Protein phosphatase subunit G5PR that regulates the JNK-mediated apoptosis signal is essential for the survival of CD4 and CD8 double-positive thymocytes

Early T lineage cells are selected in the thymus by the specific recognition of peptide components presented by MHC molecules on the surface of thymic epithelial cells and dendritic cells. As a potential regulator of the apoptotic and survival signals, the protein phosphatase 2A-component G5PR regulates Bim phosphorylation in B-cells. Here, we studied whether G5PR is involved in the regulation of the similar apoptotic pathway for cell survival during the selection of thymocytes. T-cell-specific G5PR knockout (G5pr(-/-)) mice displayed thymic atrophy, significant reduction in thymocyte numbers, particularly a 10-fold decrease in the number of CD4 and CD8 double-positive (DP) thymocytes and few mature single-positive (SP) cells. G5pr(-/-) thymocytes exhibited normal potential of proliferation and differentiation during the transition from double-negative (DN) to DP stage, but significantly increased susceptibility to apoptosis at the DP stage. G5PR deficiency did not affect on Bim activation in thymocytes, but caused hyper-activation of JNK and Caspase-3 with augmented Fas ligand (FasL) expression, indicating that G5PR regulates the thymocyte unique apoptotic signal involved in JNK-mediated Caspase-3 activation but not in Bim activation. G5PR is essential for the survival of DP cells during thymocyte development.     

Thymocyte selection is regulated by the helix-loop-helix inhibitor protein, Id3

E2A, HEB, E2-2, and daughterless are basic helix-loop-helix (bHLH) proteins that play key roles in multiple developmental pathways. The DNA binding activity of E2A, HEB, and E2-2 is regulated by a distinct class of inhibitor HLH proteins, the Id gene products. Here, we show that Id3 is required for major histocompatability (MHC) class I- and class II-restricted thymocyte positive selection. Additionally, H-Y TCR-mediated negative selection is severely perturbed in Id3 null mutant mice. Finally, we show that E2A and Id3 interact genetically to regulate thymocyte development. These observations identify the HLH inhibitory protein Id3 as an essential component required for proper thymocyte maturation.     

Hedgehog signaling regulates differentiation from double-negative to double-positive thymocyte

The hedgehog (Hh) signaling pathway is involved in the development of many tissues. Here we show that sonic hedgehog (Shh) is involved in thymocyte development. Our data suggest that termination of Hh signaling is necessary for differentiation from CD4-CD8-double-negative (DN) to CD4+CD8+ double-positive (DP) thymocyte. Shh is produced by the thymic stroma, and Patched and Smoothened (Smo), the transmembrane receptors for Shh, are expressed in DN thymocytes. A neutralizing monoclonal antibody against Shh increases differentiation of DN to DP thymocytes, and Shh protein arrests thymocyte differentiation at the CD25+ DN stage, after T cell receptor beta (TCRbeta) gene rearrangement. We show that one consequence of pre-TCR signaling is downregulation of Smo, allowing DN thymocytes to proliferate and differentiate.     

Perturbed thymopoiesis in vitro in the absence of suppressor of cytokine signalling 1 and 3

Cytokine signals are central to the differentiation of thymocytes and their stepwise progression through defined developmental stages. The intensity and duration of cytokine signals are regulated by the suppressor of cytokine signalling (SOCS) proteins. A clear role for SOCS1 during the later stages of thymopoiesis has been established, but little is known about its role during early thymopoiesis, nor the function of its closest relative, SOCS3. Here, we find that both SOCS1 and SOCS3 are expressed during early thymopoiesis, with expression coincident during the double negative (DN)2 and DN3 stages. We examined thymocyte differentiation in vitro by co-culture of SOCS-deficient bone marrow cells with OP9 cells expressing the Notch ligand Delta-like1 (OP9-DL1). Cells lacking SOCS1 were retarded at the DN3:DN4 transition and appeared unable to differentiate into double positive (DP) thymocytes. Cells lacking both SOCS1 and SOCS3 were more severely affected, and displayed an earlier block in T cell differentiation at DN2, the stage at which expression of SOCS1 and SOCS3 coincides. This indicates that, in addition to their specific roles, SOCS1 and SOCS3 share overlapping roles during thymopoiesis. This is the first demonstration of functional redundancy within the SOCS family, and has uncovered a vital role for SOCS1 and SOCS3 during two important checkpoints in early T cell development.     

Developmentally Regulated Expression of the Transmembrane Adaptor Protein TRIM in Fetal and Adult T Cells

TRIM is a recently identified transmembrane adaptor protein which is exclusively expressed in T cells and natural killer (NK) cells. In peripheral blood T cells TRIM has been reported to coprecipitate, comodulate, and cocap with the T-cell receptor (TCR), suggesting that it is an integral component of the TCR/CD3/zeta complex. Here we investigate the expression of TRIM mRNAs and proteins in developing thymocytes. Two splicing isoforms with open reading frames are observed, namely a full length (TRIM) and a truncated version (DeltaTM-TRIM). The latter lacks the extracellular and transmembrane domains as well as the first 10 cytoplasmic aminoacids and is significantly expressed only as mRNA in early fetal thymocytes. TRIM mRNA is detected in all mainstream thymocyte subsets in adult mice. TRIM protein, in contrast, first appears in the DN2 (CD44+ CD25+) subset of adult double negative (DN) cells. In fetal thymocyte development, TRIM mRNA is seen from dg 14.5 onwards whereas TRIM protein appears first on dg 16.5. In contrast to the adult, the TRIM protein was seen in a subset of fetal DN1 cells. In fetal and adult thymocytes, TRIM protein expression was highest in DN2, DN3 (CD44-25+) and in DP cells, compatible with a functional role at or around phases of thymic selection.     

Aberrant development of thymocytes in mice lacking laminin-2

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin-2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4- CD8-) and SP (CD4+ CD8-, CD4- CD8+) populations and a significant decrease in the DP (CD4+ CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice.     

Death of T cell precursors in the human thymus: a role for CD38

Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC-peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC-peptide-TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.     

Glucocorticoid (GC) sensitivity and GC receptor expression differ in thymocyte subpopulations

Positive and negative selection steps in the thymus prevent non-functional or harmful T cells from reaching the periphery. To examine the role of glucocorticoid (GC) hormone and its intracellular receptor (GCR) in thymocyte development we measured the GCR expression in different thymocyte subpopulations of BALB/c mice with or without previous dexamethasone (DX), anti-CD3 mAb, RU-486 and RU-43044 treatment. Four-color labeling of thymocytes allowed detection of surface CD4/CD8/CD69 expression in parallel with intracellular GCR molecules by flow cytometry. Double-positive (DP) CD4+CD8+ thymocytes showed the lowest GCR expression compared to double-negative (DN) CD4-CD8- thymocytes and mature single-positive (SP) cells. DX treatment caused a concentration-dependent depletion of the DP cell population and increased appearance of mature SP cells with reduced GCR levels. GCR antagonists (RU-486 or RU-43044) did not influence the effect of DX on thymocyte composition; however, RU-43044 inhibited the high-dose GC-induced GCR down-regulation in SP and DN cells. GCR antagonists alone did not influence the maturation of thymocytes and receptor numbers. Combined low-dose anti-CD3 mAb and DX treatment caused an enhanced maturation (positive selection) of thymocytes followed by the elevation of CD69+ DP cells. The sensitivity of DP thymocytes with a GCRlow phenotype to GC action and the ineffectiveness of the GCR antagonist treatment may reflect a non-genomic GC action in the thymic selection steps.