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Summary The immune system is composed of a variety of different T-cell lineages distributed through both secondary lymphoid tissue and non-lymphoid tissue. The intestinal epithelium is a critical barrier surface that contains numerous intraepithelial lymphocytes that aid in maintaining homeostasis at that barrier. This review focuses on T-cell receptor αβ (TCRαβ) CD8αα intraepithelial lymphocytes, and how recent advances in the field clarify how this unique T-cell subset is selected, matures, and functions in the intestines. We consider how the available evidence reveals a story of ontogeny starting from agonist selection of T cells in the thymus and finishing through the specific signaling environment of the intestinal epithelium. We conclude with how this story raises further key questions about the development of different ontogenic waves of TCRαβ CD8αα IEL and their importance for intestinal epithelial homeostasis.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8+ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in “hot tumors.”

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted antigen (TRA) did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. PD-1 was induced in the thymus and was required to establish cell-intrinsic tolerance to TRA in CD8+thymocytes independently of clonal deletion. PD-1 signaling in developing thymocytes was sufficient for tolerance induction, however in some cases the absence of PD-L1-mediated signaling in the periphery resulted in broken tolerance at late time points. Finally, we showed that chronic exposure to high affinity antigen supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.Graphical Abstract

BACKGROUND Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.

KEY POINTS: 25-Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine. 25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin. 25OHD inhibits the potentiating effects of PKC-mediated TRPV1 activity. 25OHD reduces T-cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity. These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well-documented effects of vitamin D upon the nuclear vitamin D receptor. The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes. ABSTRACT: The capsaicin receptor TRPV1 plays an important role in nociception, inflammation and immunity and its activity is regulated by exogenous and endogenous lipophilic ligands. As vitamin D is lipophilic and involved in similar biological processes as TRPV1, we hypothesized that it directly regulates TRPV1 activity and function. Our calcium imaging and electrophysiological data demonstrate that vitamin D (25-hydroxyvitamin D (25OHD) and 1,25-hydroxyvitamin D (1,25OHD)) can weakly activate TRPV1 at physiologically relevant concentrations (100 nM). Furthermore, both 25OHD and 1,25OHD can inhibit capsaicin-induced TRPV1 activity (IC50 = 34.3 ± 0.2 and 11.5 ± 0.9 nM, respectively), but not pH-induced TRPV1 activity, suggesting that vitamin D interacts with TRPV1 in the same region as the TRPV1 agonist capsaicin. This hypothesis is supported by our in silico TRPV1 structural modelling studies, which place 25OHD in the same binding region as capsaicin. 25OHD also attenuates PKC-dependent TRPV1 potentiation via interactions with a known PKC phospho-acceptor residue in TRPV1. To provide evidence for a physiological role for the interaction of vitamin D with TRPV1, we employed two different cellular models known to express TRPV1: mouse CD4+ T-cells and trigeminal neurons. Our results indicate that 25OHD reduces TRPV1-induced cytokine release from T-cells and capsaicin-induced calcium activity in trigeminal neurons. In summary, we provide evidence that vitamin D is a novel endogenous regulator of TRPV1 channel activity that may play an important physiological role in addition to its known effects through the canonical nuclear vitamin D receptor pathway. ispartof: JOURNAL OF PHYSIOLOGY-LONDON vol:598 issue:19 pages:4321-4338 ispartof: location:England status: published

A layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium. The MUC2 mucin is the primary constituent of intestinal mucus and plays critical protective roles against luminal microbes and other noxious agents. In this study, we investigated whether MUC2 helps maintain CD8 T cell tolerance toward intestinal luminal Ags by gavaging wild-type and Muc2−/− mice with a model Ag and monitoring immune responses posttreatment. We report that orally delivered OVA rapidly disseminates through the blood of Muc2−/− (but not control) mice and causes immune activation of Ag-specific CD8 T cells at both local and distal sites. Further, the administration of oral OVA to Muc2−/− mice led to its presentation by thymic dendritic cells and the deletion of Ag-specific thymocytes. Collectively, our findings suggest that intestinal mucus helps limit the shaping of the TCR repertoire of developing thymocytes by intestinal luminal Ags.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8+ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in “hot tumors.”

