CD4 Lineage Commitment Research Articles

Adjusting to self in the thymus: CD4 versus CD8 lineage commitment and regulatory T cell development.

During thymic development, thymocytes adjust their TCR response based on the strength of their reactivity to self-peptide MHC complexes. This tuning process allows thymocytes with a range of self-reactivities to survive positive selection and contribute to a diverse T cell pool. In this review, we will discuss recent advances in our understanding of how thymocytes tune their responsiveness during positive selection, and we present a "sequential selection" model to explain how MHC specificity influences lineage choice. We also discuss recent evidence for cell type diversity in the medulla and discuss how this heterogeneity may contribute to medullary niches for negative selection and regulatory T cell development.

ThPOK is a critical multifaceted regulator of myeloid lineage development.

The transcription factor ThPOK (encoded by Zbtb7b) is well known for its role as a master regulator of CD4 lineage commitment in the thymus. Here, we report an unexpected and critical role of ThPOK as a multifaceted regulator of myeloid lineage commitment, differentiation and maturation. Using reporter and knockout mouse models combined with single-cell RNA-sequencing, progenitor transfer and colony assays, we show that ThPOK controls monocyte-dendritic cell versus granulocyte lineage production during homeostatic differentiation, and serves as a brake for neutrophil maturation in granulocyte lineage-specified cells through transcriptional regulation of lineage-specific transcription factors and RNA via altered messenger RNA splicing to reprogram intron retention.

ETS2 repressor factor (ERF) is involved in T lymphocyte maturation acting as regulator of thymocyte lineage commitment.

Thymocyte differentiation and lineage commitment is regulated by an extensive network of transcription factors and signaling molecules among which Erk plays a central role. However, Erk effectors as well as the molecular mechanisms underlying this network are not well understood. Erf is a ubiquitously expressed transcriptional repressor regulated by Erk-dependent phosphorylation. Here, we investigated the role of Erf in T cell maturation and lineage commitment, using a double-fluorescent Erf-floxed mouse to produce thymus-specific Erf knockouts. We observed significant accumulation of thymocytes in the CD4/CD8 DP stage, followed by a significant reduction in CD4SP cells, a trend for lower CD8SP cell frequency, and an elevated percentage of γδ expressing thymocytes in Erf-deficient mice. Also, an elevated number of CD69+ TCRβ+ cells indicates that thymocytes undergoing positive selection accumulate at this stage. The expression of transcription factors Gata3, ThPOK, and Socs1 that promote CD4+ cell commitment was significantly decreased in Erf-deficient mice. These findings suggest that Erf is involved in T cell maturation, acting as a positive regulator during CD4 and eventually CD8 lineage commitment, while negatively regulates the production of γδ T cells. In addition, Erf-deficient mice displayed decreased percentages of CD4+ and CD8+ splenocytes and elevated levels of IL-4 indicating that Erf may have an additional role in the homeostasis, differentiation, and immunologic response of helper and cytotoxic T cells in the periphery. Overall, our results show, for the first time, Erf's involvement in T cell biology suggesting that Erf acts as a potential regulator during thymocyte maturation and thymocyte lineage commitment, in γδ T cell generation, as well as in Th cell differentiation.

Formaldehyde inhibits development of T lymphocytes in mice

Formaldehyde (FA) is a ubiquitous environmental pollutant and a group 1 carcinogen. CD8+ T cells are specific tumor killers in the adaptive immune system. In this study, 2.0 mg/m3 respiratory FA exposure to male mice was found to selectively reduce mature CD8+ T cells in the spleen and the CD8 T cells in the thymus. In order to monitor the exposure of FA in vitro, a fluorescent FA donor was utilized in the treatment of murine thymocytes. Cytotoxicity induced by FA was found to be not selective at single positive stage. Significant inhibition of the runt-related transcription factor-3, the critical transcriptional factors for CD8 lineage commitment, was found in FA-treated double positive cells. ThPOK, the transcriptional factor for CD4 commitment, was stimulated by FA in double positive cells. Increased expression of special AT-rich sequence-binding protein-1 and decreased expression of B-cell lymphoma/leukemia 11B, which are the upstream regulators of ThPOK and the runt-related transcription factor-3, were also observed in FA-treated double positive cells. These results demonstrate that FA could selectively inhibit the development of CD8+ T cells via the alteration of transcriptional factor expressions of thymocytes at double positive stages, which would potentially interfere with the immune defense against cancers.

