ABSTRACT: The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription.
Project description:RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, ?-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass ?-selection without TCR? gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.
Project description:alphabeta and gammadelta T cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and gammadelta T cell receptor (gammadeltaTCR) play instructional roles in specifying the alphabeta and gammadelta T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify fate and promote the development of these lineages remain poorly understood. Here, we show that differential activation of the extracellular signal-related kinase (ERK)-early growth response gene (Egr)-inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression served to regulate adoption of the gammadelta fate. Moreover, Id3 was both necessary and sufficient to enable gammadelta-lineage cells to differentiate independently of Notch signaling and become competent IFNgamma-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the gammadelta fate and its maturation in the thymus.
Project description:T-cell receptor-? (TCR?) rearrangement in CD4(+)CD8(+) double-positive immature thymocytes is a prerequisite for production of ?? T cells and invariant natural killer T cells. This developmental event is regulated by the TCR? enhancer (E?), which induces chromatin modification and recruitment of the recombination-activating proteins Rag1 and Rag2. However, the molecular mechanism underlying the activation and long-range action of E? remains incompletely understood. We show here that the chromatin-modifying factor TRIM28 is highly expressed in double-positive thymocytes and persistently phosphorylated at serine 473. TRIM28 binds to E? and induces histone 3 lysine 4 trimethylation in the E? and distant regions of the TCR? locus, coupled with recruitment of Rag proteins. T-cell-conditional ablation of TRIM28 impaired TCR? gene rearrangement and compromised the development of ?? T cells and invariant natural killer T cells. These findings establish TRIM28 as a unique regulator of thymocyte development and highlight an epigenetic mechanism involving TRIM28-mediated active chromatin modification in the TCR? locus.
Project description:A human T-cell receptor (TcR) derived from an autoreactive T-cell specific for GAD65, from a subject at high risk for autoimmune diabetes, was introduced into HLA-DR4 transgenic mice. The source of TcR was a CD4(+) T(H)1(+) T-cell clone which responded to an immunodominant epitope of the human islet protein GAD65, an epitope shared with both GAD65 and GAD67 in the mouse. The resulting HLA-DR4/GAD-TcR transgenic mice on a Rag2(o/o)/I-Ab(o/o)/B6 background exhibited a CD4(+) infiltrate into pancreatic islets that correlated with a loss of insulin in infiltrated islets. These mice also exhibited a subclinical impaired tolerance to exogenously fed glucose as assayed by an intraperitoneal glucose tolerance test. T cells containing the GAD65/67 (555-567) responsive TcR undergo strong negative selection as evidenced by a 10-fold lower thymocyte cellularity compared to non-TcR transgenic mice, and clonotype peripheral T cells represented approximately 1% of CD4(+) T cells in Rag2 sufficient mice. Upon in vitro stimulation, GAD65/67 555-567 responsive T cells secrete interferon-gamma, minimal interleukin (IL)-2 and tumor necrosis factor-alpha, and no IL-4, IL-5, IL-10, or IL-17, consistent with a T(H)1 profile. These data demonstrate that CD4(+) T cells specific for a naturally processed epitope within GAD can specifically home to pancreatic islets and lead to impaired islet beta-cell function in diabetes-associated HLA-DR4 transgenic mice on the relatively non-autoimmune C57BL/6 background. The relatively slow progression and patchy insulitis are reminiscent of the chronic pre-clinical phase similar to a majority of human at-risk subjects, and models these indolent features of human T1D.
Project description:Omenn syndrome (OS) is an atypical primary immunodeficiency characterized by severe autoimmunity because of activated T cells infiltrating target organs. The impaired recombinase activity in OS severely affects expression of the pre-T-cell receptor complex in immature thymocytes, which is crucial for an efficient development of the thymic epithelial component. Anti-CD3? monoclonal antibody (mAb) treatment in RAG2(-/-) mice was previously shown to mimic pre-TCR signaling promoting thymic expansion. Here we show the effect of anti-CD3? mAb administration in the RAG2(R229Q) mouse model, which closely recapitulates human OS. These animals, in spite of the inability to induce the autoimmune regulator, displayed a significant amelioration in thymic epithelial compartment and an important reduction of peripheral T-cell activation and tissue infiltration. Furthermore, by injecting a high number of RAG2(R229Q) progenitors into RAG2(-/-) animals previously conditioned with anti-CD3? mAb, we detected autoimmune regulator expression together with the absence of peripheral immunopathology. These observations indicate that improving epithelial thymic function might ameliorate the detrimental behavior of the cell-autonomous RAG defect. Our data provide important therapeutic proof of concept for future clinical applications of anti-CD3? mAb treatment in severe combined immunodeficiency forms characterized by poor thymus function and autoimmunity.
Project description:E2A proteins regulate multiple stages of thymocyte development and suppress T-cell lymphoma. The activity of E2A proteins throughout thymocyte development is modulated by signals emanating from the pre-TCR and TCR. Here we demonstrate that E2A is required for the complete arrest in both differentiation and proliferation observed in thymocytes with defects in proteins that mediate pre-TCR signaling, including LAT, Lck and Fyn. We show that E2A proteins are required to prevent the accumulation of TCRbeta negative cells beyond the pre-TCR checkpoint. E2A-deficient thymocytes also exhibit abnormal cell-cycle progression prior to pre-TCR expression. Furthermore, we demonstrate that E47 can act in concert with Bcl-2 to induce cell-cycle arrest in vitro. These observations indicate that E2A proteins function during early thymocyte development to block cell-cycle progression prior to the expression of TCRbeta. In addition, these data provide further insight into how deficiencies in E2A lead to T lymphoma.
Project description:In humans and mice, the early development of ?? T cells is controlled by the pre-T-cell receptor ? chain (pT?) that is covalently associated with the T-cell receptor ? (TCR?) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pT?. Using in silico and gene synteny-based approaches, we identified the pT? gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pT? functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pT? cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pT? was exclusively to enable expression of the TCR? chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pT? signaling.
Project description:Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR ? gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR ? loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the ? locus and TCR V?/J? recombination resulting in the appearance of many new ??TCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR ? enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR ? rearrangement. Our data suggest that Ikaros controls V?/J? recombination in T cells by controlling access of the transcription and recombination machinery to the TCR ? loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to ??TCR(+) CD4(+) CD8(+) thymocyte transition.
Project description:Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre-T-cell receptor (pre-TCR)- but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR-stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development.
Project description:Developing thymocytes bifurcate from a bipotent precursor into ??- or ??-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) ?-, ?-, and ?-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and ??-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-? and ??-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the ??-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a ??-biased gene-expression profile, and produced IFN-? in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong ??-TCR ligand, ??-expressing cells showed a lower propensity to differentiate along the ??-lineage. Importantly, differentiation along the ??-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-?. These results confirm a requirement for a strong ??-TCR ligand engagement to promote maturation along the ?? T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a ??-lineage choice in the case of weaker ??-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.