Objective To investigate the effect of ionizing radiation on the expression of p16, CyclinDl, and CDK4 in mouse thymocytes and splenocytes. Methods Fluorescent staining and flow cytometry analysis were employed for th...Objective To investigate the effect of ionizing radiation on the expression of p16, CyclinDl, and CDK4 in mouse thymocytes and splenocytes. Methods Fluorescent staining and flow cytometry analysis were employed for the measurement of protein expression. Results In time course experiments, it was found that the expression of p16 protein was significantly increased at 8, 24, and 48 h for thymocytes (P<0.05, P<0.01, and P<0.05, respectively) and at 24 h for splenocytes (P<0.05) after whole body irradiation (WBI) with 2.0 Gy X-rays. However, the expression of CDK4 protein was significantly decreased from 8 h to 24 h for thymocytes (P<0.05,P<0.01) and from 8 h to 72 h for splenocytes (P<0.05-P<0.01). In dose effect experiments, it was found that the expression of p16 protein in thymocytes and splenocytes was significantly increased at 24 h after WBI with 1.0, 2.0, and 4.0 Gy (P<0.05-P<0.01), whereas the expression of CDK4 protein was significantly decreased with 2.0Gy for thymocytes (P<0.05) and 0.5-6.0 Gy for splenocytes (P<0.05-P<0.01). Results also showed that the expression of CyclinDl protein decreased markedly in both thymocytes and splenocytes after exposure. Conclusion The results indicate that the expression of p 16 protein in thymocytes and splenocytes can be induced by ionizing radiation, and the p16-CyclinD1/CDK4 pathway may play an important role for G1 arrest of thymocytes induced by X-rays.展开更多
Aim: To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection.Methods: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymu...Aim: To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection.Methods: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymus were collect-ed . Blood and thymus suspension smears were prepared for quantitative histochemistry and immunohistochemistry studyunder light and electron microscopes. Results; In testes resected mice the size and the weight of thymus weremarkedly increased. The demarcation between cortex and medulla was clear. The cortex was thickened and the celldensity was increased. The ratio of cortex/medulla stereometry was increased. The total cell count, thymocyte count,the percentage of acid a-naphthyl acetate esterase (ANAE) positive thymocytes, nonlymphocytes and the rosette forma-tion of macrophages and thymocytes were all increased. The thymocytes surrounded closely to the light thymic epithelialcells, dendritic cells or macrophages. The lymphocytes, particularly the ANAE positive lymphocytes of peripheralblood were increased. Conclusion; After bilateral testicular resection, the thymus of aged male mice showed mor-phological regeneration and the thymocytes and its microenvironment appeared to be definitely improved. It is suggestedthat testicular resection may improve immune function. (Asian J Androl 2001 Dec; 3; 271 — 275)展开更多
LKB1 is a serine/threonine kinase that directly activates the energy sensor AMP-activated protein kinase （AMPK） in response to bioenergetic stress, and mainly acts as a tumor suppressor that controls cell polarity a...LKB1 is a serine/threonine kinase that directly activates the energy sensor AMP-activated protein kinase （AMPK） in response to bioenergetic stress, and mainly acts as a tumor suppressor that controls cell polarity and proliferation. Although LKB1 is expressed in multiple tissues including the thymus and the spleen, its roles in T-cell development and function remain unknown. Here, we show that T-cell-specific deletion of LKB1 resulted in reduced survival of double-positive （DP） thymocytes and impaired generation of both CD4 and CD8 single-positive thymocytes. Disruption of LKB1 not only prevented the activation of AMPK but also impaired the expression of anti-apoptotic protein BcI-XL. Importantly, ectopic expression of either BcI-XL or the constitutively active AMPK mutant significantly rescued DP thymocytes from LKB1 deficiency-induced cell death. Moreover, ectopic expression of the constitutively active AMPK mutant was found to restore the expression of BcI-XL in LKB1-deficient DP thymocytes. These findings identify LKB1 as a critical factor for the survival of DP thymocytes through regulation of AMPK activation and Bcl-XL expression.展开更多
T cells are derived from progenitor thymocytes, of which only a minority receive the appropriate TCR signal, undergo positive selection and mature. Owing to the very short lifespan of thymocytes, the prerequisite for ...T cells are derived from progenitor thymocytes, of which only a minority receive the appropriate TCR signal, undergo positive selection and mature. Owing to the very short lifespan of thymocytes, the prerequisite for posi- tive selection is survival. TCR signal-induced Bcl-2 expression is believed to play a dominant role in the survival of positively selecting thymocytes, but how Bcl-2 is directly regulated is unknown. Here we report that the immediate early gene （IEG） c-Fos can stimulate the expression of Bcl-2, depending on a specific AP-l-binding site in the Bcl-2 promoter. In c-Fos transgenic （Fos-Tg） mice, c-Fos binds to this site and promotes the expression of Bcl-2. As a result, Fos-Tg thymocytes exhibited enhanced survival, and more mature single-positive （SP） thymocytes were generated, even on a unique TCR background. The TCR repertoire remained normal in Fos-Tg mice. Our results identified c-Fos as the mediator of the stimulatory effect of TCR signaling on Bcl-2 expression. Therefore, c-Fos, as an IEG, because of its early response ability, can quickly rescue the survival of short-lived thymocytes during positive selection. Our results provide novel insight into the mechanism regulating the survival of positively selecting thymocytes.展开更多
Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exactbiological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavilydispute...Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exactbiological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavilydisputed, since data supporting both views have been reported in several independent studies. To clarify thisissue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the presentstudy, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis inthymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and afterinduction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering ofapoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptoticprocesses. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expressionwas restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolyticcleavage site and is therefore predicted to encode a monomeric protein. The biological function undernormal circumstances, however, will need further investigations for clarification. While apoptosis could notmodulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted inup-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control ofcell activation-mediated rather than apoptosis- induced signals.展开更多
Objective:To determine the effect of an immunosuppressive active component (periploside A) isolated from the stem bark of Periplocae Cortex (Periploca sepium Bge.),a Chinese medicinal herb used in the treatment of rhe...Objective:To determine the effect of an immunosuppressive active component (periploside A) isolated from the stem bark of Periplocae Cortex (Periploca sepium Bge.),a Chinese medicinal herb used in the treatment of rheumatoid arthritis for centuries in China,on positive selection of thymocytes in vitro.Methods:Female C57BL/6 mice at 6 weeks of age were housed in specific pathogen-free conditions.Double-positive thymocytes from C57BL/6 mice were induced into positive selection in vitro with or without periploside A treatment.Cell viability and expression of CD69,CD4,and CD8 were analyzed by flow cytometry.Results:Flow cytometric examination of thymocyte populations revealed that the percentage of CD8+ single-positive thymocytes was decreased by periploside A upon differentiation induced by an anti-CD3 antibody.However,the percentage of CD4+ single-positive thymocytes was decreased by periploside A upon differentiation induced by phorbol 12-myristate 13-acetate/ionomycin.Expression of CD69 plays a major role in prohibiting differentiation of thymocytes.Treatment with periploside A decreased CD69 expression in thymocytes.Conclusion:These results demonstrate that periploside A influences positive selection of thymocytes in vitro.展开更多
Supernatants from human primary thymic epithelial cell culture were collected. The proliferationpromoting effect of the supernatant on mouse thymocyte, as well as on Tcell line 85, Be 13, HDMar, Peer, Loucy, Molt4, R...Supernatants from human primary thymic epithelial cell culture were collected. The proliferationpromoting effect of the supernatant on mouse thymocyte, as well as on Tcell line 85, Be 13, HDMar, Peer, Loucy, Molt4, Reh and Jurkat, were observed. The results demonstrated that the supernatants could increase spontaneous 3 HTdR intake of mouse thymocyte, promote ConAinduced thymocyte proliferation and stimulate the proliferation of 85 cell or the cells of HDMar, Loucy and Jurkat at stationary phase, but did not display any effect on Be13, Peer, Molt4 and Reh cells.展开更多
The MTEC1 cell line, established in our laboratory, is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constitutively produce multiple cytokines. The selection of thymic microe...The MTEC1 cell line, established in our laboratory, is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constitutively produce multiple cytokines. The selection of thymic microenvironment on developing T cells was investigated in an in vitro system. Un-separated fresh thymocytes from Balb/c mice were cocul-tured with MTECl cells or/and MTEC1-SN,then,the viability, proliferation and phenotypes of cultured thymocytes were assessed. Without any exogenous stimulus, both MTECl cells and MTECl -SN were able to maintain the viability of thymocytes, while only the MTECl cells, not the MTECl -SN, could directly activate thymocytes to exhibit moderate proliferation, indicating that the proliferative signal is delivered through cell surface interactions of MTECl cells and thymocytes. Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTECl cells preferentially activate the subsets of CD4+ CDS', CD4+ CD8+ and CD4- CD8- thymocytes; whereas MTEC1- SN preferentially maintained the viability of CD4+ CD8- and CD4-CD8+ thymocyte subsets.For the Con A-activated thymocytes, both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency, phenotyped as CD4+CD8-, CD4-CD8+, and CD4- CD8- subsets. In summary, MTEC1 cells displayedselective support to the different thymocyte subsets , and the selectivity is dependent on the status of thymocytes.展开更多
Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendr...Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^+8^+ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class Ⅰ and class Ⅱ molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.展开更多
Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring ...Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring at fetal day (Fd) 14 to 17, Fd 18 to day (D) 1 after birth, D 2 to D 5 and D6 thereafter. The first three proliferation waves coincided with the generation of CD4/'CD8/' (DP), TCR+CD4/hiCD8-/lo (CD4 SP), and TCR+CD4-/loCD8int/hi (CD8 SP) thymocytes, respectively. The transition from DN to DP cells was further investigated and it was found out that there were two differential pathways via immature single positive (ISP) cells in the BALB/c mice, each functioning at different fetal ages. One is via TCR-CD4-CD8+ cells, occurring between Fd 15 and Fd 17 and the other is via TCR-CD4+CD8- cells, occurring from Fd 17 until birth. In contrast, the TCR-CD4-CD8+ pathway dominated overwhelmingly in the C57BL/6 mice. These findings shed new light on the hypothesis that the differential pathway preference varies with mouse strains. With respect to the shift in the intensity of CD4 and CD8 expression on thymocytes from fetal to adult mice, the TCR+CD4/hiCD8-/lo, and TCR+CD4-/loCD8int/hi subsets might be equivalent to the medullary type TCR+CD4/CD8 SP cells.展开更多
The aim of the present study was to determine whether the sensitivity of thymocytes to X-ray radiation depends on their proliferative states and whether radiation impairs the maturation of donor-derived thymocytes in ...The aim of the present study was to determine whether the sensitivity of thymocytes to X-ray radiation depends on their proliferative states and whether radiation impairs the maturation of donor-derived thymocytes in recipient thymus.We assigned 8-week-old C57BL/6J mice into three treatment groups：1） untreated;2） X-ray radiation;3） X-ray radiation plus bone marrow transplantation with donor bone marrow cells from transgenic mice express-ing enhanced green fluorescent protein（GFP） on a universal promoter.After 4 weeks,the size of the thymus,the number and proliferation of thymocytes and ratios of different stage thymocytes were analyzed by immunohisto-chemistry and flow cytometry.The results showed that：1） CD4＋CD8＋ thymocytes were more sensitive to X-ray radiation-induced cell death than other thymocytes;2） the proliferative capacity of CD4＋CD8＋ thymocytes was higher than that of other thymocytes;3） the size of the thymus,the number of thymocytes and ratios of thymo-cytes of different stages in irradiated mice recovered to the normal level of untreated mice by bone marrow trans-plantation;4） the ratio of GFP-positive CD4＋CD8＋ thymocytes increased significantly,whereas the ratio of GFP-positive CD4＋ or CD8＋ thymocytes decreased significantly.These results indicate that the degree of sensitivity of thymocytes to X-ray radiation depends on their proliferative states and radiation impairs the maturation of donor-derived CD4＋CD8＋ thymocytes in recipient thymus.展开更多
Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in sple...Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.展开更多
Using terminal deoxynucleotide transferase mediated dUTP digoxigenin nick end labeling (TUNEL) assay and propidium iodide DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the p...Using terminal deoxynucleotide transferase mediated dUTP digoxigenin nick end labeling (TUNEL) assay and propidium iodide DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the process of programmed cell death of thymocytes in vitro were investigated. It was noticed that thymocytes bound to MTSC4 used in this study. That the percentages of apoptotic nuclei of the bound thymocytes on MTSC4 were much higher than those of medium cultured thymocytes, while the bound thymocytes on mouse thymic epithelial cell (MTEC1) showed much lower percentages of apoptosis. FACS analysis quantitatively confirmed the observation. Phenotype analysis showed that MTSC4 induced the deletion of CD4+CD8+ cells and CD4+CD8 cells in 18 h of coculture. The results suggest that the negative selection of medullary thymocytes may be achieved by thymic dendritic cells through their enhancing effects on apoptosis.展开更多
Murine CD4+CD8- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2- cells are less functional, whereas the Qa-2+ cells are fully functional. Evidence is provided here that the transition from Qa...Murine CD4+CD8- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2- cells are less functional, whereas the Qa-2+ cells are fully functional. Evidence is provided here that the transition from Qa-2- to Qa-2+ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2+ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2+CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2+CD4SP thymocytes. The profile of cytokines secreted by MTECl-induced Qa-2+CD4SP thymocytes was Thy 0 type specified by the production of IL-2, IL-4 and IL-6. The results suggest that Qa-2-CD4SP thymocytes may give rise to the Qa-2+CD4SP thymocytes, and acquire fully functional competence in thymic medulla under the foster of local epithelial cells.展开更多
A mouse chemokine MIP-2(macrophage in-flamatory protein 2) is constitutively expressed not only by peritoneal macrophages, but also by fresh thymic stromal cells, based on RT-PCR detection. Moreover, the specific rece...A mouse chemokine MIP-2(macrophage in-flamatory protein 2) is constitutively expressed not only by peritoneal macrophages, but also by fresh thymic stromal cells, based on RT-PCR detection. Moreover, the specific receptor of MIP-2 is expressed at different levels among four main subgroups of murine thymocytes including DN, DP, CD4SP and CD8SP. By the chemotaxis assays with Boyden chamber, we proved that the recombinant mouse MIP-2 can chemoattract the four main subgroups of thymocytes in different degrees, it mainly chemoattract the DP and SP subgroups. We firstly reported that MIP-2 is involved in the regulation of the directional migration of developing thymocytes.展开更多
