserved, heterochronic RNA-binding protein that is differentially expressed among early life and adult hematopoietic progenitor cells, plays a substantial role in influencing early T-cell development and function. Here, we discuss the mechanisms by which Lin28 shapes the T-cell landscape to protect the developing fetus and newborn. Manipulation of the Lin28 gene regulatory network is being considered as one means of improving hematopoietic stem cell transplant outcomes; as such, understanding the impact of Lin28 on T-cell function is of clinical relevance., (© 2021 Federation of European Biochemical Societies.)

Negative selection of developing T cells plays a significant role in T-cell tolerance to self-antigen. This process relies on thymic antigen-presenting cells which express both self-antigens and cosignaling molecules. Inducible T-cell costimulator (ICOS) belongs to the CD28 family of cosignaling molecules and binds to ICOS ligand (ICOSL). The ICOS signaling pathway plays important roles in shaping the immune response to infections, but its role in central tolerance is less well understood. Here we show that ICOSL is expressed by subsets of thymic dendritic cells and medullary thymic epithelial cells as well as thymic B cells. ICOS expression is upregulated as T cells mature in the thymus and correlates with T-cell receptor signal strength during thymic selection. We also provide evidence of a role for ICOS signaling in mediating negative selection. Our findings suggest that ICOS may fine-tune T-cell receptor signals during thymic selection contributing to the generation of a tolerant T-cell population., (© 2021 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

CD4 + T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4 + T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4 + T cells impact follicular helper T (T FH ) cell versus non-T FH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4 + T cell chromatin landscapes that ultimately shape their effector potential., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Central tolerance aims to limit the production of T lymphocytes bearing TCR with high affinity for self-peptide presented by MHC molecules. The accumulation of thymocytes with such receptors is limited by negative selection or by diversion into alternative differentiation, including T regulatory cell commitment. A role for the orphan nuclear receptor NR4A3 in negative selection has been suggested, but its function in this process has never been investigated. We find that Nr4a3 transcription is upregulated in postselection double-positive thymocytes, particularly those that have received a strong selecting signal and are destined for negative selection. Indeed, we found an accumulation of cells bearing a negative selection phenotype in NR4A3-deficient mice as compared with wild-type controls, suggesting that Nr4a3 transcriptional induction is necessary to limit accumulation of self-reactive thymocytes. This is consistent with a decrease of cleaved caspase-3 + -signaled thymocytes and more T regulatory and CD4 + Foxp3 - HELIOS + cells in the NR4A3-deficient thymus. We further tested the role for NR4A3 in negative selection by reconstituting transgenic mice expressing the OVA Ag under the control of the insulin promoter with bone marrow cells from OT-I Nr4a3 +/+ or OT-I Nr4a3 -/- mice. Accumulation of autoreactive CD8 thymocytes and autoimmune diabetes developed only in the absence of NR4A3. Overall, our results demonstrate an important role for NR4A3 in T cell development., (Copyright © 2021 by The American Association of Immunologists, Inc.)

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo . This is associated with p16 INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16 INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16 INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy., 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 © 2021 Janelle, Neault, Lebel, De Sousa, Boulet, Durrieu, Carli, Muzac, Lemieux, Labrecque, Melichar, Mallette and Delisle.)

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., (© 2021 Australian and New Zealand Society for Immunology, Inc.)

The ability of T cells to identify foreign antigens and mount an efficient immune response while limiting activation upon recognition of self and self-associated peptides is critical. Multiple tolerance mechanisms work in concert to prevent the generation and activation of self-reactive T cells. T cell tolerance is tightly regulated, as defects in these processes can lead to devastating disease; a wide variety of autoimmune diseases and, more recently, adverse immune-related events associated with checkpoint blockade immunotherapy have been linked to a breakdown in T cell tolerance. The quantity and quality of antigen receptor signaling depend on a variety of parameters that include T cell receptor affinity and avidity for peptide. Autoreactive T cell fate choices (e.g., deletion, anergy, regulatory T cell development) are highly dependent on the strength of T cell receptor interactions with self-peptide. However, less is known about how differences in the strength of T cell receptor signaling during differentiation influences the 'function' and persistence of anergic and regulatory T cell populations. Here, we review the literature on this subject and discuss the clinical implications of how T cell receptor signal strength influences the 'quality' of anergic and regulatory T cell populations.

