Antigen and glucocorticoid hormone (GC) induce positive selection of DP thymocytes in a TcR transgenic mouse model

Thymocyte maturation in the thymus is controlled by stromal and humoral components. Among the humoral regulators locally produced glucocorticoids (GCs) seem to have a key role in the positive selection of thymocytes. Our previous studies have shown that the administration of GCs or the stimulation through the CD3 complex can induce apoptosis of double positive (DP) cells, but the combined presence of these stimuli induces positive selection. In this work our aim was to investigate the effects of antigen exposure and synthetic GC hormone (dexamethasone, DX) administration on the selection processes of DP cells in TcR transgenic mice. In our model, AND—pigeon cytochrome c (PCC)-specific I-E^k (MHC-II) restricted Vβ3, V11 TcR expressing transgenic mice were treated with PCC, with high or low dose DX, or with PCC and DX together, followed by the analysis of total thymocyte numbers, thymocyte composition, with regard to their CD69, Vβ3 and Annexin V expression. The administration of PCC and/or DX for 2 days resulted in a decreased DP cell number and a significantly increased CD4 SP cell ratio. However, in both cases the total thymocyte numbers decreased. CD69 expression increased on both DP and CD4 SP cells after PCC and/or DX treatments. We found that after DX or combined treatment, the percentage of Annexin V positive cells increased. The ratio of Vβ3 TcR bearing DP thymocytes showed no change after DX or PCC administrations alone, but it decreased significantly after combined treatment. MHC-II bound PCC peptides in the presence of GCs enhanced the maturation of Vβ3+ DP cells into CD4 SP stage, therefore, the Vβ3− cells remained mostly in the DP immature stage. These data indicate that both antigen and low dose GC alone are capable of inducing positive selection of DP cells, but together they gave a stronger effect in promoting positive selection. From these we conclude that GCs influence the maturation and selection processes of thymocytes.     

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.     

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。     

CXCR4 tropic human immunodeficiency virus type 1 induces an apoptotic cascade in immature infected thymocytes that resembles thymocyte negative selection

HIV-1 often replicates in the thymus of infected individuals, causing thymocyte depletion and thymic dysfunction. Nevertheless, the mechanisms by which thymocyte depletion occurs are not clear. Here we report that HIV-1 infection induced apoptosis primarily in productively infected thymocytes; aldrithiol-2 or Efavirenz treatment largely abrogated HIV-1-induced apoptosis. Moreover, X4-HIV-1 induced apoptosis primarily in immature CD4+ CD8+ (DP) thymocytes whereas most mature CD4 or CD8 single-positive (SP) thymocytes were resistant to X4 HIV-1-induced apoptosis despite infection. Consistent with this, we observed significant induction of several genes involved in negative selection of DP thymocytes. Furthermore, treatment of thymocytes with cycloheximide abrogated HIV-1-induced apoptosis, implying a requirement for de novo protein synthesis. Our results suggest that HIV-1-induced apoptosis of thymocytes requires the activation of caspases and the participation of mitochondrial apoptosis effectors, which serve to amplify the apoptotic signal, a process similar to that elaborated during thymocyte negative selection.     

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.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.     

CD4单克隆抗体改变新生小鼠胸腺细胞亚群的分布和功能

新生期小鼠（３～７天）腹腔注入ＣＤ４ＭｃＡｂ，可引起胸腺细胞表型和功能变化。胸腺细胞表型分析显示：胸腺细胞总数、ＤＰ和ＣＤ４ＳＰ细胞明显减少；ＣＤ８ＳＰ细胞数增加；ＤＮ细胞数目变化不大。上述变化与ＣＤ４ＭｅＡｂ和胸腺细胞表面的ＣＤ４分子的结合有关。功能试验表明：实验组小鼠胸腺细胞对有丝分裂素ＣｏｎＡ、ＰＷＭ的反应性降低，混合淋巴细胞反应（ＭＬＲ）也明显低于对照组。但是胸腺细胞对异型细胞的杀伤作用则高于对照组。功能试验的结果与表型的变化是吻合的，有剂量依赖性。ＣＤ４ＭｃＡｂ引起的胸腺细胞表型和功能的变化是可以恢复的，一般于注射ＣＤ４ＭｃＡｂ后１０天，胸腺细胞亚群分布恢复或接近正常，而功能则恢复较慢。     

Development of autoreactive diabetogenic T cells in the thymus of NOD mice

Type 1 diabetes results from destruction of pancreatic beta cells by beta cell-specific autoreactive T cells in the nonobese diabetic (NOD) mouse. Defects in thymic negative selection are thought to result in failure to delete potential beta cell-reactive T cells, contributing to the development of autoimmune diabetes. We investigated this possibility by comparing the deletion profile of double-positive (DP) thymocytes in NOD mice with diabetes-resistant strains of mice after anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway. We found that immature NOD CD4+CD8+ DP thymocytes have a lower activation threshold than C57BL/6 and Balb/c thymocytes. This was confirmed by showing that NOD DP thymocytes have a higher level of ERK and JNK phosphorylation. The low activation threshold of immature thymocytes resulted in rapid deletion of strongly activated immature DP thymocytes by negative selection, whereas weakly activated immature thymocytes differentiated more efficiently into CD69+CD3high DP thymocytes by positive selection. SP thymocytes, particularly CD4-CD8+ T cells that were efficiently generated from activated DP thymocytes, could induce severe insulitis and diabetes in NOD.scid mice. We conclude that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes in NOD mice.     