Phenotypic analysis of the medullary-type CD4 CD8+ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CD8+T cells is TCRαβ+CD3+Qa-2+HSA3G11 6C10 ...Phenotypic analysis of the medullary-type CD4 CD8+ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CD8+T cells is TCRαβ+CD3+Qa-2+HSA3G11 6C10 , whereas in the medullary-type CD8SP thymocytes, 20% are Qa-2+; 33%, HAS ; 30%, 3G11 ; and 70% are 6C10 . The disparate expression patterns of these four cell surface markers suggest that medullary-type CD8SP thymocytes may undergo phenotypic maturation process. According to the distribution of these four celi surface markers, six subgroups of CD8SP thymocytes have been identified. The precursor-progeny relationship along with developmental pathway is postulated as follows: 6C10+HSA+3G11 Qa-2 - 6C10+HSA+ 3G11+Qa-2 6C10 HSA+3G11+Qa-2 -6C10 HSA 3G11 +Qa-2 -6C10 HSA 3G11 Qa-2 -6C10 HA S 3G11 Qa-2+, the cells inthe last subgroup exit the thymus and home into periphery.展开更多
TCRαβTCD4-CD8+ thymocytes are heterogeneity. They may undergo phenotypic and functional maturation within thymic medulla. Medullary-type CD8SP thymocytes were divided into seven subsets based on phenotypic analysis,...TCRαβTCD4-CD8+ thymocytes are heterogeneity. They may undergo phenotypic and functional maturation within thymic medulla. Medullary-type CD8SP thymocytes were divided into seven subsets based on phenotypic analysis, and their precursor-progeny relationship along with the differential pathway was also delineated. To further testify the validity of the maturation pathway, we purified 6C10-CD69+ cells representing the early stage and 6C10-Qa-2+ cells representing the later stage among medullary-type CD8SP thymocytes and compared their functional maturation levels. CD8+ T cells of spleen were used as the control. It is shown that there is no obvious difference of proliferation ability among these three subsets; however, intracytoplasmic cytokine assay shows that there is a hierarchy of IFN-γ and TNFα secretion among these subsets, strikingly comparable to their phenotypic status among medullary type CD8SP thymocytes. The bioassays of IL-2 and IFN-γ in culture supernatant give the similar results.展开更多
The presence of a relatively mature CD4+CD8- (SP)T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with ...The presence of a relatively mature CD4+CD8- (SP)T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with a phenotype of CD69 +/- , HSAmed/lo and heterogeneous for Qa - 2 expression. The proliferation capability of TCRαβ+ 3G11-6C10- CD4+ CD8- thymocytes was high while using Con A stimulus. And Con A stimulation could result in secretion of IL-4, IL-10, IL-6 and a little amount of IFNγ. IL-2 was barely detectable. This is distinct from typical Th0 type cytokines. The cells of this subset were NK1.1 negative, but strongly expressed GATA-3 mRNA. The results suggest that the CD4+ subset of 3G11 - 6C10- NK1.1 - phenotype possesses immunocompetent cells with functions characteristic of Th2-like cytokines, which may indicate the cells at transitional status from ThO to Th2, with a propensity to Th2.展开更多
Thymic medullary type epithelial cell line (MTEC1), which expressed H\|2D d and Ia\+d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H\|2\+b mice reconstituted wit...Thymic medullary type epithelial cell line (MTEC1), which expressed H\|2D d and Ia\+d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H\|2\+b mice reconstituted with H\|2 b×d F1 bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, in \%in vitro\% functional assays, were analyzed to investigate whether the MTEC1 cells \%in vivo\% could induce the production of H\|2\+d restricted antigen\|specific T cells. The H\|2\+d restricted VSV\|antigen specific proliferating and IL\|2 producing T cells as well as H\|2\+d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H\|2\+d self\|antigen. On the contrary, H\|2\+d restricted antigen\|specific and H\|2\+d self\|antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of "second thymic selection" in thymic medulla has been postulated and its implication discussed.展开更多
PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-cultu...PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-culture study. The aim of this research is to investigate the character of PF18-3 mAb recognized molecule ( PF18-3 molecule) and its role in MTSC4-induced thymocyte apoptosis. The characterization of PF18-3 molecule expression has been conducted by FACS analysis. PF18-3 molecules have been found to express on MTSC4 as well as on Con A activated but not freshly isolated thymocytes. Up-regulated expression of PF18-3 molecules has been also observed on thymocytes after being co-cultured with MTSC4 for 48 h. The results from FACS analyses by Pl staining for detecting apoptosis-related hypodiploid and by PF18-3 mAb staining reveal that PF18-3 molecules expresss specifically on the apoptotic subgroup of thymocytes with high hypodiploid content. The PF18-3 molecule expressed on apoptotic thymocytes展开更多
基金This work was supported by a grant from the National Natural Science Foundation of China(No.39770193).