Germinal centers (GCs) are essential for antibody affinity maturation. GC B cells have a unique repertoire of cell surface glycans compared with naive B cells, yet functional roles for changes in glycosylation in the GC have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a downregulation in ligands for CD22, an inhibitory co-receptor of the B cell receptor. To test a functional role for downregulation of CD22 ligands in the GC, we generate a mouse model that maintains CD22 ligands on GC B cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22 dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC.

Several unique waves of γδ T cells are generated solely in the fetal/neonatal thymus, whereas additional γδ T cell subsets are generated in adults. One intriguing feature of γδ T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus; however, it is less clear whether this paradigm holds true in adult animals. In this study, we investigated the relationship between maturation and thymic export of adult-derived γδ thymocytes in mice. In the Rag2pGFP model, immature (CD24+) γδ thymocytes expressed high levels of GFP whereas only a minority of mature (CD24−) γδ thymocytes were GFP+. Similarly, most peripheral GFP+ γδ T cells were immature. Analysis of γδ recent thymic emigrants (RTEs) indicated that most γδ T cell RTEs were CD24+ and GFP+, and adoptive transfer experiments demonstrated that immature γδ thymocytes can mature outside the thymus. Mature γδ T cells largely did not recirculate to the thymus from the periphery; rather, a population of mature γδ thymocytes that produced IFN-γ or IL-17 remained resident in the thymus for at least 60 d. These data support the existence of two populations of γδ T cell RTEs in adult mice: a majority subset that is immature and matures in the periphery after thymic emigration, and a minority subset that completes maturation within the thymus prior to emigration. Additionally, we identified a heterogeneous population of resident γδ thymocytes of unknown functional importance. Collectively, these data shed light on the generation of the γδ T cell compartment in adult mice.

The thymus is the site of both {beta} and {gamma}{delta}-T cell development. After several unique waves of {gamma}{delta}-T cells are generated in, and exported from, the fetal/neonatal thymus, the adult thymus continues to produce a stream of {gamma}{delta}-T cells throughout life. One intriguing feature of {gamma}{delta} T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus, however, it is less clear whether this paradigm holds true in adult animals. To investigate the relationship between maturation and time since V(D)J recombination in adult-derived {gamma}{delta}-thymocytes, we used the Rag2pGFP model. Immature (CD24+) {gamma}{delta}-thymocytes expressed high levels of GFP while only a small minority of mature (CD24-) {gamma}{delta}-thymocytes were GFP+. Similarly, most GFP+ {gamma}{delta}-splenocytes were immature, while some were mature. Analysis of {gamma}{delta}-recent thymic emigrants (RTEs) indicated that most {gamma}{delta}-T cell RTEs were CD24+ and GFP+ and adoptive transfer experiments showed that immature {gamma}{delta}-thymocytes could be maintained in the periphery for at least 3 days over which time they matured. With respect to the mature {gamma}{delta}-thymocytes that were GFP-, parabiosis experiments demonstrated that mature {gamma}{delta}-T cells did not recirculate from the periphery. Instead, a population of mature {gamma}{delta}-thymocytes remained resident in the thymus for at least 60 days while mature {gamma}{delta}-thymocytes derived solely from adult hematopoiesis were mostly lost from the thymus within 60 days. Collectively, these data demonstrate two streams of actively developing {gamma}{delta}-T cells in adult mice: an immature subset that quickly leaves the thymus and matures in the periphery, and one that completes maturation within the thymus over a longer period of time. Furthermore, there is a fetal-derived and heterogeneous population of resident {gamma}{delta}-thymocytes of unknown functional importance.

Germinal centres (GC) are sites of B-cell expansion and selection, which are essential for antibody affinity maturation. Compared to naive B-cells, GC B-cells have notable changes in their cell surface glycans. While these changes are routinely used to identify the GC, functional roles for these changes have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a reduction in the glycan ligands for CD22, which is an inhibitory co-receptor of the B-cell receptor (BCR). To test a functional role for downregulated CD22 ligands in the GC, we generated a mouse model that maintains CD22 ligands on GC B-cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B-cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B-cells, resulting in reduced memory B-cell and plasma cell output as well as delayed affinity maturation of antibodies. The defect we observe in this model is dependent on CD22, highlighting that coordinated downregulation of CD22 ligands on B cells plays a critical function in the GC. Collectively, our study uncovers a crucial role for glycan remodeling and CD22 in B-cell fitness in the GC.

