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.     

Cross-Linking the TCR Complex Induces Apoptosis in CD4+8+ Thymocytes in the Presence of Cyclosporin A

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD⁺8⁺ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.     

Double-positive thymocytes resistant to antigen-MHC-induced negative selection lack active caspase

Thymocytes bearing autoreactive TCR are eliminated from the organism by a process termed negative selection. The molecular basis of this deletion has been recently shown to be a consequence of TCR-triggered activation of a caspase by certain peptide-MHC ligands in the immature CD4+CD8+ double-positive (DP) thymocyte subpopulation. Of note, the numerically minor TCRhigh DP thymocyte subpopulation, unlike the major TCRlow DP subset, is resistant to negative selection. Despite exposure to cognate peptide, TCRhigh DP thymocytes mature into single-positive thymocytes and are exported into the periphery. Here we investigated the mechanism by which these thymocytes escape negative selection. Using a cytochemical assay in conjunction with a caspase-specific affinity ligand, we demonstrate that the resistance of the TCRhigh DP thymocytes to negative selection correlates with the disappearance of TCR-triggered caspase activity in these cells. Thus thymocytes which have presumably begun the positive selection process inactivate the thymic caspase pathway and are no longer susceptible to negative selection.      

Induction of thymocyte positive selection does not convey immediate resistance to negative selection

The acquisition of functional competence represents a critical phase during intrathymic development of T cells. Thymocytes reaching this stage represent cells which have been positively selected on the basis of major histocompatibility complex reactivity, but which have also been purged of potentially autoreactive T-cell receptor specificities by negative selection. While the developmental window in which thymocytes are subjected to positive selection is now well defined, the precise developmental timing of negative selection, in relation to positive selection events, is less clear. Moreover, the underlying mechanism allowing single-positive thymocytes to respond to T-cell receptor ligation by activation rather than death, remains controversial. Here we have analysed the developmental timing of negative selection in relation to positive selection, using measurement of thymocyte susceptibility to dendritic cell presentation of the superantigen staphylococcal enterotoxin B (SEB). We show that thymocytes which have received initial positive selection signals, namely CD4+ CD8+ CD69+ thymocytes, like their CD4+ CD8+ CD69minus sign precursors, are susceptible to negative selection, indicating that induction of positive selection does not convey immediate resistance to negative selection. In contrast, newly generated CD4+ CD8minus sign CD69+ cells are not only resistant to deletion by SEB, but respond to SEB-mediated T-cell receptor-ligation by activation, indicating that the acquisition of functional competence occurs at the newly generated CD4+ CD8minus sign CD69+ stage. Finally, by using direct retroviral infection of primary CD4+ CD8+ thymocytes, we also show that Notch-1 activation in CD4+ CD8+ thymocytes does not correlate with, nor convey resistance to superantigen-mediated negative selection. Thus, our data suggest that although Notch-1 has been implicated in resistance to thymocyte apoptosis, the acquisition of resistance to negative selection occurs independently of Notch-1 signalling.     

CD28协同刺激分子对T细胞受体介导的胸腺细胞亚群凋亡的影响

目的：分析anti-TCRαβmAb anti-CD28mAb诱导小鼠胸腺淋巴细胞不同亚群的凋亡及凋亡程度，分析CD28协同刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响。方法：新鲜分离胸腺细胞，加入anti-TCRαβmAb-anti-TCRαβmAb anti-CD28mAb等培养20h，进行多重染色，流式细胞仪分析。结果：与胸腺细胞自发凋亡的结果相比较；（1）双信号刺激可明显增加胸腺细胞凋亡的数目，尤其是CD4^ CD8^ 胸腺细胞的凋亡数目。（2）凋亡的CD4^ CD8^ 亚群，CD4^ CD8^-亚群细胞表面CD28的表达均增多。结论：CD28共刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响与细胞的成熟程度有关，CD28共刺激分子能明显增强不成熟皮质胸腺细胞的凋亡。     