文摘Objective To investigate the effect of ionizing radiation on the expression of p16, CyclinDl, and CDK4 in mouse thymocytes and splenocytes. Methods Fluorescent staining and flow cytometry analysis were employed for the measurement of protein expression. Results In time course experiments, it was found that the expression of p16 protein was significantly increased at 8, 24, and 48 h for thymocytes (P<0.05, P<0.01, and P<0.05, respectively) and at 24 h for splenocytes (P<0.05) after whole body irradiation (WBI) with 2.0 Gy X-rays. However, the expression of CDK4 protein was significantly decreased from 8 h to 24 h for thymocytes (P<0.05,P<0.01) and from 8 h to 72 h for splenocytes (P<0.05-P<0.01). In dose effect experiments, it was found that the expression of p16 protein in thymocytes and splenocytes was significantly increased at 24 h after WBI with 1.0, 2.0, and 4.0 Gy (P<0.05-P<0.01), whereas the expression of CDK4 protein was significantly decreased with 2.0Gy for thymocytes (P<0.05) and 0.5-6.0 Gy for splenocytes (P<0.05-P<0.01). Results also showed that the expression of CyclinDl protein decreased markedly in both thymocytes and splenocytes after exposure. Conclusion The results indicate that the expression of p 16 protein in thymocytes and splenocytes can be induced by ionizing radiation, and the p16-CyclinD1/CDK4 pathway may play an important role for G1 arrest of thymocytes induced by X-rays.
文摘Aim: To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection.Methods: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymus were collect-ed . Blood and thymus suspension smears were prepared for quantitative histochemistry and immunohistochemistry studyunder light and electron microscopes. Results; In testes resected mice the size and the weight of thymus weremarkedly increased. The demarcation between cortex and medulla was clear. The cortex was thickened and the celldensity was increased. The ratio of cortex/medulla stereometry was increased. The total cell count, thymocyte count,the percentage of acid a-naphthyl acetate esterase (ANAE) positive thymocytes, nonlymphocytes and the rosette forma-tion of macrophages and thymocytes were all increased. The thymocytes surrounded closely to the light thymic epithelialcells, dendritic cells or macrophages. The lymphocytes, particularly the ANAE positive lymphocytes of peripheralblood were increased. Conclusion; After bilateral testicular resection, the thymus of aged male mice showed mor-phological regeneration and the thymocytes and its microenvironment appeared to be definitely improved. It is suggestedthat testicular resection may improve immune function. (Asian J Androl 2001 Dec; 3; 271 — 275)
基金Acknowledgments We thank X Wu （Fudan University） for LckCre mouse and K Wong （Dana-Farber Cancer Institute） for LKB1 mouse, R Bosselut （National Institutes of Health） and D Li （Shanghai Institutes for Biological Sciences） for instructive comments on the manuscript We are grateful to our colleagues F Liu for animal husbandry, W Bian for cell sorting and X Wang for real-time PCR analysis. This research was supported in part by the National Natural Science Foundation of China （30872290, 30925031）, the Ministry of Science and Technology （2006CB504303, 2007CB815802, 2009ZX 10004-105）, the Hi-Tech Research and Development Program of China （2007AA02Z167）, the National Basic Research Program of China （2007CB914504） and the Chinese Academy of Sciences （KSCX 1-YW-R-43, KSCX2-YW-R-10）.
文摘LKB1 is a serine/threonine kinase that directly activates the energy sensor AMP-activated protein kinase （AMPK） in response to bioenergetic stress, and mainly acts as a tumor suppressor that controls cell polarity and proliferation. Although LKB1 is expressed in multiple tissues including the thymus and the spleen, its roles in T-cell development and function remain unknown. Here, we show that T-cell-specific deletion of LKB1 resulted in reduced survival of double-positive （DP） thymocytes and impaired generation of both CD4 and CD8 single-positive thymocytes. Disruption of LKB1 not only prevented the activation of AMPK but also impaired the expression of anti-apoptotic protein BcI-XL. Importantly, ectopic expression of either BcI-XL or the constitutively active AMPK mutant significantly rescued DP thymocytes from LKB1 deficiency-induced cell death. Moreover, ectopic expression of the constitutively active AMPK mutant was found to restore the expression of BcI-XL in LKB1-deficient DP thymocytes. These findings identify LKB1 as a critical factor for the survival of DP thymocytes through regulation of AMPK activation and Bcl-XL expression.
