Different Stages Of Thymocyte Development Research Articles

Humanized Mice Reveal New Insights Into the Thymic Selection of Human Autoreactive CD8+ T Cells.

Thymic selection constitutes the first checkpoint in T-cell development to purge autoreactive T cells. Most of our understanding of this process comes from animal models because of the challenges of studying thymopoiesis and how T cell receptor (TCR) specificity impacts thymocyte phenotype in humans. We developed a humanized mouse model involving the introduction of autoreactive TCRs and cognate autoantigens that enables the analysis of selection of human T cells in human thymic tissue in vivo. Here, we describe the thymic development of MART1-specific autoreactive CD8+ T cells that normally escape deletion and how their phenotype and survival are affected by introduction of the missing epitope in the hematopoietic lineage. Expression of the epitope in a fraction of hematopoietic cells, including all major types of antigen-presenting cells (APCs), led to profound yet incomplete deletion of these T cells. Upregulation of PD-1 upon antigen encounter occurred through the different stages of thymocyte development. PD-1 and CCR7 expression were mutually exclusive in both transgenic and non-transgenic thymocytes, challenging the view that CCR7 is necessary for negative selection in humans. In the presence of antigen, MART1-reactive T cells down-regulated TCR, CD3, CD8, and CD4 in the thymus and periphery. Moreover, expression of secondary TCRs influences MHC class I-restricted T cells to develop as CD4+, particularly regulatory T cells. This new model constitutes a valuable tool to better understand the development of autoreactive T cells identified in different human autoimmune diseases and the role of different APC subsets in their selection.

Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells

Members of the Runx family of transcriptional regulators are required for the appropriate expression of CD4 and CD8 at discrete stages of T cell development. The roles of these factors in other aspects of T cell development are unknown. We used a strategy to conditionally inactivate the genes encoding Runx1 or Runx3 at different stages of thymocyte development, demonstrating that Runx1 regulates the transitions of developing thymocytes from the CD4(-)CD8(-) double-negative stage to the CD4(+)CD8(+) double-positive (DP) stage and from the DP stage to the mature single-positive stage. Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4(+) helper and CD8(+) cytotoxic T cell lineages, respectively. Runx1-deficient CD4(+) T cells had markedly reduced expression of the interleukin 7 receptor and exhibited shorter survival. In addition, inactivation of both Runx1 and Runx3 at the DP stages resulted in a severe block in development of CD8(+) mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells.

The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells

Members of the Runx family of transcriptional regulators are required for the appropriate expression of CD4 and CD8 at discrete stages of T cell development. The roles of these factors in other aspects of T cell development are unknown. We used a strategy to conditionally inactivate the genes encoding Runx1 or Runx3 at different stages of thymocyte development, demonstrating that Runx1 regulates the transitions of developing thymocytes from the CD4−CD8− double-negative stage to the CD4+CD8+ double-positive (DP) stage and from the DP stage to the mature single-positive stage. Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages, respectively. Runx1-deficient CD4+ T cells had markedly reduced expression of the interleukin 7 receptor and exhibited shorter survival. In addition, inactivation of both Runx1 and Runx3 at the DP stages resulted in a severe block in development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells.

HIV Infection Rapidly Induces and Maintains a Substantial Suppression of Thymocyte Proliferation

The supply of naive T cells by the thymus normally requires precursor T cell proliferation within the thymus and would be particularly important in the setting of HIV infection when both naive and memory T cells are progressively depleted. As a robust, quantitative index of intrathymic proliferation, the ratio of different T cell receptor excision circles (TRECs), molecular markers of distinct T cell receptor rearrangements occurring at different stages of thymocyte development, was measured in peripheral blood-mononuclear cells (PBMCs). This ratio has the virtue that it is a “signature” of thymic emigrants throughout their entire life and, thus, can be measured in peripheral cell populations that are easy to obtain. Using the new assay, we evaluated the effect of HIV infection on intrathymic precursor T cell proliferation by longitudinal analysis of PBMCs from recently infected individuals. Our findings reveal a substantial reduction in intrathymic proliferation. The analysis also indicates the existence of a compensatory mechanism acting to sustain the numbers of recent thymic emigrants (RTEs) in the periphery.

Involvement of p38 mitogen–activated protein kinase in different stages of thymocyte development

AbstractPositive selection of thymocytes during T-cell development is mediated by T-cell receptor (TCR)–activated signals. For different mitogen-activated protein kinases (MAPKs) activated by TCR complex, a selective involvement of extracellular signal–regulated kinase, but not p38 MAPK, in positive selection has been suggested. Using transgenic mice with dominant-negative mutation of both MAP kinase kinase 3 (MMK3) and MKK6, we obtained mice with different extents of inhibition of p38 MAPK activation. Partial inhibition of p38 MAPK impaired CD4−CD8− thymocyte development and T-cell proliferation, but not positive selection. Interference with thymocyte positive selection was observed in mice with effective suppression of p38 MAPK. Our results suggest that, in addition to early thymocyte development, p38 is involved in positive selection.