TCR抗体诱导不成熟胸腺细胞亚群凋亡的敏感性研究

为比较小鼠不同胸腺细胞亚群对抗TCR抗体诱导凋亡的敏感性，用体外抗TCR抗体刺激分离胸腺细胞．BALB／c小鼠体内注射抗TCR抗体，FACS检测胸腺细胞。结果显示，CD4^ CD8^ DP胸腺细胞和CD4^-CD8^ CD3^-TCR-细胞对抗TCR抗体诱导的凋亡敏感，但CD4^-CD8^-CD3^-TCR-胸腺前体细胞自发凋亡率低，且抗TCR抗体诱导的凋亡．表明胸腺细胞对凋亡的敏感点产生于CD4^-CD8^ CD3^-TCR-细胞表达后，胸腺细胞的凋亡敏感性受发育调节。     

Apoptosis of thymic lymphoma clones by thymic epithelial cells: a putative model for 'death by neglect'

We have previously described an in vitro system in which thymic epithelial cells induce apoptosis in CD4+ 8+ thymocytes or thymic lymphoma cells, in the absence of an exogenous antigen. A thymic epithelial cell line (TEC) recapitulated the response, by inducing apoptosis in CD4+ 8+ thymocytes of the thymic lymphoma clone, PD1.6. The present study pursues the involvement of the T-cell receptor (TcR) in the response of PD1.6 to TEC. TcR cross-linking did not cause apoptosis of PD1.6, although it induced tyrosine phosphorylation of p95vav. In contrast, TEC did not induce phosphorylation of p95vav but induced apoptosis of PD1.6 cells. These results suggest that TcR-evoked signals are not involved in TEC-induced apoptosis of PD1.6. Intracellular calcium chelation, using BAPTA-loaded PD1.6 cells, diminished TEC-induced apoptosis. Protein kinase C depletion in PD1.6 cells augmented their apoptotic response to TEC. Thus, the response of PD1.6 to TEC is calcium-dependent and inhibited by PKC. Likewise, the apoptotic response of PD1.6 to A23187 was abrogated by PKC activation. PD1.6 cells may represent an immature double positive thymocyte population, which does not undergo negative selection. The interaction of PD1.6 with TEC may thus serve as a model for the TcR-independent 'Death by Neglect', which takes place in the thymus during thymocyte development.     

Inhibition of thymopoiesis of CD34+ cell maturation by HIV-1 in an in vitro CD34+ cell and thymic epithelial organ culture model

The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells. When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3- population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed. When CTE cells were preincubated with T- and M-tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+CD3- cells become HIV-infected. In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25-CD3-. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.     

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes

T-cell differentiation in the thymus depends on positive selection of CD4+CD8+ double positive (DP) thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows maturation of only those thymocytes that are capable of self-peptide-MHC recognition. Thymocytes that fail to bind self-peptide-MHC die by apoptosis. An important question in thymocyte differentiation is whether co-stimulation is required for positive selection and on which cells co-stimulatory molecules may be expressed in the thymus. The vascular cell adhesion molecule (VCAM-1) and the intercellular cell adhesion molecule (ICAM-1) are known to be potent co-stimulatory molecules in activation of peripheral T-cells by interacting with the integrins VLA-4 and LFA-1, respectively. We were prompted to investigate whether VCAM-1 and ICAM-1 may also act as co-stimulators during selection of thymocytes. By using recombinant proteins of murine VCAM-1 and ICAM-1 fused to the Fc region of human IgG1 (rVCAM-1, rICAM-1) we examined the capacity of VCAM-1 and ICAM-1 to act as co-stimulatory molecules in positive selection in vitro. Triggering the CD3/TCR complex together with co-stimulation applied by rVCAM-1 or rICAM-1 induced the generation of CD4+ single positive (SP) thymocytes from CD4+CD8+ DP thymocytes whereas either signal alone did not result in generation of CD4+ SP thymocytes. VCAM-1 and ICAM-1 act therefore as co-stimulatory molecules in thymocyte positive selection in vitro. The generation of CD4+ SP cells is accompanied by cell survival both when it was co-stimulated with rVCAM-1 and with rICAM-1. Importantly we show here that VCAM-1 expression in the murine thymus is restricted to cortical F4/80 positive hematopoietic antigen presenting cells (hAPC) present exclusively in the cortex whereas expression of ICAM-1 has been reported on the epithelium both in cortex and medulla. This suggests that not only the cortical epithelium may use the co-stimulatory molecule ICAM-1 to mediate positive selection, but also cortical hAPCs may contribute to positive selection of thymocytes by using the co-stimulator VCAM-1.     

