Intrathymic T cell development and selection proceeds normally in the absence of glucocorticoid receptor signaling

Glucocorticoids are believed to play a role in T cell development and selection, although their precise function is controversial. Glucocorticoid receptor (GR)-deficient mice were used to directly investigate this problem. GR-deficient thymocytes were resistant to dexamethasone-mediated apoptosis, confirming the absence of glucocorticoid responsiveness. An absence of GR signaling had no impact on thymocyte development either in vivo or in vitro. T cell differentiation, including positive selection, was normal as assessed by normal development of CD4+CD8+, alphabetaTCR+CD4+, and alphabetaTCR+CD8+ thymocytes. Negative selection, mediated by the superantigen staphylococcal enterotoxin B (SEB), or anti-CD3/CD28, was also normal in the absence of GR signaling. In contrast to earlier reports, these data demonstrate that GR signaling is not essential for intrathymic T cell development or selection.     

Mutually antagonistic signals regulate selection of the T cell repertoire

The sensitivity of T cells to agonist-induced death during development contrasts with their proliferative responses after agonist challenge in the periphery. The means by which TCR engagement results in these distinct outcomes is incompletely understood. It has been previously hypothesized that glucocorticoids (GC) modulate the threshold for thymocyte activation by blunting the consequences of TCR engagement. In support of this possibility, inhibition of GC production in fetal thymic organ culture was shown to result in CD4(+)CD8(+) thymocyte apoptosis. This was dependent upon MHC diversity, implying that endogenous GC might regulate antigen-specific selection. Similarly, mice expressing reduced GC receptor (GR) levels due to the presence of an antisense transgene have fewer CD4(+)CD8(+) thymocytes, which was due to an impaired transition from CD4(-)CD8(-) precursors and increased apoptosis. Here we ask how manipulating peptide diversity in the context of reduced GC signaling might affect T cell development and function. In mice with impaired GR expression there was a rescue of thymocyte cellularity and proportions as the diversity of peptides presented by self-MHC was reduced. Furthermore, whereas more CD4(+) T cells survived the selection process in mice expressing single-peptide-MHC class II complexes and reduced GR levels, these cells largely failed to recognize the same MHC molecules bound with foreign peptides. Together, these results support a role for endogenous GC in balancing TCR-mediated signals during thymic selection.     

T cell development and function in CrkL-deficient mice

The adapter protein CrkL has been implicated in multiple signal transduction pathways in hematopoietic cells. In T lymphocytes, the recruitment of CrkL-C3G complexes has been correlated with hyporesponsiveness, implicating CrkL as a potential negative regulator. To test this hypothesis we examined T cell activation in CrkL-deficient mice. The CrkL(-/-) genotype was partially embryonic lethal. In viable CrkL(-/-) mice, peripheral blood counts were normal. The thymus from CrkL(-/-) mice had 40% fewer cells compared to littermates, but the proportion of thymocyte subsets was comparable. There was no discernable alteration in T cell function as reflected by T cell numbers, expression of memory markers, IL-2 production, proliferation, and differentiation into Th1/Th2 phenotypes. Immunization induced comparable levels of IgG2a and IgG1 antibodies. Chimeric mice, generated by transfer of CrkL(-/-) fetal liver cells into irradiated RAG2(-/-) recipients, also showed normal T cell function, arguing against selection via partial embryonic lethality. Our results indicate that CrkL is not absolutely required for T cell development or function, and argue against it being an essential component of a negative regulatory pathway in TCR signaling.     

RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family with unknown physiological function. Here we demonstrate that Rhoh-/- mice have impaired T cell receptor (TCR)-mediated thymocyte selection and maturation, resulting in T cell deficiency. RhoH deficiency resulted in defective CD3zeta phosphorylation, impaired translocation of the signaling molecule Zap70 to the immunological synapse and reduced activation of Zap70-mediated signaling in thymic and peripheral T cells. Proteomic analyses demonstrated that RhoH is a component of TCR signaling and is required for recruitment of Zap70 to the TCR through interaction with RhoH noncanonical immunoreceptor tyrosine-based activation motifs (ITAMs). In vivo reconstitution studies also demonstrated that RhoH function depends on phosphorylation of the RhoH ITAMs. These findings suggest that RhoH is a critical regulator of thymocyte development and TCR signaling by mediating recruitment and activation of Zap70.     

Brca1 required for T cell lineage development but not TCR loci rearrangement

Brca1 (breast cancerl, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell-specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1-/- mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53-/- backgrounds, completely restored survival and development of Brca1-/- thymocytes; peripheral T cell numbers were not totally restored in Brcal-/- p53-/- mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1-/- thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.     