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4 + T cells. Further, we describe a relationship between CD5 levels on naïve human CD4 + T cells and binding affinity to foreign peptide, in addition to a predominance of CD5 hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5 lo and CD5 hi naïve human CD4 + T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4 + T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Medullary thymic epithelial cells (mTEC) contribute to the development of T cell tolerance by expressing and presenting tissue-restricted antigens (TRA), so that developing T cells can assess the self-reactivity of their antigen receptors prior to leaving the thymus. mTEC are a heterogeneous population of cells that differentially express TRA. Whether mTEC subsets induce distinct autoreactive T cell fates remains unclear. Here, we establish bacterial artificial chromosome (BAC)-transgenic mouse lines with biased mTEC lo or mTEC hi expression of model antigens. The transgenic lines support negative selection of antigen-specific thymocytes depending on antigen dose. However, model antigen expression predominantly by mTEC lo supports TCRαβ + CD8αα intraepithelial lymphocyte development; meanwhile, mTEC hi -restricted expression preferentially induces T reg differentiation of antigen-specific cells in these models to impact control of infectious agents and tumor growth. In summary, our data suggest that mTEC subsets may have a function in directing distinct mechanisms of T cell tolerance.

It is becoming increasingly clear that unconventional T cell subsets, such as NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells, each play distinct roles in the immune response. Subsets of these cell types can lack both CD4 and CD8 coreceptor expression. Beyond these known subsets, we identify CD4 - CD8 - TCRαβ + , double-negative (DN) T cells, in mouse secondary lymphoid organs. DN T cells are a unique unconventional thymic-derived T cell subset. In contrast to CD5 high DN thymocytes that preferentially yield TCRαβ + CD8αα intestinal lymphocytes, we find that mature CD5 low DN thymocytes are precursors to peripheral DN T cells. Using reporter mouse strains, we show that DN T cells transit through the immature CD4 + CD8 + (double-positive) thymocyte stage. Moreover, we provide evidence that DN T cells can differentiate in MHC-deficient mice. Our study demonstrates that MHC-independent thymic selection can yield DN T cells that are distinct from NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells., (Copyright © 2020 by The American Association of Immunologists, Inc.)

T cell development is a dynamic process accompanied by extensive thymocyte migration, cellular interactions, and T cell receptor (TCR) signaling. In particular, thymic selection processes that ensure a functional, self-tolerant repertoire require TCR interactions with self-peptide presented by major histocompatibility complex molecules expressed by specialized thymic antigen-presenting cells. The quantity and quality of these TCR signals influence T cell fate. Two-photon microscopy, which enables live imaging of cells in intact tissue, has emerged as a powerful tool to gain insights into thymocyte migration and TCR signaling during T cell development in situ. Here we describe the generation of non-irradiated, low-density chimeric mice by neonatal injection of adult bone marrow engineered to express fluorescent TCR signaling reporters for imaging by two-photon microscopy. We also describe how the thymic lobes from chimeric mice are prepared for live imaging of thymocyte behavior and TCR signaling events. While we focus on imaging TCR signals associated with T cell development in the thymus, these techniques can also be adapted to study TCR signaling in mature T cells in peripheral lymphoid organs.

Individual CD4 + T cells can become one of a number of helper (Th) lineages with distinct effector functions. However, whether biases in Th potential exist prior to antigen encounter is unknown. Studies have identified cell-intrinsic functional heterogeneity among naïve T cells that can be parsed based on the strength of T-cell receptor (TCR) interactions with self-peptide. Here, using CD5 levels as a surrogate for the strength of these basal TCR signals, we sought to identify pre-existing effector biases in the CD4 + T-cell lineage. We show that ex vivo-activated CD5 lo CD4 + T cells produce greater amounts of the Th1 cytokine interferon-gamma (IFNγ) than their CD5 hi counterparts. In addition, a greater percentage of CD5 lo effector CD4 + T cells produce IFNγ in both polyclonal and monoclonal CD4 + T-cell populations after antigen challenge in vivo. These results suggest that differential IFNγ production potential exists among CD4 + T cells prior to activation and independent of TCR affinity for foreign antigen., (© 2019 Australian and New Zealand Society for Immunology Inc.)