Molecular cues dictating positive and negative selection in the thymus

Abstract T cells develop in the thymus where CD4+CD8+ double positive (DP) thymocytes undergo positive and negative selection to generate mature CD4+ and CD8+ single positive (SP) T cells. Positive selection allows for the generation of a diverse repertoire of T cells capable of recognizing peptide:MHC (pMHC) complexes, while negative selection ensures that the mature T cell repertoire is self-tolerant. The cell types involved in thymic selection are well understood and recent work has begun to elucidate temporal and spatial aspects involved in selection. However, the intracellular cues regulating development and CD4 vs. CD8 lineage commitment are incompletely understood. Our work focuses on utilizing a single cell RNA sequencing (scRNA-seq) approach to elucidate the molecular cues guiding thymic selection of CD4+ and CD8+ T cells.

HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment.

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.

T-bet-expression in dendritic cells is essential for T. gondii clearance

Abstract The host’s type I immune response is essential for resistance to diverse groups of intracellular pathogens, including Mycobacterium, Leishmania, Salmonella, and Toxoplasma gondii. Type I immunity is traditionally characterized by the transcription factor T-bet, encoded by Tbx21, which has been considered the master regulator of determining the lineage commitment of CD4+ TH1 cells and their production of the effector molecule IFN-γ. However, recent studies by our and other groups have shown that T-bet expression in CD4+ T cells is dispensable for IFN-γ production, and that T-bet-independent T cell-derived IFN-γ is sufficient to cause immunopathology during infection. Despite comparable levels of IFN-γ generated by Tbx21−/− mice, they remain highly susceptible to infection. Furthermore, Tbx21−/− mice succumb significantly faster to T. gondii infection than animals lacking B and T cells, yet retain an intact myeloid innate compartment, suggesting that T-bet deficiency in innate immune cells results in loss of host resistance to intracellular parasite, and rapid host mortality during infection. Thus, we investigated T-bet expression levels in myeloid innate immune cells and observed that T-bet expression in monocyte-derived CD11c+ DCs occurs in an IFN-γ-dependent manner. We also established that T-bet expression in DCs, but not macrophages, is required for resistance to intracellular T. gondii infection. Overall, we revealed an essential role for T-bet expression in DCs for controlling host immunity to intracellular parasite infection.

Arsenic exposure impels CD4 commitment in thymus and suppress T cell cytokine secretion by increasing regulatory T cells

Arsenic is globally infamous for inducing immunosuppression associated with prevalence of opportunistic infection in exposed population, although the mechanism remains elusive. In this study, we investigate the effect of arsenic exposure on thymocyte lineage commitment and the involvement of regulatory T cells (Treg) in arsenic-induced immunosuppression. Male Balb/c mice were exposed to 0.038, 0.38 and 3.8 ppm sodium arsenite for 7, 15 and 30 days through oral gavage. Arsenic exposure promoted CD4 lineage commitment in a dose dependent manner supported by the expression of ThPOK in thymus. Arsenic also increased splenic CD4+ T cells and promoted their differentiation into Treg cells. In parallel, arsenic exposure induced immunosuppression characterized by low cytokine secretion from splenocytes and increased susceptibility to Mycobacterium fortuitum (M. fortuitum) infection. Therefore, we linked arsenic-induced rise in Treg cells with suppressed Th1 and Th2 related cytokines, which has been reversed by inhibition of Treg cells in-vivo using wortmannin. Other parameters like body weight, kidney and liver function, histoanatomy of thymus and spleen as well as thymocyte and splenocytes viability were unaltered by arsenic exposure. Taken together our findings indicated that environmentally relevant dose of arsenic enhanced differentiation of Treg cells which in turn induce immunosuppression in experimental animals.