Peripheral nerve injury frequently evokes chronic neuropathic pain. This is initiated by a transient inflammatory response that leads to persistent excitation of dorsal root ganglion (DRG) neurons by inflammatory cytokines such as interleukin 1β(IL-1β). In non-neuronal cells such as lymphocytes, interleukin 1 exerts actions at attomolar (aM; 10-18 M) concentrations. We now report that DRG neurons in defined-medium, neuron-enriched culture display increased excitability following 5-6 d exposure of 1aM IL-1β. This response is mediated in part by type 1 interleukin receptors and involves decreased function of putative KCa1.1 channels. This finding provides new insights into the neuroimmune interactions responsible for neuropathic pain.

Negative selection against highly self-reactive thymocytes is critical for preventing autoimmunity. Thymocyte deletion, anergy induction, and agonist selection are all forms of negative selection that can occur following a high-affinity TCR signal. Of Bim and Nur77, two TCR-induced proteins with proapoptotic function, Bim has been shown to be important for clonal deletion in several model systems, whereas Nur77 was often dispensable. However, Nur77 has been reported to influence other aspects of T cell development by mechanisms that may not be related to its proapoptotic function. In this study, we examined the role of Nur77 during thymocyte development in the presence and absence of Bim to separate apoptotic from nonapoptotic functions of Nur77. Polyclonal Bim−/− and Bim−/−Nur77−/− mice exhibited comparable accumulation of high-affinity signaled CD4+CD8+ double-positive thymocytes and CD8+ and CD4+ single-positive thymocytes. However, combined Bim and Nur77 deficiency increased the frequency of thymic Foxp3+ T regulatory cells and Foxp3−FR4hiCD73hi anergic phenotype CD4+ T cells compared with Bim−/− mice, suggesting that Nur77 expression impairs the development of nonconventional tolerance-inducing cell fates. Using the OT-I RIP-mOVA model, we found that Nur77 deficiency did not substantially impact clonal deletion nor did it exacerbate the defect in clonal deletion in the absence of Bim. However, additional loss of Nur77 in the absence of Bim led to diabetes induction, suggesting that Nur77 promotes tolerance in this context. Together, these data reveal novel nondeletional roles for Nur77 that differ between T cell subsets and have implications for self-tolerance.

Golec et al. show that RasGRP1, a critical Ras activator in thymocytes, is required for TCRαβ+CD8αα IEL development by regulating the survival of a heterogeneous population of thymic progenitors that receive a strong TCR signal. Therefore, RasGRP1 is necessary for thymic selection events stemming from strong or weak TCR signals., Strong T cell receptor (TCR) signaling largely induces cell death during thymocyte development, whereas weak TCR signals induce positive selection. However, some T cell lineages require strong TCR signals for differentiation through a process termed agonist selection. The signaling relationships that underlie these three fates are unknown. RasGRP1 is a Ras activator required to transmit weak TCR signals leading to positive selection. Here, we report that, despite being dispensable for thymocyte clonal deletion, RasGRP1 is critical for agonist selection of TCRαβ+CD8αα intraepithelial lymphocyte (IEL) progenitors (IELps), even though both outcomes require strong TCR signaling. Bim deficiency rescued IELp development in RasGRP1−/− mice, suggesting that RasGRP1 functions to promote survival during IELp generation. Additionally, expression of CD122 and the adhesion molecules α4β7 and CD103 define distinct IELp subsets with differing abilities to generate TCRαβ+CD8αα IEL in vivo. These findings demonstrate that RasGRP1-dependent signaling underpins thymic selection processes induced by both weak and strong TCR signals and is differentially required for fate decisions derived from a strong TCR stimulus.