T cell development depends on sequential interactions of thymocytes with cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells. PSMB11 is a catalytic proteasomal subunit present exclusively in cTECs. Because proteasomes regulate transcriptional activity, we asked whether PSMB11 might affect gene expression in cTECs. We report that PSMB11 regulates the expression of 850 cTEC genes that modulate lymphostromal interactions primarily via the WNT signaling pathway. cTECs from Psmb11 -/- mice 1) acquire features of medullary thymic epithelial cells and 2) retain CD8 thymocytes in the thymic cortex, thereby impairing phase 2 of positive selection, 3) perturbing CD8 T cell development, and 4) causing dramatic oxidative stress leading to apoptosis of CD8 thymocytes. Deletion of Psmb11 also causes major oxidative stress in CD4 thymocytes. However, CD4 thymocytes do not undergo apoptosis because, unlike CD8 thymocytes, they upregulate expression of chaperones and inhibitors of apoptosis. We conclude that PSMB11 has pervasive effects on both CD4 and CD8 thymocytes via regulation of gene expression in cTECs., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Lineage-committed αβ and γδ T cells are thought to originate from common intrathymic multipotent progenitors following instructive T cell receptor (TCR) signals. A subset of lymph node and mucosal Vγ2 + γδ T cells is programmed intrathymically to produce IL-17 (Tγδ17 cells), however the role of the γδTCR in development of these cells remains controversial. Here we generated reporter mice for the Tγδ17 lineage-defining transcription factor SOX13 and identified fetal-origin, intrathymic Sox13 + progenitors. In organ culture developmental assays, Tγδ17 cells derived primarily from Sox13 + progenitors, and not from other known lymphoid progenitors. Single cell transcriptome assays of the progenitors found in TCR-deficient mice demonstrated that Tγδ17 lineage programming was independent of γδTCR. Instead, generation of the lineage committed progenitors and Tγδ17 cells was controlled by TCF1 and SOX13. Thus, T lymphocyte lineage fate can be prewired cell-intrinsically and is not necessarily specified by clonal antigen receptor signals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neonatal and adult T cells differ in their effector functions. Although it is known that cell-intrinsic differences in mature T cells contribute to this phenomenon, the factors involved remain unclear. Given emerging evidence that the binding strength of a TCR for self-peptide presented by MHC (self-pMHC) impacts T cell function, we sought to determine whether altered thymic selection influences the self-reactivity of the TCR repertoire during ontogeny. We found that conventional and regulatory T cell subsets in the thymus of neonates and young mice expressed higher levels of cell surface CD5, a surrogate marker for TCR avidity for self-pMHC, as compared with their adult counterparts, and this difference in self-reactivity was independent of the germline bias of the neonatal TCR repertoire. The increased binding strength of the TCR repertoire for self-pMHC in neonates was not solely due to reported defects in clonal deletion. Rather, our data suggest that thymic selection is altered in young mice such that thymocytes bearing TCRs with low affinity for self-peptide are not efficiently selected into the neonatal repertoire, and stronger TCR signals accompany both conventional and regulatory T cell selection. Importantly, the distinct levels of T cell self-reactivity reflect physiologically relevant differences based on the preferential expansion of T cells from young mice to fill a lymphopenic environment. Therefore, differences in thymic selection in young versus adult mice skew the TCR repertoire, and the relatively higher self-reactivity of the T cell pool may contribute to the distinct immune responses observed in neonates., (Copyright © 2017 by The American Association of Immunologists, Inc.)