HDAC3 is required to suppress the CD8-lineage program in DP thymocytes for CD4-lineage T cell commitment

Abstract MHC-restricted CD4 and CD8 T cells are vital components of the immune system. The decision to generate CD4 or CD8 T cells occurs in the thymus. After positive selection, intermediate thymocytes downregulate CD8 to test coreceptor specificity. Thymocytes develop into CD4 T cells if they experience persistent TCR signaling, while termination of TCR signaling leads to IL-7-mediated CD8-lineage commitment via Runx3. While the general signaling and transcriptional mechanisms are known, the epigenetic mechanisms controlling error-free lineage commitment are largely unknown. We found histone deacetylase 3 (HDAC3) to be critical for generating CD4 T cells, as its deletion leads to the generation of only CD8SP cells. Introduction of an OT-II TCR transgene revealed redirection of MHC class II-restricted cells to the CD8 lineage upon HDAC3 deletion. We found evidence that HDAC3-deficient DP thymocytes are primed for the CD8-lineage, as they exhibit hyperacetylation at CD8-lineage promoting genes and show elevated expression of phosphorylated-STAT5 (pSTAT5). During lineage choice, OT-II expressing HDAC3-deficient cells inappropriately upregulate Runx3. Therefore, HDAC3 functions as a brake in DP thymocytes whose release leads to a developmental bias for CD8 T cells.

Strong TCR signaling prolongs lineage uncertainty during MHC-I specific positive selection (HEM8P.241)

Abstract CD4/CD8 lineage choice in the thymus is primarily determined by the duration of TCR signaling during positive selection. Positive selection signaling by MHC-I ligands is of relatively short duration because CD8 coreceptor expression is terminated. However, the duration of TCR signaling during MHC-I specific positive selection should, in theory, also be affected by TCR affinity as well as coreceptor signal strength, but how TCR affinity and coreceptor signal strength affect MHC-I specific positive selection is not really known. To address this point, we used RAG-deficient mice expressing a panel of MHC-I restricted TCR transgenes with TCRs of different affinities for in vivo self ligands, and used genetically engineered mouse models that modulate the intensity of TCR signaling. Our data reveal that TCR signal intensity dictates how long MHC-I signaled thymocytes remain lineage-uncommitted before they express Runx3 and undergo CD8 lineage commitment. Furthermore, our data reveal that lineage errors by very high affinity TCR undergoing MHC-I specific positive selection do not normally occur because CD8 coreceptors have evolved to provide only weak Lck/coreceptor signals. We conclude that TCR signal intensity dramatically affects the timing of lineage fate during MHC-I specific positive selection and that error-free CD8 lineage choice is dependent on weak signaling by the CD8 coreceptor.

Lineage-Plasticity and Inflammatory Change of Human FoxP3+ Regulatory T Cells in Acute Viral Infection: Implication in Immune-Mediated Tissue Injury