Developmental shift in TcR-mediated rescue of thymocytes from glucocorticoid-induced apoptosis

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.     

Killing of immature CD4+CD8+ thymocytes in vivo by anti-CD3 or 5′-(N-ethyl)-carboxamido-adenosine is blocked by glucocorticoid receptor antagonist RU-486

Negative selection in thymus occurs by apoptosis in CD4⁺CD8¹ cells. These immature thymocytes are readily killed, both in vitro and in vivo, by glucocorticoid treatment. Increased levels of intracellular cAMP in vitro also induce apoptosis of thymocytes and T cell receptor (TcR) stimulation potentiate cAMP responses through receptors linked to adenylic cyclase. Presently, we have tested the possibility that TcR-mediated apoptosis in vivo may require the glucocorticoid receptor (GR) as a downstream, intracellular mediator. Use of the GR antagonist RU-486, 24 h before and simultaneous with, anti-CD3 or 5′-(N-ethyl)-carboxamide-adenosine (NECA) treatment, resulted in a selective inhibition of CD4⁺CD8⁺ thymocyte death. In addition, a low dose of glucocorticoid potentiated thymocyte death induced by anti-CD3 monoclonal antibodies. These data support a model in which thymic negative selection depends on a defined set of transduction signals which potentiate the GR to become responsive to endogenous levels of glucocorticoid.     

Accelerated transition from the double-positive to single-positive thymocytes in G alpha i2-deficient mice

Deletion of alpha i2 subunit of heterotrimeric G proteins induces a 2-4-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes as compared with wild-type littermates, but how G alpha i2 is involved in thymocyte development is unknown. To determine a role for G alpha i2 in a specific developmental stage of thymocyte differentiation, we studied the ontogeny of thymocytes in G alpha i2-deficient mice. Our data show that an accelerated transition from the double-positive (DP) to SP thymocytes, rather than impairment in thymic emigration, accounts for a high proportion of the SP thymocytes in the absence of G alpha i2. Lack of G alpha i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The augmented response may be the reason behind the expedited transition from the DP to SP thymocytes in the animal. In accordance with this, effects of G alpha i2 deficiency on CD8 or CD4 SP thymocyte differentiation required engagement of the TCRs with either MHC class I or MHC class II molecule. The abnormal thymocyte development resulted in an increase in positive selection, altered usage of TCR Vbeta gene, aberrant development of CD4+ CD25+ T regulatory cells and untimely thymic involution, the contribution of which to colitis development in the animal is discussed. These findings reveal a previously unappreciated role for G alpha i2 protein in clonal selection and functionality of thymocytes.     

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.     

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.     

TCR signaling inhibits glucocorticoid-induced apoptosis in murine thymocytes depending on the stage of development

Signaling by either the TCR or glucocorticoid receptor (GR) induces apoptosis in thymocytes. Interestingly, it has been shown previously that hybridoma T cells escape apoptosis induced by either TCR or GR when both of these receptors signal simultaneously. Whether such mutual antagonism is present in primary thymocytes was the subject of the present study. Both glucocorticoids (GC) and anti-TCR/CD28 (or anti-CD3/CD28) mAb induced apoptosis in total thymocytes. When these signals were present at the same time, GC-induced apoptosis was partially inhibited by TCR/CD3 signaling. Costimulation by anti-CD28 enhanced the inhibitory effects of anti-CD3 on GC-induced apoptosis about 30-fold. However, subset analysis revealed that most cells rescued from GC-induced apoptosis were mature CD4+ and CD8+ thymocytes, and these cells were resistant to TCR/CD3-induced apoptosis in the absence of GC. Similar results were obtained with mature splenic CD4+ and CD8+ T cells. TCR/CD3 signaling alone, while inducing apoptosis in CD4+(CD8+)TCRlow thymocytes, rescued a small subset of CD4+(CD8+)TCRlow thymocytes from GC-induced apoptosis. Thus, TCR signaling increasingly reverses GC-induced apoptosis as thymocyte development progresses. As GC are infinitely present in vivo, these findings support a model wherein TCR signaling may be required to prevent GC-induced apoptosis both under basal and immune challenging conditions.     

The frequency of double-positive thymocytes expressing an alphabeta TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing alphabeta T-cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T-cell compartment size. Scid recipients were inoculated with various ratios of TCR Calpha(0/0) and wild-type bone marrow (BM) stem cells. Increasing the frequency of TCR Calpha(0/0) thymocytes at steady-state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12-14 weeks following BM inoculation, the frequency of TCR Calpha(0/0) DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild-type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency-dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady-state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady-state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.     

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.     

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.