It is well established that Hoxa genes play a critical role in the proliferative capacity of adult hematopoietic stem and progenitor cells, but the importance of Hoxa genes in later stages of hematopoietic differentiation is less clear. Previously, we observed that B-cell numbers were reduced in adult mice in which Hoxa deletion was induced. In the current study, we investigated the requirement of Hoxa genes at different stages of B-cell development. Using an MxCre-inducible conditional knock-out mouse model, we showed that immature B-cell fractions and early lymphoid progenitors were markedly reduced in the absence of Hoxa, whereas mature B-cell populations were found at levels comparable to controls. Deletion of Hoxa genes in B-cell lineage-committed cells, however, did not affect B-cell development despite sustained Hoxa gene expression in immature CD19 + B-cell subsets. Together, these results suggest that the effect of Hoxa on B-cell development originates in early lymphoid progenitor cells., (Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Vaccination with antigen-pulsed CD40-activated B (CD40-B) cells can efficiently lead to the in vivo differentiation of naive CD8 + T cells into fully functional effectors. In contrast to bone marrow-derived dendritic cell (BMDC) vaccination, CD40-B cell priming does not allow for memory CD8 + T-cell generation but the reason for this deficiency is unknown. Here, we show that compared to BMDCs, murine CD40-B cells induce lower expression of several genes regulated by T-cell receptor signaling, costimulation, and inflammation (signals 1-3) in mouse T cells. The reduced provision of signals 1 and 2 by CD40-B cells can be explained by a reduction in the quality and duration of the interactions with naive CD8 + T cells as compared to BMDCs. Furthermore, CD40-B cells produce less inflammatory mediators, such as IL-12 and type I interferon, and increasing inflammation by coadministration of polyriboinosinic-polyribocytidylic acid with CD40-B-cell immunization allowed for the generation of long-lived and functional CD8 + memory T cells. In conclusion, it is possible to manipulate CD40-B-cell vaccination to promote the formation of long-lived functional CD8 + memory T cells, a key step before translating the use of CD40-B cells for therapeutic vaccination., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

Recently, tissue slices have been adapted to study both mouse and human T cell development. Thymic slices combine and complement the strengths of existing organotypic culture systems to study thymocyte differentiation. Specifically, the thymic slice system allows for high throughput experiments and the ability to introduce homogenous developmental intermediate populations into an environment with a well-established cortex and medulla. These qualities make thymic slices a highly versatile and technically accessible model to study thymocyte development. Here we describe methods to prepare, embed, and slice thymic lobes to study T cell development in situ.

Humanized mice represent an important model to study the development and function of the human immune system. While it is known that mouse thymic stromal cells can support human T-cell development, the extent of interspecies cross-talk and the degree to which these systems recapitulate normal human T-cell development remain unclear. To address these questions, we compared conventional and non-conventional T-cell development in a neonatal chimera humanized mouse model with that seen in human fetal and neonatal thymus samples, and also examined the impact of a human HLA-A2 transgene expressed by the mouse stroma. Given that dynamic migration and cell-cell interactions are essential for T-cell differentiation, we also studied the intrathymic migration pattern of human thymocytes developing in a murine thymic environment. We found that both conventional T-cell development and intra-thymic migration patterns in humanized mice closely resemble human thymopoiesis. Additionally, we show that developing human thymocytes engage in short, serial interactions with other human hematopoietic-derived cells. However, non-conventional T-cell differentiation in humanized mice differed from both fetal and neonatal human thymopoiesis, including a marked deficiency of Foxp3(+) T-cell development. These data suggest that although the murine thymic microenvironment can support a number of aspects of human T-cell development, important differences remain, and additional human-specific factors may be required.

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell-cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte-thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The classic anti-viral cytokine interferon (IFN)-β can be induced during parasitic infection, but relatively little is know about the cell types and signaling pathways involved. Here we show that inflammatory monocytes (IMs), but not neutrophils, produce IFN-β in response to T. gondii infection. This difference correlated with the mode of parasite entry into host cells, with phagocytic uptake predominating in IMs and active invasion predominating in neutrophils. We also show that expression of IFN-β requires phagocytic uptake of the parasite by IMs, and signaling through Toll-like receptors (TLRs) and MyD88. Finally, we show that IMs are major producers of IFN-β in mesenteric lymph nodes following in vivo oral infection of mice, and mice lacking the receptor for type I IFN-1 show higher parasite loads and reduced survival. Our data reveal a TLR and internalization-dependent pathway in IMs for IFN-β induction to a non-viral pathogen.

