Establishment of CD8+ T Cell Thymic Central Tolerance to Tissue-Restricted Antigen Requires PD-1.

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 Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1-deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1-deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag 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.

Destined for the intestine: thymic selection of TCRαß CD8αα intestinal intraepithelial lymphocytes.

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 αß (TCRαß) 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 TCRαß CD8αα IEL and their importance for intestinal epithelial homeostasis.

γδ Thymocyte Maturation and Emigration in Adult Mice.

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.

Coordinated changes in glycosylation regulate the germinal center through CD22.

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.

Cutting Edge: Intestinal Mucus Limits the Clonal Deletion of Developing T Cells Specific for an Oral Antigen.

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.

Deciphering the Immunomodulatory Capacity of Oncolytic Vaccinia Virus to Enhance the Immune Response to Breast Cancer.

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."

Role of the Orphan Nuclear Receptor NR4A Family in T-Cell Biology.

The nuclear orphan receptors NR4A1, NR4A2, and NR4A3 are immediate early genes that are induced by various signals. They act as transcription factors and their activity is not regulated by ligand binding and are thus regulated via their expression levels. Their expression is transiently induced in T cells by triggering of the T cell receptor following antigen recognition during both thymic differentiation and peripheral T cell responses. In this review, we will discuss how NR4A family members impact different aspects of the life of a T cell from thymic differentiation to peripheral response against infections and cancer.

Nur77 Regulates Nondeletional Mechanisms of Tolerance in T Cells.

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.

Thymic progenitors of TCRαß+ CD8αα intestinal intraepithelial lymphocytes require RasGRP1 for development.

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 TCRαß+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 α4ß7 and CD103 define distinct IELp subsets with differing abilities to generate TCRαß+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.

Alterations in the Thymic Selection Threshold Skew the Self-Reactivity of the TCR Repertoire in Neonates.

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.

RasGRP1 and RasGRP3 Are Required for Efficient Generation of Early Thymic Progenitors.

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.

Differential roles for Bim and Nur77 in thymocyte clonal deletion induced by ubiquitous self-antigen.

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 HY(cd4) 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.

RasGRP1, but not RasGRP3, is required for efficient thymic ß-selection and ERK activation downstream of CXCR4.

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 "ß-selection" before maturing into CD4(+)CD8(+) 'double positive' (DP) thymocytes. ß-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 ß-selection. Additionally, it has long been known that ERK is activated during ß-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the ß-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 TCRß chain. As a result of impaired ß-selection, the pool of TCRß(+) 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 ß-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the ß-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Loss of Timp3 gene leads to abdominal aortic aneurysm formation in response to angiotensin II.

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.

Examination of thymic positive and negative selection by flow cytometry.

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 ß 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 αßTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αßTCR 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.

Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens.

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.

Calnexin deficiency leads to dysmyelination.

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.

Bim-mediated apoptosis is not necessary for thymic negative selection to ubiquitous self-antigens.

T cell education in the thymus is critical for establishing a functional, yet self-tolerant, T cell repertoire. Negative selection is a key process in enforcing self-tolerance. There are many questions that surround the mechanism of negative selection, but it is currently held that apoptosis initiated by Bim and/or Nur77 is critical for negative selection. Recent studies, however, have questioned the necessity of Bim in maintaining both central and peripheral T cell tolerance. To reconcile these apparently contradictory findings, we examined the role of Bim in negative selection in the well-characterized, physiological HY(cd4) mouse model. We found that while Bim expression was required for CD4(+)CD8(+) double-positive thymocyte apoptosis, it was not required for negative selection. Furthermore, Bim deficiency did not alter the frequency or affinity of male reactive cells that escape negative selection in an oligoclonal repertoire. Collectively, these studies indicate that negative selection occurs efficiently in the absence of apoptosis and suggest that the current paradigm of negative selection requiring apoptosis be revisited.

Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.

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 HY(cd4) 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.

Transcriptional analysis of clonal deletion in vivo.

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.