文摘T cells are derived from progenitor thymocytes, of which only a minority receive the appropriate TCR signal, undergo positive selection and mature. Owing to the very short lifespan of thymocytes, the prerequisite for posi- tive selection is survival. TCR signal-induced Bcl-2 expression is believed to play a dominant role in the survival of positively selecting thymocytes, but how Bcl-2 is directly regulated is unknown. Here we report that the immediate early gene （IEG） c-Fos can stimulate the expression of Bcl-2, depending on a specific AP-l-binding site in the Bcl-2 promoter. In c-Fos transgenic （Fos-Tg） mice, c-Fos binds to this site and promotes the expression of Bcl-2. As a result, Fos-Tg thymocytes exhibited enhanced survival, and more mature single-positive （SP） thymocytes were generated, even on a unique TCR background. The TCR repertoire remained normal in Fos-Tg mice. Our results identified c-Fos as the mediator of the stimulatory effect of TCR signaling on Bcl-2 expression. Therefore, c-Fos, as an IEG, because of its early response ability, can quickly rescue the survival of short-lived thymocytes during positive selection. Our results provide novel insight into the mechanism regulating the survival of positively selecting thymocytes.
基金This work was supported by grant No. R05-2001-000-00464-0 from the Basic Research Program of the Korea Science and Engineering Foundation. The authors are very thankful to M.L. Cowan for corrections and suggestions to the text.
文摘Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exactbiological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavilydisputed, since data supporting both views have been reported in several independent studies. To clarify thisissue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the presentstudy, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis inthymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and afterinduction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering ofapoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptoticprocesses. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expressionwas restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolyticcleavage site and is therefore predicted to encode a monomeric protein. The biological function undernormal circumstances, however, will need further investigations for clarification. While apoptosis could notmodulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted inup-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control ofcell activation-mediated rather than apoptosis- induced signals.
基金This study was supported by the National Natural Science Foundation of China(30901909).
文摘Objective:To determine the effect of an immunosuppressive active component (periploside A) isolated from the stem bark of Periplocae Cortex (Periploca sepium Bge.),a Chinese medicinal herb used in the treatment of rheumatoid arthritis for centuries in China,on positive selection of thymocytes in vitro.Methods:Female C57BL/6 mice at 6 weeks of age were housed in specific pathogen-free conditions.Double-positive thymocytes from C57BL/6 mice were induced into positive selection in vitro with or without periploside A treatment.Cell viability and expression of CD69,CD4,and CD8 were analyzed by flow cytometry.Results:Flow cytometric examination of thymocyte populations revealed that the percentage of CD8+ single-positive thymocytes was decreased by periploside A upon differentiation induced by an anti-CD3 antibody.However,the percentage of CD4+ single-positive thymocytes was decreased by periploside A upon differentiation induced by phorbol 12-myristate 13-acetate/ionomycin.Expression of CD69 plays a major role in prohibiting differentiation of thymocytes.Treatment with periploside A decreased CD69 expression in thymocytes.Conclusion:These results demonstrate that periploside A influences positive selection of thymocytes in vitro.
文摘Supernatants from human primary thymic epithelial cell culture were collected. The proliferationpromoting effect of the supernatant on mouse thymocyte, as well as on Tcell line 85, Be 13, HDMar, Peer, Loucy, Molt4, Reh and Jurkat, were observed. The results demonstrated that the supernatants could increase spontaneous 3 HTdR intake of mouse thymocyte, promote ConAinduced thymocyte proliferation and stimulate the proliferation of 85 cell or the cells of HDMar, Loucy and Jurkat at stationary phase, but did not display any effect on Be13, Peer, Molt4 and Reh cells.
基金grants from the China medical board, USA,and from the national foundation of natural scirnces,China
文摘The MTEC1 cell line, established in our laboratory, is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constitutively produce multiple cytokines. The selection of thymic microenvironment on developing T cells was investigated in an in vitro system. Un-separated fresh thymocytes from Balb/c mice were cocul-tured with MTECl cells or/and MTEC1-SN,then,the viability, proliferation and phenotypes of cultured thymocytes were assessed. Without any exogenous stimulus, both MTECl cells and MTECl -SN were able to maintain the viability of thymocytes, while only the MTECl cells, not the MTECl -SN, could directly activate thymocytes to exhibit moderate proliferation, indicating that the proliferative signal is delivered through cell surface interactions of MTECl cells and thymocytes. Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTECl cells preferentially activate the subsets of CD4+ CDS', CD4+ CD8+ and CD4- CD8- thymocytes; whereas MTEC1- SN preferentially maintained the viability of CD4+ CD8- and CD4-CD8+ thymocyte subsets.For the Con A-activated thymocytes, both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency, phenotyped as CD4+CD8-, CD4-CD8+, and CD4- CD8- subsets. In summary, MTEC1 cells displayedselective support to the different thymocyte subsets , and the selectivity is dependent on the status of thymocytes.