Inhibition of Rho at different stages of thymocyte development gives different perspectives on Rho function

Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2. Thymocyte development is regulated by a complex signalling network [1], one component of which is the GTPase Rho. The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function [2,3]. To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus [4–6]. In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold. Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter [7]. In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck–C3 mice, in which the reduction predominantly results from defects in cell survival of CD25+ thymocyte progenitors, the CD2–C3 transgenic mice had a pre-T-cell differentiation block at the CD25+ stage after rearrangement of the T-cell receptor (TCR) β chains. Analysis of CD2–C3 mice demonstrated that Rho acts as an intracellular switch for TCR β selection, the critical thymic-differentiation checkpoint. These results show that Rho-mediated survival signals for CD25+ pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo.

Enhancement of activation-induced cell death by fibronectin in murine CD4+ CD8+ thymocytes.

Development of T cells in the thymus is achieved through the interactions of thymocytes with their microenvironments. This study focused on the function of fibronectin (FN), a major extracellular matrix molecule in the thymus, in the cell death induced by activation via the T-cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti-CD3 monoclonal antibody (mAb), especially when a high concentration of anti-CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence-activated cell sorter (FACS) analysis revealed that the activation-induced cell death (AICD) caused by anti-CD3 mAb alone, or by a combination of anti-CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)-4 and VLA-5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA-4 was predominantly expressed at the CD4- CD8- stage, and thereafter the expression was reduced, whereas VLA-5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide but not in the presence of the connecting segment-1 (CS-1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA-5.

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites.

The site of negative selection in the thymus has been inferred from a range of different experiments. Analysis of thymic deletion of V beta 5+, V beta 11+ or V beta 17a+ cells in H-2E transgenic mice led to the theory that negative selection occurs predominantly in the medulla (specifically, through presentation by medullary dendritic cells). Other experiments investigated whether transgenic TCR are deleted at the double-positive (DP) or single-positive stage following encounter with peptide ligand: by flow cytometric analysis deletion is generally found to occur at the DP thymocyte stage and as these cells are found predominantly in the cortex, it has been inferred that this is the key site of negative selection. The visualization of apoptotic thymocytes in situ has recently been reported for specific examples of negative selection. Using a panel of TCR transgenic lines in which negative selection occurs at different stages of thymocyte development, we have used TUNEL staining to analyse the anatomical sites of thymocyte apoptosis. For the first time we have been able to compare directly the sites of deletion induced by the endogenous cognate peptides or by endogenous superantigen. We show that generalization from the medullary deletion of V beta 5+, V beta 11+ or V beta 17a+ cells by the endogenous superantigens Mtv 8 and 9 from limited examples of cortical deletion by exogenous peptide administered to TCR transgenic mice is over-simplified. Apoptotic thymocytes in mice lacking Mtv superantigens are indeed localized in the cortex. However, when deletion is induced by cognate self peptide, apoptosis can occur in the cortex, the medulla or at the junction between the two.

Regulated activity of the IgH intron enhancer (E mu) in the T lymphocyte lineage.

The activity of the IgH (E mu) enhancer in the T lymphocyte lineage has been investigated using both transgenic mice and transfection studies. Thymocyte fractionation experiments indicate that a transgene consisting of the bacterial chloramphenicol acetyl transferase (CAT) gene, linked to E mu and the SV40 early promoter (E mu-CAT), is expressed only in thymocytes with a mature medullary phenotype and not in immature cells. Transfection of this same construct into two thymoma cell lines representing different stages of thymocyte development mimics the pattern of activity observed in vivo. Further transfection experiments suggest that this pattern of expression might be attributed to the differential activity of the E2E3 and octanucleotide motifs of E mu during development. In contrast, an Ig lambda transgene (linked to E mu and an Ig V lambda promoter) is expressed in the majority of thymocytes. We envisage that the different patterns of expression of the two transgenes reflect interactions between their respective promoters and the factors which are bound to E mu at different stages of thymocyte development. Although differing in their pattern of expression within the thymus, the two transgenes share the property of extinction in peripheral T lymphocytes. These results indicate that the expression of E mu-linked transgenes in the thymus cannot simply be explained by activation of the enhancer in a lymphoid progenitor cell prior to B/T lineage divergence. Rather, the enhancer (or components of it) must be independently activated (and inactivated) during T lymphocyte development. Furthermore, this activity is consistent with the developmental timing of Ig DH-JH rearrangements in these cells.