FoxP3+ CD4+CD25hi regulatory T (Treg) cells play a major role in maintaining the immune homeostasis by preventing the activation of self-reactive T cells as well as in controlling a series of immune responses in viral infections. Recent studies suggest that lineage-commitment of CD4+T cells, including Treg cells, is not a fixed fate, rather a status with a wide range of plasticity. Functional changes and lineage-plasticity of Treg cells during acute viral infection, especially of human, have not been reported so far. Herein, we investigated whether Treg cells show the functional plasticity and whether such changes can affect the regulation of immunopathology in a human acute viral infection. As a model of human acute viral infection, we used a cohort of patients with acute hepatitis A (AHA), since tissue (liver) injury in AHA is mediated exclusively by activated T cells, not by direct cytopathic effect of virus.To assess the plasticity of Treg cell lineage, first, we examined the production of a variety of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining and multicolor flow cytometry. We found that a significant proportion of FoxP3+ CD4+CD25hi Treg cells produced TNF-α following TCR stimulation in patients with AHA, but not in heathy subjects. Analyses at multiple time points during the course of infection showed that TNF-α production from Treg cells decreased in convalescent phase. Likewise, we observed that liver-infiltrating Treg cells also produced TNF-α after TCR stimulation. Moreover, highly-purified CD4+CD25hiCD127lo/-Treg cells could also produce TNF-α following TCR stimulation, indicating that Treg cells of AHA patients can produce TNF-α in direct response to TCR stimulation.Next, to exclude the possibility that TNF-α might be secreted from transiently FoxP3-expressing activated non-Treg CD4+ T cells, we examined the expression level of CD127 on TNF-α-secreting FoxP3+ CD4+ T cells. TNF-α+ Treg cells expressed CD127 in the level similar to conventional TNF-α- counterpart, and CD127 expression levels of both Treg populations were much lower than FoxP3- CD4+ T cells. Furthermore, DNA methylation analysis of Treg cell-specific demethylated region (TSDR) after sorting TNF-α+ Treg cells revealed completely demethylated pattern in highly conserved CpG island of FOXP3 gene. These findings support that TNF-α is produced from bona fide Treg cells, not from FoxP3-expressing activated non-Treg CD4+T cells.In analysis of immunophenotypes, TNF-α+ Treg cells were enriched in CD45RA-FoxP3lo population, implying their reduced in vivo suppressive activity. Along with the lower level of FoxP3, TNF-α+ Treg cells showed lower level of CD39 expression, a surrogate marker of Treg cell suppressive activity, compared to TNF-α- Treg cells. Furthermore, TNF-α+Treg cells showed a robust evidence of lineage-plasticity toward Th17 lineage, expressing a key transcription factor RORγt. Consistently, they expressed CCR6 and co-produced IL-17A following TCR stimulation, which are the hallmark of Th17 effector function.To analyze the clinical implication of attenuate suppressive function and plasticity shown by TNF-α+ Treg cells, we examined correlation between production of proinflammatory cytokines from Treg cells and severity of liver damage in AHA. As a result, proportion of TNF-α-producing Treg cells closely and linearly correlated with severity of liver damage, suggesting the critical role of TNF-α+ Treg cells in the immunopathogenesis of AHA. However, Treg cell suppression assay in the absence or presence of anti-TNF-α antibody showed that Treg cell suppressive function was not affected by TNF-α blockade. This indicates that attenuated function of TNF-α+Treg cells is not attributed simply to production of a kind of inflammatory cytokine, rather to more complicated reprogramming mechanism.Taken together, these data provide a clear evidence of attenuated suppressive activity and Th17-toward lineage plasticity of FoxP3+ Treg cells, represented by TNF-α production, in a human acute viral infection. Also, we suggest one possible mechanism that lineage plasticity and inflammatory changes of Treg cells could be implicated in the immunopathogenesis of human diseases. DisclosuresNo relevant conflicts of interest to declare.

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.

Apoptotic Programs Are Determined during Lineage Commitment of CD4+ T Effectors: Selective Regulation of T Effector-Memory Apoptosis by Inducible Nitric Oxide Synthase

Lineage-committed T effectors generated in response to Ag during the inflammatory phase are destined to die during termination of the immune response. We present evidence to suggest that molecular signatures of lineage commitment are reflected in apoptotic cascades activated in CD4(+) T effectors. Exemplifying this, ablation of inducible NO synthase (iNOS) protected effector-memory T (TEM) cells, but not T(Naive) or central-memory T cells, activated in vitro, from apoptosis triggered by cytokine deprivation. Furthermore, attrition of T effectors generated in the secondary, but not the primary, response to Ag was substantially reduced in mice, which received iNOS inhibitors. Distinct patterns of iNOS expression were revealed in wild-type TEM effectors undergoing apoptosis, and ablation of iNOS protein in primary and TEM wild-type effectors confirmed observations made in iNOS(-/-) cells. Describing molecular correlates of this dependence, mitochondrial damage, activation of the protein Bax, and release from mitochondria of the apoptosis-inducing factor were selectively abrogated in iNOS(-/-) TEM effectors. Suggesting that iNOS dependence was linked to the functional identity of T cell subsets, both iNOS induction and apoptosis were compromised in IFN-γ(-/-) TEM effectors, which mirrored the response patterns of iNOS(-)(/)(-) TEM. Collectively, these observations suggest that programs regulating deletion and differentiation are closely integrated and likely encoded during lineage commitment of T effectors.