Specialized microenvironments within the thymus are comprised of unique cell types with distinct roles in directing the development of a diverse, functional, and self-tolerant T cell repertoire. As they differentiate, thymocytes transit through a number of developmental intermediates that are associated with unique localization and migration patterns. For example, during one particular developmental transition, immature thymocytes more than double in speed as they become mature T cells that are among the fastest cells in the body. This transition is associated with dramatic changes in the expression of chemokine receptors and their antagonists, cell adhesion molecules, and cytoskeletal components to direct the maturing thymocyte population from the cortex to medulla. Here we discuss the dynamic changes in behavior that occur throughout thymocyte development, and provide an overview of the cell-intrinsic and extrinsic mechanisms that regulate human thymocyte migration.

The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap70 catalytic activity in a model of synchronized thymic selection, we showed that CD4(+)CD8(+) thymocytes integrate multiple, transient, Zap70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas 1 h of signaling was sufficient for negative selection. Titration of Zap70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, which revealed heterogeneity, even among CD4(+)CD8(+) thymocytes expressing identical TCRs undergoing positive selection.

Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3-4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca(2+)]i) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca(2+)]i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.

The recognition by the T cell receptor (TCR) of self-peptides presented by the major histocompatibility complex (MHC) on antigen-presenting cells, such as dendritic cells and thymic epithelial cells, controls T cell fate in the thymus, with weak TCR signals inducing survival (positive selection) and stronger signals inducing death (negative selection). In vitro studies indicate that peptide ligands that induce positive selection stimulate a low, but sustained, pattern of TCR signaling; however, the temporal pattern of TCR signaling in MHC class I-restricted thymocytes (thymocytes that are presented with peptides by MHC class I) in the thymus, under conditions that support positive selection, is unknown. We addressed this question by examining intracellular Ca(2+) dynamics and migratory changes in thymocytes undergoing positive and negative selection in thymic slices. Brief, serial signaling events that were separated by migratory periods and low cytosolic Ca(2+) concentrations correlated with the positive selection of MHC class I-restricted thymocytes, whereas sustained Ca(2+) signaling and the arrest of thymocytes were associated with negative selection. Low-avidity peptides and the presentation of peptides by cortical thymic epithelial cells, rather than dendritic cells, failed to induce strong migratory arrest of thymocytes, which led to transient TCR signaling. Thus, we provide a comparison of positive and negative selection signals in situ and suggest that the absence of strong stop signals distinguishes between positive and negative selection.

The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.

Two-photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two-photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal of immunologists. In this unit, we provide several protocols for preparation of various organs for imaging by two-photon microscopy that are intended to introduce the new user to some basic aspects of this method.

The movement of proteins within cells can provide dynamic indications of cell signaling and cell polarity, but methods are needed to track and quantify subcellular protein movement within tissue environments. Here we present a semiautomated approach to quantify subcellular protein location for hundreds of migrating cells within intact living tissue using retrovirally expressed fluorescent fusion proteins and time-lapse two-photon microscopy of intact thymic lobes. We have validated the method using GFP-PKCζ, a marker for cell polarity, and LAT-GFP, a marker for T-cell receptor signaling, and have related the asymmetric distribution of these proteins to the direction and speed of cell migration. These approaches could be readily adapted to other fluorescent fusion proteins, tissues and biological questions.

alphabeta and gammadelta T cells originate from a common, multipotential precursor population in the thymus, but the molecular mechanisms regulating this lineage-fate decision are unknown. We have identified Sox13 as a gammadelta-specific gene in the immune system. Using Sox13 transgenic mice, we showed that this transcription factor promotes gammadelta T cell development while opposing alphabeta T cell differentiation. Conversely, mice deficient in Sox13 expression exhibited impaired development of gammadelta T cells but not alphabeta T cells. One mechanism of SOX13 function is the inhibition of signaling by the developmentally important Wnt/T cell factor (TCF) pathway. Our data thus reveal a dominant pathway regulating the developmental fate of these two lineages of T lymphocytes.