Cytotoxic T cell (CTL) covers several subtypes, which are CD8+, CD4 and CD4-CD8-. CTL derives from T cell repertoire in lymphoid hematopoietic stem cells. It matures in thymus and is activated in peripheral lymphoid tissues. Effector CTL kills the target cells by 2 ways. One is apoptotic effect mediated by FasL-Fas pathway and the other one is cytolytic effect mediated by granzymes. CTL has aroused great attention due to its significance in anti-tumor and anti-virus.
Animals ; Fas Ligand Protein ; Humans ; Membrane Glycoproteins ; immunology ; Perforin ; Pore Forming Cytotoxic Proteins ; T-Lymphocytes, Cytotoxic ; immunology ; fas Receptor ; immunology

OBJECTIVETo investigate the mechanism of immune escape in renal cell carcinoma(RCC).

METHODSFas and FasL expressions were examined by immunohistochemical technique in 44 RCC patients, with the Ki67 expression and apoptosis of tumor infiltrating lymphocytes(TIL) monitored simultaneously. Cytokines including IL2 and IFN alpha were used to induce the expression of the renal carcinoma cell lines 786-0 cells. Combination treatment of 786-0 with cytokines and Anti-Fas monoclonal antibody (FasAb) was used to induce apoptosis. FasL function was assessed by in vitro co-culture assays using renal cancer cells 786-0 and Fas-sensitive Jurkat T-cells.

RESULTS(1) Fas expression rate in RCC(22.8%) was lower than that in the controlled normal kidney tissues(53.8%, P < 0.01). FasL expression rate in RCC (46.5%) was higher than that in the controlled normal kidney tissues (23.2%, P < 0.01). That of Ki67 was 32.8%, with the expressions of Fas and Ki67 showing a negative correlation (r = -0.62, P < 0.05). In contrast, the expressions of FasL and Ki67 showed a positive correlation. (r = 0.93, P < 0.01). The Fas expression of stage I was significantly higher than that of stages III and IV. The expression rate of FasL in RCC was significantly increased with RCC stage (P < 0.01). (2) The apoptotic rate of TIL in RCC (33.9%) was significantly higher than that of the normal kidney tissues (3.5%, P < 0.01). The expression of FasL and the apoptotic rate of TIL in RCC gave a positive correlation (r = 0.96, P < 0.01). (3) Fas expression rate in 786-0 cells was 13.7%. The apoptotic rate mediated by FAsAb was 9.6%. IFN alpha was able to up-regulate the Fas expression and subsequently augment the FasAb-mediated apoptosis in 786-0 cells. But IL2 did not show similar effects. (4) The FasL expression rate of 786-0 was 18.6%. FasL expressed by 786-0 cells was able to induce apoptosis of Jurkat T-cells in co-culture assays and the apoptosis of Jurkat T-cells was significantly lowered after blocking the effect of FasL with Fas-neutralizing antibody NOK-2, giving the apoptotic rates of 14.9% and 2.0%, respectively, the difference therein is statistically significant (P < 0.01).

CONCLUSIONDown-regulation of Fas expression and up-regulation of FasL-expression are the mechanisms through which the RCC cells escape from immune attack.

Adult ; Aged ; Carcinoma, Renal Cell ; immunology ; Fas Ligand Protein ; Female ; Humans ; Ki-67 Antigen ; immunology ; Kidney Neoplasms ; immunology ; Male ; Membrane Glycoproteins ; immunology ; Middle Aged ; fas Receptor ; immunology

OBJECTIVE: To observe the expression of Fas receptor and ligand in human laryngocarcinoma cell line, Hep-2 and to investigate the possible mechanism of immune escape through Fas/FasL pathway in Hep-2 cell. METHOD: The mRNA and protein expressions of Fas and FasL in Hep-2 cell were analyzed by RT-PCR and flow cytometry (FCM). Growth curve of Jurkat cell was drawer based on the results of MMT, and apoptosis of Jurkat cell were determined by FCM and Hoechst 33342 staining after coculturing with Hep-2 cell. RESULT: The expressions of Fas and FasL in Hep-2 cell line were evaluated by flow cytometry and the mean fluorescence intensity were (32.91 +/- 5.6) and (25.57 +/- 7.1) respectively. After coincubation with Hep-2 cell (1 X 10(9)/L), the apoptosis rates of Jurkat cells were (38.95 +/- 0.11) % and (13.28 +/- 0.14) %, with planting concentration at 1 x 10(8)/L and 5 x 10 (8)/L respectively. In contrast, the apoptosis rate of Jurkat cultured separately was (7.53 +/- 0.17)%. The proliferation of Jurkat cell was obviously inhibited after coculture. However, the apoptosis rate was significantly decreased after adding neutralizing antibody of FasL. CONCLUSION Laryngocarcinoma cell could induce apoptosis of T lymphocytes through Fas-FasL system, thus it provided a potential mechanism to escape from immune surveillance of host.
Apoptosis ; Cell Line ; immunology ; Cell Line, Tumor ; Fas Ligand Protein ; metabolism ; Flow Cytometry ; Humans ; T-Lymphocytes ; immunology ; Tumor Escape ; fas Receptor ; metabolism