基金This work was funded by grants from Major State Bascic Research Development Program of China(No.G1999053904)National Natural Science Foundation of China(No.30271200)Young Scentist Cooperation Research Fund(No.39928019).
文摘Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^+8^+ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class Ⅰ and class Ⅱ molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.
基金supported by grants from National 973 Program in China(No.G1999053904)National Natural Sciences Foundation of China(No.39730410)
文摘Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring at fetal day (Fd) 14 to 17, Fd 18 to day (D) 1 after birth, D 2 to D 5 and D6 thereafter. The first three proliferation waves coincided with the generation of CD4/'CD8/' (DP), TCR+CD4/hiCD8-/lo (CD4 SP), and TCR+CD4-/loCD8int/hi (CD8 SP) thymocytes, respectively. The transition from DN to DP cells was further investigated and it was found out that there were two differential pathways via immature single positive (ISP) cells in the BALB/c mice, each functioning at different fetal ages. One is via TCR-CD4-CD8+ cells, occurring between Fd 15 and Fd 17 and the other is via TCR-CD4+CD8- cells, occurring from Fd 17 until birth. In contrast, the TCR-CD4-CD8+ pathway dominated overwhelmingly in the C57BL/6 mice. These findings shed new light on the hypothesis that the differential pathway preference varies with mouse strains. With respect to the shift in the intensity of CD4 and CD8 expression on thymocytes from fetal to adult mice, the TCR+CD4/hiCD8-/lo, and TCR+CD4-/loCD8int/hi subsets might be equivalent to the medullary type TCR+CD4/CD8 SP cells.
基金supported by an operating grant(No.BK2008440) to DSM from the Science and Technology Department of Jiangsu province,China
文摘The aim of the present study was to determine whether the sensitivity of thymocytes to X-ray radiation depends on their proliferative states and whether radiation impairs the maturation of donor-derived thymocytes in recipient thymus.We assigned 8-week-old C57BL/6J mice into three treatment groups：1） untreated;2） X-ray radiation;3） X-ray radiation plus bone marrow transplantation with donor bone marrow cells from transgenic mice express-ing enhanced green fluorescent protein（GFP） on a universal promoter.After 4 weeks,the size of the thymus,the number and proliferation of thymocytes and ratios of different stage thymocytes were analyzed by immunohisto-chemistry and flow cytometry.The results showed that：1） CD4＋CD8＋ thymocytes were more sensitive to X-ray radiation-induced cell death than other thymocytes;2） the proliferative capacity of CD4＋CD8＋ thymocytes was higher than that of other thymocytes;3） the size of the thymus,the number of thymocytes and ratios of thymo-cytes of different stages in irradiated mice recovered to the normal level of untreated mice by bone marrow trans-plantation;4） the ratio of GFP-positive CD4＋CD8＋ thymocytes increased significantly,whereas the ratio of GFP-positive CD4＋ or CD8＋ thymocytes decreased significantly.These results indicate that the degree of sensitivity of thymocytes to X-ray radiation depends on their proliferative states and radiation impairs the maturation of donor-derived CD4＋CD8＋ thymocytes in recipient thymus.
文摘Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.
文摘Using terminal deoxynucleotide transferase mediated dUTP digoxigenin nick end labeling (TUNEL) assay and propidium iodide DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the process of programmed cell death of thymocytes in vitro were investigated. It was noticed that thymocytes bound to MTSC4 used in this study. That the percentages of apoptotic nuclei of the bound thymocytes on MTSC4 were much higher than those of medium cultured thymocytes, while the bound thymocytes on mouse thymic epithelial cell (MTEC1) showed much lower percentages of apoptosis. FACS analysis quantitatively confirmed the observation. Phenotype analysis showed that MTSC4 induced the deletion of CD4+CD8+ cells and CD4+CD8 cells in 18 h of coculture. The results suggest that the negative selection of medullary thymocytes may be achieved by thymic dendritic cells through their enhancing effects on apoptosis.
基金Project supported by the National Natural Science Foundation of China and China Medical Board, Rockefeller Foundation.
文摘Murine CD4+CD8- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2- cells are less functional, whereas the Qa-2+ cells are fully functional. Evidence is provided here that the transition from Qa-2- to Qa-2+ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2+ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2+CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2+CD4SP thymocytes. The profile of cytokines secreted by MTECl-induced Qa-2+CD4SP thymocytes was Thy 0 type specified by the production of IL-2, IL-4 and IL-6. The results suggest that Qa-2-CD4SP thymocytes may give rise to the Qa-2+CD4SP thymocytes, and acquire fully functional competence in thymic medulla under the foster of local epithelial cells.
基金This work was supported by the International Center for Genetic Engineering and Biotechnology (ICGEB) (Grant No. 98/016) and the National "973" Project of China (Grant No. G1999053904).