Gene expression analysis of thymocyte selection in vivo

Self versus non-self discrimination is a key feature of immunorecognition. Through TCR-activated apoptotic mechanisms, autoreactive thymocytes are purged at the CD4(+)CD8(+) double-positive (DP) precursor stage prior to maturation to CD4(+) or CD8(+) single-positive (SP) thymocytes. To investigate this selection process in vivo, gene expression analysis by oligonucleotide array was performed in TCR transgenic mice. In total, 244 differentially expressed DP thymocyte genes induced or repressed by TCR triggering in vivo were identified. Genes involved in the biological processes of apoptosis, DNA recombination, antigen processing and adhesion are coordinately engaged. Moreover, analysis of gene expression in thymocyte subsets revealed that TCR ligand-induced expression profiles vary according to their developmental stage, with 48 genes showing DP preference and nine showing SP thymocyte preference. Finally, our data suggest that both the extrinsic and the intrinsic apoptosis pathways are operating in thymic selection.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

Enhancement of CD3-mediated thymocyte apoptosis by the cross-linkage of heat-stable antigen.

Heat-stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4-CD8- double-negative and CD4+CD8+ double-positive thymocytes but not CD4+ or CD8+ single-positive thymocytes. Effects of anti-HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single-cell suspension culture system. Immobilized R13 enhanced the CD3-mediated DNA fragmentation and killing of thymocytes but not the dexamethasone-induced or phorbol myristate acetate-induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3-mediated apoptosis when HSA and T-cell receptor (TCR)/CD3 were co-cross-linked by a cross-reactive secondary antibody. Even without the cross-reactive secondary antibody, soluble R13 enhanced CD3-mediated apoptosis, although a greater than 100-fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3-mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation-induced apoptosis of thymocytes and the involvement of HSA in negative selection.     

Sex steroids induce apoptosis of CD8+CD4+ double-positive thymocytes via TNF-alpha

T cell production by the thymus, thymic size, cellularity and output all decrease drastically after puberty. Among the candidates that may mediate this decrease are the sex steroids: hypersecretion or pharmacological administration of these hormones has long been known to induce thymic hypocellularity, and their depletion yields thymic hypercellularity. Here we show that a typical sex steroid, testosterone, specifically targets CD8+CD4+ double-positive (DP) thymocytes for apoptosis via TNF-alpha. Anti-TNF-alpha monoclonal antibodies abrogated testosterone-induced DP apoptosis, and TNF-alpha-/- DP thymocytes were largely resistant to testosterone-mediated apoptosis in vivo. Testosterone accomplished this effect by upregulating TNF-alpha production and by simultaneously sensitizing DP thymocytes to TNF-alpha. Thus, TNF-alpha is the critical mediator of sex steroid-induced apoptosis in thymocytes, and its manipulation should provide a point of intervention to modulate T cell production in sex hormone disorders.     