B-Raf-mediated signaling pathway regulates T cell development

The activities of the Raf kinase family proteins control extracellular signal-regulated kinase (ERK) activation in many aspects of cellular responses. However, the relative contributions of individual isozymes to cellular functions including T cell responses are still unclear. In addition to Raf-1, another Raf family kinase, B-Raf, is expressed in murine thymocytes and peripheral T cells, and its activation was induced by TCR stimulation. Here, we investigated the function of B-Raf in development of T cells by generating chimeric mice in which a T cell-compromised host was reconstituted with fetal liver-derived cells from embryonic lethal B-Raf-deficient mice. Although B-Raf was dispensable for normal T cell lineage differentiation at the CD4(-)CD8(-) double-negative stage, thymocytes in the chimeric mice derived from B-Raf(-/-) cells exhibited a drastic arrest of differentiation at the CD4(+)CD8(+) double-positive stage, suggesting that B-Raf is crucial for T cell development, especially for the transition to CD4(+) and CD8(+) single-positive thymocytes. Regarding intracellular signaling, we found that activation of ERK following TCR stimulation was impaired in the thymocytes from the chimeric mice. In conclusion, we present first evidence for the important role of B-Raf-mediated signaling in T cell development.     

Altered epithelial cell proportions in the fetal lung of glucocorticoid receptor null mice

Glucocorticoids provide important signals for maturation of the fetal lung and antenatal glucocorticoids are used to reduce the respiratory insufficiency suffered by preterm infants. To further understand the role of glucocorticoids in fetal lung maturation, we have analyzed mice with a targeted null mutation for the glucocorticoid receptor (GR) gene, which severely retards lung development. The lungs of fetal GR-null mice have increased lung weight and DNA content, are condensed and hypercellular, with reduced septal thinning leading to a 6-fold increase in the airway to capillary diffusion distance. In fetal GR-null mice, mRNA levels of the type II epithelial cell surfactant protein genes A and C were reduced by approximately 50%. Analysis of epithelial cell types by electron microscopy revealed that the proportions of type II cells were increased by approximately 30%, whereas the proportions of type-I cells were markedly reduced (by approximately 50%). Similarly, we found a 50% reduction in mRNA levels for T1alpha and aquaporin-5, two type I cell-specific markers, and a 20% reduction in aquaporin-1 mRNA levels. This demonstrates that during murine embryonic development, receptor-mediated glucocorticoid signaling facilitates the differentiation of epithelial cells into type I cells, but is not obligatory for type II cell differentiation.     

Tespa1 is involved in late thymocyte development through the regulation of TCR-mediated signaling

Signaling via the T cell antigen receptor (TCR) during the CD4(+)CD8(+) double-positive developmental stage determines thymocyte selection and lineage commitment. Here we describe a previously uncharacterized T cell-expressed protein, Tespa1, with critical functions during the positive selection of thymocytes. Tespa1(-/-) mice had fewer mature thymic CD4(+) and CD8(+) T cells, which reflected impaired thymocyte development. Tespa1 associated with the TCR signaling components PLC-γ1 and Grb2, and Tespa1 deficiency resulted in attenuated TCR signaling, as reflected by defective activation of the Erk-AP-1 and Ca(2+)-NFAT pathways. Our findings demonstrate that Tespa1 is a component of the TCR signalosome and is essential for T cell selection and maturation through the regulation of TCR signaling during T cell development.     

CD4(+)CD8(+)TCR(low) thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid-induced apoptosis

While signaling by either the TCR or glucocorticoid receptor (GR) can induce apoptosis in thymocytes, recent studies have shown that combining these signals results in survival of CD4(+)CD8(+) thymocytes. Although glucocorticoids (GC) in this way may directly affect T cell selection, no data are available addressing GR expression in thymocyte subsets and in individual cells within subsets. We studied GR expression by combining immunofluorescence cell surface staining for CD4, CD8 and TCR with intracellular staining of GR in four-color cytometry. Significant differences of GR expression were observed in various thymocyte subsets, although a homogeneous distribution of GR expression in individual thymocyte subsets emerged. The highest GR expression was found in CD4(-)CD8(-)TCR(-) thymocytes, and decreased during development via the CD4(-)CD8(+)TCR(-) subpopulation into the CD4(+)CD8(+)TCR(low) subset. Interestingly, the latter population, although expressing less than half the GR density of CD4(-)CD8(-)TCR(-) cells, is the most sensitive subset to GC-induced apoptosis. Up-regulation of TCR expression by the CD4(+)CD8(+)TCR(low) subset to CD4(+)CD8(+)TCR(high) cells was accompanied by a parallel increase in GR expression. The latter finding and the presence of a homogeneous distribution of GR in each thymocyte subset provides an experimental basis for the concept that GR can antagonize TCR-mediated signals at a constant rate relative to TCR expression.     

The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development

The glucocorticoids (GC) and retinoids (RA) modulate branching morphogenesis and cytodifferentiation in the developing lung. We investigated downstream target genes that link glucocorticoid stimulation to the achievement of a mature lung in glucocorticoid receptor (GR) knockout mice. All GR(null) mice and approximately 80% of mice homozygous for a hypomorphic allele (GR(hypo)) die shortly after birth of respiratory failure. cDNA microarray analysis showed organ-specific upregulation of the retinoic acid responsive gene midkine (MK) and its chondroitin-sulfate binding partner PG-M/versican at fetal day 18 and at neonatal day 1 in lungs of GR(hypo) mice, and at neonatal day 1 in lungs of GR(null) mice. By contrast, lung MK and PG-M/versican were downregulated in these mice at fetal day 16.5. In situ hybridization studies showed a dramatic decrease in MK and PG-M/versican RNA between days 16.5 and 17.5 in GR(WT) but not in GR(null) mice. Continued diffuse and robust expression of MK protein was observed in GR(null) mice at neonatal day 1. These findings suggest that MK may contribute to the dysmature lung phenotype in GR-deficient mice. Exposure of cultured day 21 fetal rat lung cells to GC downregulated MK, whereas RA enhanced MK expression. Our findings demonstrate the coincident modulation of expression of MK at the same developmental time point by both GC and RA, providing a potential mechanism for the integration of GC and RA effects on fetal lung development.     