The autoimmune disease of type 1 diabetes (T1D) results in the immune destruction of β-cells. Recent studies suggest supplementation of vitamin D along can significantly improve patients’ β-cell function and glycemic control possibly by dampening naïve T-cell activation. However, the underlying cellular mechanism for this effect has not been elucidated completely, especially as naïve T-cells possess absent or very low VDR expression. Therefore, the effects of Vitamin D on naïve T-cells may involve a VDR-independent pathway. Interestingly, TRPV1 channel activation is necessary for naïve T-cell activation. Our initial calcium imaging and electrophysiological data show that Vitamin D (25OHD) can partially activate TRPV1. 25OHD can inhibit capsaicin induced TRPV1 activity. We propose that vitamin D is a partial agonist of TRPV1, through direct binding to TRPV1 and modulating naïve T-cell activation. Furthermore, our flow cytometry studies confirm both 25OHD and 1,25OHD significantly reduce TNFα/INFγ and IL2/IL4 production of mouse CD4+ naïve T-cells after 24 hours activation. Our results support the concept that naïve T-cell activation can be dampened by vitamin D in a VDR-independent manner, via an as yet undescribed mechanism involving the modulation of TRPV1 activity. Moreover, in silico and point-muatgenic experiments indicate 25-OHD binds to the same region as known TRPV1 agonist and antagonists. These novel findings provide evidence of an additional pathway for the action of Vitamin D action and advance our knowledge of the underlying cellular mechanism by which vitamin D may beneficially regulate naive T-cell activation in autoimmune disease such as T1D. Disclosure W. Long: Research Support; Self; JDRF. M. Fatehi: None. S. Soni: None. R. Panigrahi: None. R.G. Kelly: None. K. Philippaert: None. A.J. Barr: None. M. Held: None. Y. Yu: None. S.A. Campbell: None. K. Ondrusova: None. T. Baldwin: None. J. Lemieux: None. P.E. Light: None. Funding JDRF

Negative selection, primarily mediated through clonal deletion of self-reactive thymocytes, is critical for establishing self-tolerance and preventing autoimmunity. Recent studies suggest that the molecular mechanisms of negative selection differ depending on the thymic compartment and developmental stage at which thymocytes are deleted. Using the physiological HYcd4 TCR transgenic model of negative selection against ubiquitous self-antigen, we previously found that one of the principal mediators implicated in clonal deletion, Bim, is required for caspase-3 activation but is ultimately dispensable for negative selection. On the basis of these data, we hypothesized that Nur77, another molecule thought to be a key mediator of clonal deletion, could be responsible for Bim-independent deletion. Despite comparable Nur77 induction in thymocytes during negative selection, Bim deficiency resulted in an accumulation of high-affinity–signaled thymocytes as well as impairment in caspase-mediated and caspase-independent cell death. Although these data suggested that Bim may be required for Nur77-mediated cell death, we found that transgenic Nur77 expression was sufficient to induce apoptosis independently of Bim. However, transgenic Nur77-induced apoptosis was significantly inhibited in the context of TCR signaling, suggesting that endogenous Nur77 could be similarly regulated during negative selection. Although Nur77 deficiency alone did not alter positive or negative selection, combined deficiency in Bim and Nur77 impaired clonal deletion efficiency and significantly increased positive selection efficiency. Collectively, these data shed light on the different roles for Bim and Nur77 during ubiquitous Ag-mediated clonal deletion and highlight potential differences from their reported roles in tissue-restricted Ag-mediated clonal deletion.

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.

How the T cell receptor (TCR) can initiate developmental programs resulting in opposing thymocyte fates (survival versus death) is a central question in immunology. Much work supports the idea that the binding strength (affinity) between TCR and self-peptide major histocompatibility complex (pMHC) plays a pivotal role in this fate decision. In thymocytes, weak binding induces survival and further development (positive selection), whereas no binding or strong binding generally leads to death (death by neglect or negative selection, respectively). However, there is growing evidence that strong interactions between the TCR and self-pMHC lead to the positive selection of specific T cell subsets (agonist selection) rather than negative selection. This article provides insight on how TCR affinity for self-pMHC regulates different thymocyte fates and the signaling mediators that orchestrate thymocyte development.

The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes first encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HYcd4 model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c+ cortical dendritic cells, and elimination of such cells impaired deletion.