Altered expression of galectin-3 induces cortical thymocyte depletion and premature exit of immature thymocytes during Trypanosoma cruzi infection

During acute infection with Trypanosoma cruzi, the causative agent of Chagas' disease, the thymus undergoes intense atrophy followed by a premature escape of CD4+CD8+ immature cortical thymocytes. Here we report a pivotal role for the endogenous lectin galectin-3 in accelerating death of thymocytes and migration of these cells away from the thymus after T. cruzi infection. We observed a pronounced increase in galectin-3 expression that paralleled the extensive depletion of CD4+CD8+ immature thymocytes after infection. In vitro, recombinant galectin-3 induced increased levels of death in cortical immature thymocytes. Consistent with the role of galectin-3 in promoting cell death, thymuses from gal-3-/- mice did not show cortical thymocyte depletion after parasite infection in vivo. In addition, galectin-3 accelerated laminin-driven CD4+CD8+ thymocyte migration in vitro and in vivo induced exportation of CD4+CD8+ cells from the thymus to the peripheral compartment. Our findings provide evidence of a novel role for galectin-3 in the regulation of thymus physiology and identify a potential mechanism based on protein-glycan interactions in thymic atrophy associated with acute T. cruzi infection.     

Different role of Apaf-1 in positive selection,negative selection and death by neglect in foetal thymic organ culture

Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1-/-) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4+CD8+ (DP) thymocytes from Apaf-1-/- mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1-/- mice were crossed with the T-cell receptor (TCR)-alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha-/-Apaf-1-/- and TCR-alpha-/-Apaf-1+/+ mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1-/- Db/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33+ thymocytes and an increased percentage of the autoreactive CD8+M33+ thymocytes in male, but not female Apaf-1-/- Db/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development.     

Reactive oxygen species induced by the deletion of peroxiredoxin II (PrxII) increases the number of thymocytes resulting in the enlargement of PrxII-null thymus

In the thymus, CD4+ or CD8+ single-positive (SP) thymocytes develop and mature by positive and negative selection or undergo "death by neglect". CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)-null mice. The level ofROS in PrxII-null thymocytes is higher than that in wild-type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII-null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double-positive (DP) thymocytes in thymus of PrxII-null mice is lower than that in wild-type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII-null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII-null thymocytes than in wild-type thymocytes. In the presence of low serum concentrations, PrxII-deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin- or OKT3-stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.     

Low glucocorticoid receptor (GR), high Dig2 and low Bcl-2 expression in double positive thymocytes of BALB/c mice indicates their endogenous glucocorticoid hormone exposure

Several studies have shown that of the four major thymocyte subsets, the CD4/CD8 double positive (DP) thymocytes are the most sensitive to in vivo glucocorticoid hormone (GC)-induced apoptosis. Our aim was to analyse fine molecular differences among thymocyte subgroups that could underlie this phenomenon. Therefore, we characterised the glucocorticoid hormone receptor (GR) expression of thymocyte subgroups both at the mRNA and protein levels by real-time PCR and flow cytometry, and correlated these features to their apoptotic sensitivity. We also investigated the time-dependent effects of the GC agonist dexamethasone (DX) with or without GC antagonist (RU486) treatments on GR mRNA/protein expression. We also analysed the expression of two apoptosis-related gene products: dexamethasone-induced gene 2 (Dig2) mRNA and Bcl-2 protein. We found that DN thymocytes had the highest GR expression, followed by CD8 single positive (SP), CD4 SP and DP thymocytes in 4-week-old BALB/c mice, both at the mRNA and protein levels, respectively. In DP cells, the Dig2 expression was significanty higher, while the Bcl-2 expression was significantly lower than in DN, CD4 SP and CD8 SP thymocytes. Single high dose DX treatment caused time-dependent depletion of DP thymocytes due to their higher apoptosis rate, which could not be abolished with RU486 pretreatment. After a single high dose DX treatment, there was a transient, significant increase of the GR mRNA and protein level of unsorted thymocytes after 8 and 16 h, followed by a significant decrease at 24 h, respectively. The time-dependent GR expression changes after DX administration could not be inhibited by the GC antagonist RU486. Twenty-four hours after exposure to high dose DX the DN, CD4 SP and CD8 SP cells showed a significant decrease of GR mRNA and protein expression, whereas the DP thymocytes, showed no significant alteration of GR mRNA or protein expression. The kinetical analysis of GR expression and apoptotic marker changes upon single high dose GC analogue administration revealed a two-phase process in thymocytes: early events, within 4–8 h, include GR upregulation and early apoptosis induction, while the late events appear most prominently at 16–20 h, when the GR is already downregulated and apoptotic cell ratio reaches its peak, with marked DP cell depletion. The low GR, high Dig2 and low Bcl-2 expression, coupled with the absence of homologous downregulation of GR after exogenous GC analogue treatment, could contribute to the high GC sensitivity of DP thymocytes. The downregulated GR and Bcl-2 together with the upregulated Dig2 level in DP cells indicates the significance of intrathymic GC effects at this differentiation stage. Since GR expression changes and apoptotic events could not be completely inhibited by GC antagonist, we propose the involvement of non-genomic GR mechanisms in these processes.     