文摘A mouse chemokine MIP-2(macrophage in-flamatory protein 2) is constitutively expressed not only by peritoneal macrophages, but also by fresh thymic stromal cells, based on RT-PCR detection. Moreover, the specific receptor of MIP-2 is expressed at different levels among four main subgroups of murine thymocytes including DN, DP, CD4SP and CD8SP. By the chemotaxis assays with Boyden chamber, we proved that the recombinant mouse MIP-2 can chemoattract the four main subgroups of thymocytes in different degrees, it mainly chemoattract the DP and SP subgroups. We firstly reported that MIP-2 is involved in the regulation of the directional migration of developing thymocytes.
文摘Phenotypic analysis of the medullary-type CD4 CD8+ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CD8+T cells is TCRαβ+CD3+Qa-2+HSA3G11 6C10 , whereas in the medullary-type CD8SP thymocytes, 20% are Qa-2+; 33%, HAS ; 30%, 3G11 ; and 70% are 6C10 . The disparate expression patterns of these four cell surface markers suggest that medullary-type CD8SP thymocytes may undergo phenotypic maturation process. According to the distribution of these four celi surface markers, six subgroups of CD8SP thymocytes have been identified. The precursor-progeny relationship along with developmental pathway is postulated as follows: 6C10+HSA+3G11 Qa-2 - 6C10+HSA+ 3G11+Qa-2 6C10 HSA+3G11+Qa-2 -6C10 HSA 3G11 +Qa-2 -6C10 HSA 3G11 Qa-2 -6C10 HA S 3G11 Qa-2+, the cells inthe last subgroup exit the thymus and home into periphery.
基金supported by the National Natural Science Foundation of China(Grant No.39230320)the National"973"Project
文摘TCRαβTCD4-CD8+ thymocytes are heterogeneity. They may undergo phenotypic and functional maturation within thymic medulla. Medullary-type CD8SP thymocytes were divided into seven subsets based on phenotypic analysis, and their precursor-progeny relationship along with the differential pathway was also delineated. To further testify the validity of the maturation pathway, we purified 6C10-CD69+ cells representing the early stage and 6C10-Qa-2+ cells representing the later stage among medullary-type CD8SP thymocytes and compared their functional maturation levels. CD8+ T cells of spleen were used as the control. It is shown that there is no obvious difference of proliferation ability among these three subsets; however, intracytoplasmic cytokine assay shows that there is a hierarchy of IFN-γ and TNFα secretion among these subsets, strikingly comparable to their phenotypic status among medullary type CD8SP thymocytes. The bioassays of IL-2 and IFN-γ in culture supernatant give the similar results.
基金Project supported by the National Natural Science Foundation of China (Grant No. 39730410).
文摘The presence of a relatively mature CD4+CD8- (SP)T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with a phenotype of CD69 +/- , HSAmed/lo and heterogeneous for Qa - 2 expression. The proliferation capability of TCRαβ+ 3G11-6C10- CD4+ CD8- thymocytes was high while using Con A stimulus. And Con A stimulation could result in secretion of IL-4, IL-10, IL-6 and a little amount of IFNγ. IL-2 was barely detectable. This is distinct from typical Th0 type cytokines. The cells of this subset were NK1.1 negative, but strongly expressed GATA-3 mRNA. The results suggest that the CD4+ subset of 3G11 - 6C10- NK1.1 - phenotype possesses immunocompetent cells with functions characteristic of Th2-like cytokines, which may indicate the cells at transitional status from ThO to Th2, with a propensity to Th2.
文摘Thymic medullary type epithelial cell line (MTEC1), which expressed H\|2D d and Ia\+d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H\|2\+b mice reconstituted with H\|2 b×d F1 bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, in \%in vitro\% functional assays, were analyzed to investigate whether the MTEC1 cells \%in vivo\% could induce the production of H\|2\+d restricted antigen\|specific T cells. The H\|2\+d restricted VSV\|antigen specific proliferating and IL\|2 producing T cells as well as H\|2\+d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H\|2\+d self\|antigen. On the contrary, H\|2\+d restricted antigen\|specific and H\|2\+d self\|antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of "second thymic selection" in thymic medulla has been postulated and its implication discussed.
文摘PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-culture study. The aim of this research is to investigate the character of PF18-3 mAb recognized molecule ( PF18-3 molecule) and its role in MTSC4-induced thymocyte apoptosis. The characterization of PF18-3 molecule expression has been conducted by FACS analysis. PF18-3 molecules have been found to express on MTSC4 as well as on Con A activated but not freshly isolated thymocytes. Up-regulated expression of PF18-3 molecules has been also observed on thymocytes after being co-cultured with MTSC4 for 48 h. The results from FACS analyses by Pl staining for detecting apoptosis-related hypodiploid and by PF18-3 mAb staining reveal that PF18-3 molecules expresss specifically on the apoptotic subgroup of thymocytes with high hypodiploid content. The PF18-3 molecule expressed on apoptotic thymocytes
