Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2(-/-) double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.     

CD28 ligation costimulates cell death but not maturation of double-positive thymocytes due to defective ERK MAPK signaling

The differentiation of double-positive (DP) CD4(+)CD8(+) thymocytes to single-positive CD4(+) or CD8(+) T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.     

EphA receptors inhibit anti-CD3-induced apoptosis in thymocytes

The EphA receptor tyrosine kinases interact with membrane-bound ligands of the ephrin-A subfamily. Interaction induces EphA receptor oligomerization, tyrosine phosphorylation, and, as a result, EphA receptor signaling. EphA receptors have been shown to regulate cell survival, migration, and cell-cell and cell-matrix interactions. However, their functions in lymphoid cells are only beginning to be described. We show in this study that functional EphA receptors are expressed by murine thymocytes, including CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) subpopulations. We demonstrate that activation of EphA receptors by the ephrin-A1 ligand inhibits the anti-CD3-induced apoptosis of CD4(+)CD8(+) double-positive thymocytes. Furthermore, ephrin-A1 costimulation suppresses up-regulation of both the IL-2R alpha-chain (CD25) and early activation Ag CD69 and can block IL-2 production by CD4(+) single-positive cells. In agreement, EphA receptor activation in thymocytes also inhibits TCR-induced activation of the Ras-MAPK pathway. Our findings suggest that EphA receptor activation is antithetical to TCR signaling in thymocytes, and that the level of engagement by ephrin-A proteins on thymic APCs regulates thymocyte selection.     

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Changes in the Thymus of Peripubertal Rats Induced by Centrally Applied Somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.     

RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.     

Glucocorticoid resistance in thymocytes from mice expressing a T cell receptor transgene

A majority of thymocytes undergo apoptosis during differentiation due to lack of survival signals provided by T cell receptor (TCR) activation. As glucocorticoids (GC) have been suggested to be involved in this process, we have investigated the GC sensitivity in thymocytes from mice expressing a transgenic selecting TCR. We now report that immature CD4(+)CD8(+) double-positive thymocytes from these mice are comparatively more resistant to corticosterone-induced apoptosis. This is associated with reduced glucocorticoid receptor (GR) expression, increased levels of membrane CD28, increased NF-kappaB DNA binding activity, and increased binding to the CD28 response element in the interleukin-2 gene promoter. Analysis of NF-kappaB/Rel proteins from nuclear extracts demonstrated altered levels of some of these proteins. Our results suggest that TCR recognition of self major histocompatibility antigens generates intracellular signals which alter the thymocyte GC sensitivity and thereby protect them against apoptosis induced by endogenous GC.     

Pre-TCR signaling and CD8 gene bivalent chromatin resolution during thymocyte development

The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.     

Peripheral T cells become sensitive to glucocorticoid- and stress-induced apoptosis in transgenic mice overexpressing SRG3.

Immature double-positive thymocytes are sensitive to glucocorticoid (GC)-induced apoptosis, whereas mature single-positive T cells are relatively resistant. Thymocytes seem to acquire resistance to GCs during differentiation into mature single-positive thymocytes. However, detailed knowledge concerning what determines the sensitivity of thymocytes to GCs and how GC sensitivity is regulated in thymocytes during development is lacking. We have previously reported that the murine SRG3 gene (for SWI3-related gene) is required for GC-induced apoptosis in a thymoma cell line. Herein, we provide results suggesting that the expression level of SRG3 protein determines the GC sensitivity of T cells in mice. SRG3 associates with the GC receptor in the thymus, but rarely in the periphery. Transgenic overexpression of the SRG3 protein in peripheral T cells induces the formation of the complex and renders the cells sensitive to GC-induced apoptosis. Our results also show that blocking the formation of the SRG3-GC receptor complex with a dominant negative mutant form of SRG3 decreases GC sensitivity in thymoma cells. In addition, mice overexpressing the SRG3 protein appear to be much more susceptible to stress-induced deletion of peripheral T cells than normal mice, which may result in an immunosuppressive state in an animal.     

Thymocytes express the golli products of the myelin basic protein gene and levels of expression are stage dependent

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5(+) cortical/MTS10(+) medullary epithelial cells and NLDC145(+) dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. GOLLI: mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4(-)CD8(-) (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4(-)CD8(-) (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8(-) or CD4(+) (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.     

8B4/20, a private CD43 epitope on developing human thymocytes, is involved in thymocyte maturation

The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3-/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We therefore used specific Ab to mimic putative 8B4/20 interactions with natural ligand and examined the effect on isolated thymocytes. Treatment with 8B4/20 had no effect on in vitro apoptosis of isolated thymocytes. In contrast, 8B4/20 ligation enhanced the conversion of isolated thymocytes to differentiated phenotypes. Increased numbers were found in 8B4/20-treated cultures of CD3high and single-positive thymocytes and decreased numbers of CD3-/low and double-positive thymocytes, strongly suggesting that engagement of 8B4/20 delivers a positive signal that favors completion of the thymocyte maturation program. The ability of 8B4/20 mAb to drive thymocyte maturation in vitro suggests that CD43 molecules bearing the 8B4/20 epitope participate in early events of thymic selection.     