Galactoside-specific plant lectin,Viscum album agglutinin-I induces enhanced proliferation and apoptosis of murine thymocytes in vivo

Galactoside-specific plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to act as a biomodulator with proinflammatory and apoptosis-inducing effects, however its cellular targets and mechanism of immunobiological action in vivo are less well understood. Therefore, in the present work the short- and long-term in vivo effects of VAA-I on thymocyte subpopulations and peripheral T cells were tested using a murine (Balb/c) model. Cell surface CD4/CD8 staining and flow cytometry allowed us to follow the changes of thymocyte subpopulations: CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+ or CD8+ single positive (SP) and mature peripheral T cells after single or repeated injections with low doses of VAA-I. The apoptosis of the cells was detected by flow cytometry using propidium iodide (PI) and Annexin V staining. To detect the short-term effects of the lectin, the animals were investigated 24 h after a single injection of 1 or 30 ng/kg body weight (BW) VAA-I+/-1 mg/kg Dexamethasone (DX). The total number of mature CD8+ SP thymocytes increased significantly with an enhancement of the ratio of apoptotic cells. In contrast, in the blood samples an elevated CD4/CD8 ratio was found. In the next trial, Balb/c mice were treated twice weekly with 1 or 30 ng/kg VAA-I+/-1 mg/kg DX for 3 weeks. The total cell count of thymocytes showed significant increases after both doses of VAA-I, but an elevated percentage of apoptotic cells was found only after treatment with 30 ng/kg VAA-I. SP thymocytes revealed higher increases in lectin-induced apoptosis than DN or DP cells. In addition, both lectin doses significantly inhibited the DX-induced reduction of all thymocyte subpopulations investigated. In conclusion, our data suggest that VAA-I is able to modulate the maturation of thymocytes in vivo.     

Developmental regulation of bcl-2 expression in the thymus.

An important factor in shaping the T-cell receptor (TcR) repertoire during thymocyte development is the susceptibility of double-positive (CD4+ CD8+) thymocytes to induction of apoptosis (negative selection) when the TcR is engaged by 'self'-antigens. Recent evidence has suggested that this susceptibility to apoptosis may be influenced by the expression of bcl-2, a proto-oncogene known to increase the resistance to apoptosis in various cell systems. Using a semi-quantitative polymerase chain reaction (PCR) technique in conjunction with staged embryonic material and purified thymocyte subpopulations we have investigated patterns of bcl-2 expression during normal T-cell development. Our results show that while bcl-2 alpha gene expression is readily detectable in immature CD3-CD4-CD8- thymocytes and in mature single-positive TcRhi cells, it is drastically reduced in TcR negative double-positive (CD3- CD4+ CD8+) cortical thymocytes of intermediate maturity. Careful mapping of bcl-2 alpha re-expression in relation to the onset of TcR expression within the population of embryonic thymocytes indicates that bcl-2 alpha is up-regulated as soon as TcR molecules are expressed on the surface of CD4+ CD8+ thymocytes. Therefore, thymocytes susceptible to apoptosis on TcR ligation express bcl-2 alpha mRNA suggesting that changing levels of bcl-2 expression are unlikely to be the only determinant regulating susceptibility to apoptosis in the thymus. The possible implications of these changes in bcl-2 expression regarding other facets of thymocyte development will be discussed.