T cell development is dependent on the migration of progenitor cells from the bone marrow to the thymus. Upon reaching the thymus, progenitors undergo a complex developmental program that requires inputs from various highly conserved signaling pathways including the Notch and Wnt pathways. To date, Ras signaling has not been implicated in the very earliest stages of T cell differentiation, but members of a family of Ras activators called RasGRPs have been shown to be involved at multiple stages of T cell development. We examined early T cell development in mice lacking RasGRP1, RasGRP3, and RasGRPs 1 and 3. We report that RasGRP1- and RasGRP3-deficient thymi show significantly reduced numbers of early thymic progenitors (ETPs) relative to wild type thymi. Furthermore, RasGRP1/3 double-deficient thymi show significant reductions in ETP numbers compared with either RasGRP1 or RasGRP3 single-deficient thymi, suggesting that both RasGRP1 and RasGRP3 regulate the generation of ETPs. In addition, competitive bone marrow chimera experiments reveal that RasGRP1/3 double-deficient progenitors intrinsically generate ETPs less efficiently than wild type progenitors. Finally, RasGRP1/3-deficient progenitors show impaired migration toward the CCR9 ligand, CCL25, suggesting that RasGRP1 and RasGRP3 may regulate progenitor entry into the thymus through a CCR9-dependent mechanism. These data demonstrate that, in addition to Notch and Wnt, the highly conserved Ras pathway is critical for the earliest stages of T cell development and further highlight the importance of Ras signaling during thymocyte maturation.

In the past few years, there has been a flurry of discoveries and advancements in our understanding of how the thymus prepares T cells to exist at peace in normal healthy tissue: that is, to be self-tolerant. In the thymus, one of the main mechanisms of T-cell central tolerance is clonal deletion, although the selection of regulatory T cells is also important and is gaining enormous interest. In this Review, we discuss the emerging consensus about which models of clonal deletion are most physiological, and we review recent data that define the molecular mechanisms of central tolerance.

During late stages of thymic development, T cells must chose between different fates, dictated by their TCR specificity. Typically, this is thought of as a choice between three alternatives (being positive selection for useful T cells vs negative selection or neglect for harmful or useless T cells). However, there is growing evidence for a fourth alternative, in which T cells are positively selected by agonist ligands, which would normally be expected to induce T cell deletion. In this review, we will discuss where and when agonist selection is induced and whether this should be considered as a novel form of thymic selection or as an alternative differentiation state for Ag-exposed T cells.

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 the TCR for self-peptide presented by MHC (self-pMHC) impacts T cell function, we sought to determine if altered thymic selection thresholds influence 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 to their adult counterparts and that this difference in self-reactivity was independent of their distinct 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 positive and agonist selection. Importantly, the differences in CD5 levels reflect physiologically relevant differences based on the preferential expansion of T cells from young mice to fill a lymphopenic environment. Therefore, differences in the thymic selection threshold in young versus adult mice skews the TCR repertoire, and the relatively higher self-reactivity of the T cell pool may contribute to the distinct immune responses observed in neonates.

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic alpha-chain and an associated beta-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the alpha-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GIIalpha is capable of associating with CD45 in a competitive situation where multiple GIIalpha isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GIIalpha acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation.

The mammalian immune system is a complex network of many cell types and proteins that collectively coordinate a protective response against foreign entities. The immune system can be divided into two broad categories: innate and adaptive immunity. Adaptive immunity, which is primarily mediated by T and B lymphocytes, first arose in jawed vertebrates and has several distinct features from the more ancient innate immune system (Pancer and Cooper 2006). While innate immunity is characterized by non-specific recognition of conserved molecular patterns leading to a rapid effector response, the adaptive immune response is delayed by the required expansion of lymphocytes bearing receptors specific for a particular antigen. After the primary immune response, a small fraction of activated lymphocytes remain as memory cells, which respond to subsequent encounters with the same antigen in a more rapid and robust manner.

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-)CD8(-) 'double negative' (DN) thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+)CD8(+) 'double positive' (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+) DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3(-/-) male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3(-/-)Ang II aortas, additional deletion of Mmp2 in these mice (Timp3(-/-)/Mmp2(-/-)) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3(-/-)/Mmp2(-/-) mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3(-/-) and Timp3(-/-)/Mmp2(-/-) mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders(1-4). T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC(5). Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events(6). For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes(7-9). Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY(cd4) model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice(10). Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY(cd4) mouse model. While negative selection in HY(cd4) mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