Interchangeability of Themis1 and Themis2 in thymocyte development reveals two related proteins with conserved molecular function

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.     

Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3(high)CD69+ thymocytes and gammadeltaTCR+ thymocytes preferentially respond to CCL25.

CCR9 mediates chemotaxis of thymocytes in response to CCL25/thymus-expressed chemokine, and its mRNA is selectively expressed in thymus and small intestine, the two known sites of T lymphopoiesis. To examine the expression of CCR9 during lymphocyte development, we generated polyclonal Ab that recognizes murine CCR9. CCR9 was expressed on the majority of immature CD4+CD8+ (double-positive) thymocytes, but not on immature CD4(-)CD8(-) (double-negative) thymocytes. CCR9 was down-regulated during the transition of double-positive thymocytes to the CD4+ or CD8+ (single-positive) stage, and only a minor subset of CD8+ lymph node T cells expressed CCR9. All CCR9+ thymocyte subsets migrated in response to CCL25; however, CD69+ thymocytes demonstrated enhanced CCL25-induced migration compared with CD69(-) thymocytes. Ab-mediated TCR stimulation also enhanced CCL25 responsiveness, indicating that CCL25-induced thymocyte migration is augmented by TCR signaling. Approximately one-half of all gammadeltaTCR+ thymocytes and peripheral gammadeltaTCR+ T cells expressed CCR9 on their surface, and these cells migrated in response to CCL25. These findings suggest that CCR9 may play an important role in the development and trafficking of both alphabetaTCR+ and gammadeltaTCR+ T cells.     

Oxidized glucocorticoids counteract glucocorticoid-induced apoptosis in murine thymocytes in vitro.

11Beta-hydroxyglucocorticoids (HGCs) are known to induce apoptosis in immature T cells. Here we show that 11-oxoglucocorticoids (OGCs), which are oxidized metabolites of HGCs, counteract the apoptosis-inducing effects of HGC in murine thymocytes in vitro. Corticosterone at concentrations ranging from 0.1-100 microM induced apoptosis in thymocytes obtained from C57BL/6J mice aged 4 weeks, as demonstrated by cell staining with anti-phosphatidylserine antibody, a decrease in mitochondrial membrane potential, and DNA fragmentation. Co-culture of the cells with 10-100 microM of OGCs, dehydrocorticosterone, cortisone, and prednisone significantly inhibited thymocyte apoptosis induced by 1 microM corticosterone, (p<0.006). Among the other 6 physiological metabolites of the HGCs we tested, 20alpha-dehydrocortisol also showed considerable inhibitory effect on corticosterone-induced thymocyte apoptosis. Corticosterone-treatment of thymocytes in vitro decreased the number of CD4 and CD8 double positive cells, while co-culturing the cells with dehydrocorticosterone significantly attenuated this corticosterone effect (p<0.0001). Numbers of double-negative cells and single-positive cells were not significantly affected by corticosterone, dehydrocorticosterone, or both together. These results raised the possibility that OGCs and probably other HGC metabolites can regulate apoptotic cell death of immature double-positive thymocytes induced by HGC.     

Activated p56lck directs maturation of both CD4 and CD8 single-positive thymocytes

p56(lck) is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56(lck) in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56(lck) (p56(lck)F505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56(lck)F505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56(lck) constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56(lck) in regulating TCR signaling.     

Inefficient cell spreading and cytoskeletal polarization by CD4+CD8+ thymocytes: regulation by the thymic environment

CD4(+)CD8(+) double-positive (DP) thymocytes express a lower level of surface TCR than do mature T cells or single-positive (SP) thymocytes. Regulation of the TCR on DP thymocytes appears to result from intrathymic signaling, as in vitro culture of these cells results in spontaneous TCR up-regulation. In this study, we examined cell spreading and cytoskeletal polarization responses that have been shown to occur in response to TCR engagement in mature T cells. Using DP thymocytes stimulated on lipid bilayers or nontransgenic thymocytes added to anti-CD3-coated surfaces, we found that cell spreading and polarization of the microtubule organizing center and the actin cytoskeleton were inefficient in freshly isolated DP thymocytes, but were dramatically enhanced after overnight culture. SP (CD4(+)) thymocytes showed efficient responses to TCR engagement, suggesting that releasing DP thymocytes from the thymic environment mimics some aspects of positive selection. The poor translation of a TCR signal to cytoskeletal responses could limit the ability of DP thymocytes to form stable contacts with APCs and may thereby regulate thymocyte selection during T cell development.