Strict Major Histocompatibility Complex Molecule Class-Specific Binding by Co-Receptors Enforces MHC-Restricted αβ TCR Recognition during T Lineage Subset Commitment

Since the discovery of co-receptor dependent αβTCR recognition, considerable effort has been spent on elucidating the basis of CD4 and CD8 lineage commitment in the thymus. The latter is responsible for generating mature CD4 helper and CD8αβ cytotoxic T cell subsets. Although CD4+ and CD8+ T cell recognition of peptide antigens is known to be MHC class II- and MHC class I-restricted, respectively, the mechanism of single positive (SP) thymocyte lineage commitment from bipotential double-positive (DP) progenitors is not fully elucidated. Classical models to explain thymic CD4 vs. CD8 fate determination have included a stochastic selection model or instructional models. The latter are based either on strength of signal or duration of signal impacting fate. More recently, differential co-receptor gene imprinting has been shown to be involved in expression of transcription factors impacting cytotoxic T cell development. Here, we address commitment from a structural perspective, focusing on the nature of co-receptor binding to MHC molecules. By surveying 58 MHC class II and 224 MHC class I crystal structures in the Protein Data Bank, it becomes clear that CD4 cannot bind to MHC I molecules, nor can CD8αβ or CD8αα bind to MHC II molecules. Given that the co-receptor delivers Lck to phosphorylate exposed CD3 ITAMs within a peptide/MHC (pMHC)-ligated TCR complex to initiate cell signaling, this strict co-receptor recognition fosters MHC class-restricted SP thymocyte lineage commitment at the DP stage even though both co-receptors are expressed on a single cell. In short, the binding preference of an αβTCR for a peptide complexed with an MHC molecule dictates which co-receptor subsequently binds, thereby supporting development of that subset lineage. How function within the lineage is linked further to biopotential fate determination is discussed.

CAMP response element modulator α controls IL2 and IL17A expression during CD4 lineage commitment and subset distribution in lupus

Appropriate expression of IL-2 plays a central role during the priming and differentiation of T cells. A tight balance between IL-2 and the effector cytokine IL-17A is essential for immune homeostasis. Epigenetic mechanisms have been documented as a key component of cytokine regulation during lineage commitment. The molecular mechanisms that induce chromatin remodeling are less well understood. We investigated epigenetic regulators that mediate the diametric expression of IL-2 and IL-17A in naive, central memory, and effector memory CD4(+) T cells. We demonstrate that cAMP response modulator (CREM)α contributes to epigenetic remodeling of IL2 in effector memory T cells through the recruitment of DNMT3a. CREMα also reduces CpG-DNA methylation of the IL17A promoter. CREMα expression is regulated at the epigenetic level by CpG-DNA methylation, which allows increased CREMα expression in effector memory CD4(+) T cells. T cells from patients with systemic lupus erythematosus (SLE) express increased levels of CREMα and exhibit a phenotype that is similar to effector memory CD4(+) T cells with epigenetically predetermined expression patterns of IL-2 and IL-17A. We conclude that CREMα mediates epigenetic remodeling of the IL2 and IL17A gene during T-cell differentiation in favor of effector memory T cells in health and disease.

Epigenetic Silencing of Cd8 Genes by ThPOK-Mediated Deacetylation during CD4 T Cell Differentiation

Intrathymic CD4/CD8 differentiation is a process that establishes the mutually exclusive expression profiles of the CD4 and CD8 T cell lineage. The RUNX3-mediated silencing of CD4 in CD8 lineage cells has been well documented; however, it is unclear how CD8 is silenced during CD4 lineage differentiation. In this study, we report that, by directly binding the CD8 locus, ThPOK works as a negative regulator that mediates the deacetylation of Cd8 genes and repositions the CD8 alleles close to heterochromatin during the development of the CD4 lineage. The ectopic expression of ThPOK resulted in increased recruitment of histone deacetylases at Cd8 loci; the enhanced deacetylation of Cd8 genes eventually led to impaired Cd8 transcription. In the absence of ThPOK, the enhanced acetylation and transcription of Cd8 genes were observed. The results of these studies showed that Cd8 loci are the direct targets of ThPOK, and, more importantly, they provide new insights into CD8 silencing during CD4 lineage commitment.

The BTB-ZF Family of Transcription Factors: Key Regulators of Lineage Commitment and Effector Function Development in the Immune System

Successful immunity depends upon the activity of multiple cell types. Commitment of pluripotent precursor cells to specific lineages, such as T or B cells, is obviously fundamental to this process. However, it is also becoming clear that continued differentiation and specialization of lymphoid cells is equally important for immune system integrity. Several members of the BTB-ZF family have emerged as critical factors that control development of specific lineages and also of specific effector subsets within these lineages. For example, BTB-ZF genes have been shown to control T cell versus B cell commitment and CD4 versus CD8 lineage commitment. Others, such as PLZF for NKT cells and Bcl-6 for T follicular helper cells, are necessary for the acquisition of effector functions. In this review, we summarize current findings concerning the BTB-ZF family members with a reported role in the immune system.