Positive and negative selection of thymocytes in the thymus are critical for the development of a mature and self-tolerant T-cell repertoire. The proapoptotic Bcl-2 family member Bim is important for negative selection by inducing apoptosis in thymocytes receiving a strong signal through their antigen receptor. However, in the case of ubiquitous self-antigens (UbA), Bim is not required for the clonal deletion of self-reactive thymocytes, suggesting the existence of nonapoptotic clonal deletion mechanisms. Unlike UbA, clonal deletion to tissue-restricted antigens (TRAs) requires positive selection and CCR7-mediated migration to the medulla. This led us to hypothesize that Bim is required for the latter. To study the role of Bim in clonal deletion to TRA, we constructed bone marrow (BM) chimeras using OT-I Bim-deficient or -sufficient donor bone marrow and recipients that express membrane bound chicken ovalbumin under control of the rat insulin promoter (Rip-mOVA). We found that clonal deletion to TRA was completely abrogated in the absence of Bim and large numbers of mature OT-I CD8 T cells survived in the periphery. Despite the large numbers of autoreactive T cells, the chimeras did not develop diabetes and OT-I Bim-deficient T cells from these chimeras were functionally impaired. Collectively, these data provide unique evidence of a differential, thymocyte-intrinsic, molecular requirement downstream of the T-cell receptor (TCR) for clonal deletion to UbA versus TRA and highlight the profound ability of other tolerance mechanisms to control T-cell autoreactivity in the absence of thymic clonal deletion.

Clonal selection of T cells occurs in the thymus and is responsible for generating a useful and functional repertoire of T cells. Aberrations in clonal selection result in altered T-cell homeostasis in the secondary lymphoid organs ranging from an absence of T cells to an overabundance of autoreactive T cells. The advent of new technologies facilitating the manipulation of the mouse genome has helped refine our understanding of the molecular and genetic pathways involved in clonal selection and has also revealed a high degree of complexity. Herein, we attempt to review recent advances in thymic selection processes, achieved mostly through genetic manipulations.

Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx(-/-)) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx(-/-) mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.

Engagement of the TCR on CD4+CD8+ thymocytes initiates either a program of survival and differentiation (positive selection) or death (clonal deletion), which is dictated in large part by the affinity of the TCR for self-peptide-MHC complexes. Although much is known about the factors involved in positive selection, little is understood about the molecular mechanism leading to clonal deletion. To gain further insight into this process, we used a highly physiological TCR transgenic mouse model to compare gene expression changes under conditions of nonselection, positive selection, and negative selection. We identified 388 genes that were differentially regulated in negative selection compared with either nonselection or positive selection. These regulated genes fall into many functional categories including cell surface and intracellular signal transduction, survival and apoptosis, transcription and translation, and adhesion and migration. Additionally, we have compared our transcriptional profile to profiles of negative selection in other model systems in an effort to identify those genes with a higher probability of being functionally relevant. These included three up-regulated genes, bim, nur77, and ian1, and one down-regulated gene, lip1. Collectively, these data provide a framework for understanding the molecular basis of clonal deletion.

Publisher Summary T lymphocytes play a critical role in adaptive immunity to pathogens. Their ability to function properly in this role, while not causing an immune reaction to host tissues, reflects cellular properties that are established as they develop in the thymus. For this reason, immunologists have long studied the thymus, and many mouse models that facilitate that these analyses exist. This chapter discusses the utility and limitations of transgenic and gene-deficient mouse strains to model thymic processes, specifically negative selection. Clonal deletion is a fundamental process necessary to maintain tolerance to self-antigens. As demonstrated by the occurrence of multi organ autoimmunity in the face of impaired negative selection to tissue-specific antigens in autoimmune regulator-deficient mice or humans, understanding the factors controlling deletion is of great importance. When one is using mice as a model system to study negative selection, it is critical to choose an appropriate model as data concerning the role of a molecule in negative selection from one model are not always the same as those in another model. Given that the medulla is replete with potent antigen-presenting cells, late deletion models appear to be the most physiological. Further work examining the molecular pathways leading to deletion, specifically those involved in delivering the apoptotic signal, is greatly needed at this juncture.