TCR-mediated activation promotes GITR upregulation in T cells and resistance to glucocorticoid-induced death

T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR-/-). We also compared the action of GC on GITR+ and GITR- T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR-/- mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR-/- cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10(-8) to 10(-6) M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.     

Absence of thymus crosstalk in the fetus does not preclude hematopoietic induction of a functional thymus in the adult

Cortical and medullary thymic epithelial cells provide essential signals for a normal programme of T‐cell development. Current models of thymus development suggest that thymocyte‐derived signals play an important role in establishing thymic microenvironments, a process termed thymus crosstalk. Studies on CD3εtg26 mice lacking intrathymic T‐cell progenitors provided evidence that normal development of the thymic cortex depends upon thymocyte‐derived signals. Importantly, the reported failure to effectively reconstitute adult CD3εtg26 mice raised the possibility that such crosstalk must occur within a developmental window, and that closure of this window during the postnatal period renders thymic epithelium refractory to crosstalk signals and unable to effectively impose T‐cell selection. We have re‐investigated the timing of provision of crosstalk in relation to development of functional thymic microenvironments. We show that transfer of either fetal precursors or adult T‐committed precursors into adult CD3εtg26 mice initiates key parameters of successful thymic reconstitution including thymocyte development and emigration, restoration of cortical and medullary epithelial architecture, and establishment of thymic tolerance mechanisms including maturation of Foxp3⁺ Treg and autoimmune regulator‐expressing medullary epithelium. Collectively, our data argue against a temporal window of thymocyte crosstalk, and instead demonstrates continued receptiveness of thymic epithelium for the formation of functionally competent thymic microenvironments.     

Stress-free T-cell development: glucocorticoids are not obligatory

A role for glucocorticoids in thymopoiesis has been suggested by studies using glucocorticoid receptor (GR) anti-sense transgenic mice, glucocorticoid synthesis inhibitors and GR antagonists. Unfortunately, no consensus has been reached on exactly how glucocorticoids influence T-cell development. The most recent approach, using GR knockout (GR(-/-)) mice, indicates that GR signaling is, in fact, dispensable in this entire process.     

Possible involvement of glial cell line-derived neurotrophic factor and its receptor,GFRalpha1, in survival and maturation of thymocytes

The glial cell line-derived neurotrophic factor (GDNF) and its receptors (GFR) play important roles in the promotion of survival and differentiation of central and peripheral neuronal populations. We show that GFRalpha1, a component of GDNF receptor, was expressed in thymocytes at an early stage of thymocyte-development and was involved in the survival of thymocyte precursors. GFRalpha1and GDNF were expressed in thymus, but not in spleen or lymph nodes in adult mice. During embryonic thymocyte development, GFRalpha1 was predominantly expressed on thymocytes from days 14.5 to 16.5 of gestation, and thereafter its expression gradually declined. In adult thymus, GFRalpha1 was expressed only on CD4(-)CD8(-) double-negative (DN) thymocytes, but not on CD4(+)CD8(+) double-positive or single-positive thymocytes. It was strongly expressed on RAG2(-/-) thymocytes arrested at the DN stage, and ist expression was reduced during their differentiation after in vivo anti-CD3 antibody stimulation. Additionally, fetal thymocyte precursors grew in serum-free medium of the fetal thymus organ culture system in the presence of recombinant GDNF (rGDNF), while the cells without rGDNF died. These results suggested that GDNF/GFRalpha1 are involved in the survival of both the nervous system and DN immature thymocytes.     

Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.     

Distinct roles of the interleukin-7 receptor α chain in fetal and adult thymocyte development revealed by analysis of interleukin-7 receptor α-deficient mice

Mouse mutants lacking expression of the IL-7 receptor (IL-7R) α chain are defective in thymopoiesis. The adult thymus has multiple defects, including reduced cell numbers and proportions of the more mature thymocyte subsets, a complete absence of CD25⁺ cells and a reduced level of RAG1 and RAG2 expression. We show here that, in contrast to the profound developmental arrest observed in the adult thymus, fetal thymocytes from IL-7Rα^−/− mice have normal proportions of all of the major thymocyte subpopulations, including CD25⁺ thymocytes and the most mature single-positive subsets. Moreover, normal levels of RAG1 and RAG2 were observed. Total thymocyte numbers, however, remained reduced. These data suggest that the IL-7Rα chain is a key regulator of both survival and proliferation during thymocyte development but that it is not essential for the production of T cells during fetal thymopoiesis.