HIV-1 Nef Disrupts Maturation of CD4+ T Cells through CD4/Lck Modulation

The HIV-1 Nef protein is a major determinant of HIV-1 pathogenicity. It has been found to induce thymocyte depletion, but the mechanisms involved are not completely understood. Also, nothing is known about its effects on thymocyte selection. We used the CD4C/HIV(Nef) transgenic (Tg) mice, which develop a profound CD4(+) T cell lymphopenia, to study their thymic development. We report that HIV-1 Nef causes depletion of double-positive thymocytes and impairs selection and lineage commitment of CD4(+) single-positive thymocytes. This latter defect could be relieved by increasing the affinity of the TCR-MHC interaction or by allowing CD4(+) T cell maturation to proceed in absence of the CD4 tail, in double-Tg (Nef × CD4(tailless)) mice or in the presence of constitutively active Tg Lck(Y505F). These rescue strategies also resulted in reversal of peripheral CD4(+) T cell lymphopenia. Our data indicate that impairment of Lck-mediated CD4 coreceptor signaling by Nef is an important in vivo mechanism of HIV-1 pathogenesis.

On the issue of peptide recognition in T cell development.

CD4-CD8 double positive (DP) thymocytes undergo a differentiation process in the thymus where they are selected based on their ability to recognize peptide antigens presented on self major histocompatibility complex (MHC) molecules. The first stage of this process is positive selection, a quality-control mechanism which ensures that the T cell receptors (TCR) presented on developing thymocytes can transmit signals via peptides presented on either MHC class I (MHC1) or MHC class II (MHC2) molecules. Work over the past decade has revealed that the peptides that drive positive selection of both CD4 and CD8 lineage cells deliver only weak TCR signals. In line with these observations, specialized protein degradation machineries have been discovered in the thymic cortex that presumably generate specialized low-affinity peptide repertoires for presentation on MHC1 and MHC2 molecules. TCR signals transduced through these weak-affinity ligands in the early stages of positive selection alter the kinetics of expression of CD4 and CD8 molecules and play a crucial role in commitment of thymocytes to either the CD4 or CD8 lineages. In this work, we review the experiments that explore the peptide repertoires that are presented to developing thymocytes during positive selection, the observed signaling patterns that lead to CD4 versus CD8 lineage commitment, and speculate about how specialized organization of the signaling machinery in DP thymocytes may allow for efficient transduction of weak signals during the course of positive selection.

Coordinate Regulation of TCR signaling, Notch, and Gata3 in T Cell Development (138.27)

Abstract The transcription factors, Notch and Gata3, are both involved in the intrathymic development of CD4 lineage T cells. It has been proposed that Gata3 is critical in CD4 T cell maturation after lineage commitment. To the contrary, we find that disruption of the Gata3 gene re-directs a fraction of MHC II-restricted thymocytes to the CD8 lineage. Moreover, Gata3 binds regulatory regions of the ThPOK (Zbtb7b) gene in vivo and is required for the ThPOK expression that enforces CD4 lineage commitment (Nature Immunol. 9:1122). Collectively, these findings place Gata3 developmentally upstream of the CD4/CD8 lineage decision and raise the question of how Gata3 itself is regulated. Our data and that of others suggest that Gata3 expression is regulated by TCR signaling, but Gata3 can also promote TCR signaling/expression. Therefore, Gata3 may operate in a self-reinforcing feedback loop during positive selection to achieve the levels of TCR signaling required for ThPOK induction and CD4 lineage commitment. In the differentiation of peripheral Th2 cells, Gata3 transcription can be regulated by Notch. We find that Notch and Gata3 promote TCR signal transduction prior to positive selection, as well as the selection/maturation of CD4/CD8 T lineages. Thus, we have designed genetic and expression studies to investigate whether Notch regulates Gata3 in thymocytes. Intramural Research Program of the National Institute of Allergy and Infectious Diseases.