Sequential rearrangement of the T cell receptor for antigen (TCR) beta and alpha chains is a hallmark of thymocyte development. This temporal control is lost in TCR transgenics because the alpha chain is expressed prematurely at the CD4- CD8- double negative (DN) stage. To test the importance of this, we expressed the HY alpha chain at the physiological CD4+ CD8+ double positive (DP) stage. The reduced DP and increased DN cellularity typically seen in TCR transgenics was not observed when the alpha chain was expressed at the appropriate stage. Surprisingly, antigen-driven selection events were also altered. In male mice, thymocyte deletion now occurred at the single positive or medullary stage. In addition, no expansion of CD8 alpha alpha intestinal intraepithelial lymphocytes (IELs) was observed, despite the fact that HY transgenics have been used to model IEL development. Collectively, these data establish the importance of proper timing of TCR expression in thymic development and selection and emphasize the need to use models that most accurately reflect the physiologic process.

CD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis. The remaining pool of CD45 was fully glycosylated and began to arrive at the cell surface at approximately 15 min. The rapidly expressed population of CD45 possessed exclusively endoglycosidase H-sensitive N-linked carbohydrate. Additionally, this rapidly expressed pool of CD45 appeared on the cell surface in a brefeldin A (BFA)-insensitive manner, suggesting that it reached the cell surface independent of the Golgi complex. The remaining CD45 trafficked through the Golgi complex, and transport proceeded via a BFA-sensitive mechanism. These data suggest that CD45 is able to reach the cell surface via two distinct routes. The first is a conventional Golgi-dependent pathway that allows fully processed CD45 to be expressed. The second utilizes an ill defined mechanism that is independent of the Golgi, is BFA-resistant, and allows for the expression of CD45 with immature carbohydrate on the cell surface.

Glucosidase II (GII) stably interacts with the external domain of CD45 in a carbohydrate-dependent manner. We have found that the association occurs in immature cells, but is significantly reduced in mature T cells. Using mannose-binding protein (MBP), in both FACS analysis and pull-down assays, we find that MBP can specifically recognize cell surface CD45 from immature, but not mature T cells. Analysis of thymocytes reveals increased MBP binding and GII association with CD45 in double-positive thymocytes compared with either double-negative or single-positive thymocytes. As well, the same pool of CD45 recognized by MBP can also associate with GII. Initial analysis of the basis of the interaction between CD45 and MBP suggests MBP binds two different glycoforms of CD45 based on the differential competition with glucose. Finally, inhibition of GII activity in cells that do not normally express MBP ligands results in significant increases in cell surface MBP ligands, including CD45. Taken together, these data suggest that the glucose content of the cell surface CD45 changes as thymocytes undergo maturation to mature T cells, and may be regulated by GII interactions. Such changes in the cell surface carbohydrate on CD45 may affect the development of thymocytes, perhaps via binding of CD45 on thymocytes to lectins on stromal cells.

Lymphopenia driven T cell activation is associated with autoimmunity. That lymphopenia does not always lead to autoimmunity suggests that control mechanisms may exist. We assessed the importance of the co-inhibitory receptor programmed death-1 (PD-1) in the control of lymphopenia-driven autoimmunity in newly generated T cells vs. established peripheral T cells and in thymic selection. PD-1 was not required for negative selection in the thymus or for maintenance of self tolerance following transfer of established PD-1−/− peripheral T cells to a lymphopenic host. In contrast, PD-1 was essential for systemic self tolerance in newly generated T cells under lymphopenic conditions, as PD-1−/− recent thymic emigrants (RTE), generated after transfer of PD-1−/− hematopoietic stem cell (HSC) precursors or thymocytes into lymphopenic adult Rag−/− recipients, induced a rapidly lethal multi-organ inflammatory disease. Disease could be blocked by using lymph node deficient recipients, indicating that lymphopenia driven PD-1−/− T cell activation required access to sufficient lymph node stroma. These data suggested that PD-1−/− mice themselves might be substantially protected from autoimmunity because their T cell repertoire is first generated early in life, a period naturally deficient in lymph node stroma. Consistent with this idea, neonatal Rag−/− recipients of PD-1−/− HSC were resistant to disease. Thus, a critical role of PD-1 resides in the control of RTE in lymphopenia. The data suggest that PD-1 and a paucity of lymphoid stroma cooperate to control autoimmunity in newly generated T cells. Clinical therapies for autoimmune disease employing lymphoablation and hematopoietic stem cell transplantation will need to take into account functional polymorphisms in the PD-1 pathway, if the treatment is to ameliorate rather than exacerbate autoimmunity.