In order to investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in CTLL-2 cells (mouse CTL cell line), and to explore a new way for enhancing the ability of T cells against Leukemia in donor lymphocytes infusion, CTLL-2 cells were transfected with pEGFP-RZ596 and pEGFPC1 (mock-transfected) via electroporation. Fas expression on CTLL-2 cells was detected by RT-PCR and Western blot. The killing effect of CTL against WEHI-3 (mouse acute myelomonocytic leukemia cell line) highly expressing FasL in vitro was detected by MTT assay. The caspase-3 proteolytic activity and the apoptosis rate of CTLL-2 cells were detected by means of BD AproAlert Caspase-3 Colorimetric kit and FITC labeled Annexin-V apoptosis detecting kit respectively. The results showed that the anti-Fas ribozyme could be successfully introduced into mouse CTLL-2 cells; Fas expression on the surface of cells transfected with the ribozyme was obviously decreased, in comparison with control and mock-transfected cells; after cocultured with WEHI-3 cells, the viability of CTLL-2 cells transfeced with the ribozyme was significantly increased, as compared with other two groups; caspase-3 activity and apoptosis rate of the ribozyme-transfeced cells were significantly decreased, the killing effect of CTLL-2 transfected with the ribozyme was stronger than that of other groups. It is concluded that anti-Fas ribozyme can remarkably decrease Fas expression on CTLL-2 cells, so as to avoid Fas-mediated apoptosis by Fas ligand on WEHI-3 cells, and to enhance their killing activity against WEHI-3 cells, as a result, the immune escape of acute myelomonocytic leukemia was depressed.
Animals ; Cell Line ; Fas Ligand Protein ; immunology ; Leukemia, Myelomonocytic, Acute ; immunology ; Mice ; RNA, Catalytic ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Tumor Cells, Cultured ; Tumor Escape ; genetics ; immunology

To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in preventing graft-versus-host disease (GVHD), the CD34+ cells transfected with FasL or not, used as stimulus cells, were mixed with allo-antigen specific T lymphocytes in presence or absence of IFN-gamma and IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry (FCM). The affects of these two cytokines on CD34+ cells in the graft were also compared. The ratio of apoptosis of T cells was 12.1+/-1.5% when CD34+ cells transfected with FasL was used as stimulus cells, much higher than that of CD34+ cells non-transfected (3.2+/-1.1%, P<0.01). And in presence of IFN-gamma or IL-2, the ratio reached 20.1+/-2.3%, 17.6+/-1.3% respectively (P<0.01). However, IFN-gamma up-regulated Fas expression of CD34+ cells and increased the sensibility of CD34+ cells to soluble FasL (sFasL); IL-2 showed no such effect. It is possible to induce apoptosis of the allo-antigen specific T cells of grafts activated by allo-antigen by exogenous Fas ligand expressed on recipient cells and this might provide a new approach for preventing GVHD and IL-2 may be more suitable for clinical application.
Antigens, CD34 ; biosynthesis ; immunology ; Apoptosis ; Cytotoxicity, Immunologic ; DNA, Complementary ; genetics ; Fas Ligand Protein ; Graft vs Host Disease ; prevention & control ; Interferon-gamma ; biosynthesis ; immunology ; Interleukin-2 ; biosynthesis ; immunology ; Membrane Glycoproteins ; biosynthesis ; immunology ; T-Lymphocytes ; cytology ; physiology ; fas Receptor ; biosynthesis ; immunology

OBJECTIVETo investigate the feasibility of strategy of allograft tolerance induction by injection of FasL-decorated donor splenocytes.

METHODSChimeric FasL with core streptavidin (SA-FasL) was efficiently displayed on the surface of splenocytes by the technology of ProtEx™. Heterotopic heart transplant procedures were performed from donor WF rats to recipient ACI rats, F344 rats were used as third-party. Intraperitoneal injection of ACI rats with "decorated" WF splenocytes was used as the approach to induce tolerance in this study. According to different therapeutic strategies, three groups were set up: SA-FasL group (n = 23), SA group (n = 20) and naive splenocytes only group (n = 8). No treatment group was regarded as control (n = 10). Adoptive transfer was underwent with injection of splenocytes from tolerant recipients into naive ACI followed by heart transplant procedures. Mixed lymphocyte reaction (MLR) and third party transplantation were performed to detect allogenic tolerance.

RESULTSThe injection of ACI rats with WF rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. There were 70% cardiac allografts in SA-FasL group achieved long term survival, and it was significantly higher than the rats in other groups (P < 0.05). Adoptive transfer of splenocytes from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary WF grafts. Donor specific tolerance was identified by MLR and third-party transplant.

CONCLUSIONThe direct display of SA-FasL on the cell membrane in a rapid and efficient manner provides a practical and clinically applicable means of immunomodulation for tolerance induction with considerable therapeutic potential for transplantation.

Animals ; Fas Ligand Protein ; genetics ; immunology ; Heart Transplantation ; immunology ; Male ; Rats ; Rats, Inbred ACI ; Rats, Inbred F344 ; Rats, Inbred WF ; Spleen ; cytology ; metabolism ; Tissue Donors ; Transplantation Tolerance ; immunology

OBJECTIVETo analyze the significance of Fas-FasL in NOD insulitis and to explore the mechanism of the autoimmune diabetes.

METHODSThirty-two female NOD mice, 3-32 weeks of age, were selected. The blood glucose concentrations were recorded. The pathological data were obtained from the HE staining of the pancreatic sections and the immunohistochemical staining, in which insulin, Fas, FasL, CD8 were detected.

RESULTSDiabetes was found from the age of 14 weeks. In normal islets, insulin + cells accounted for (59.37 +/- 1.21)%, and some islet cells were observed expressing Fas. At the age of 6 weeks, insulitis lesions could be found. The average score of insulitis tended to rise with the increasing age (P < 0.0005). Meanwhile, insulin + cells decreased (P < 0.0005), and correlated negatively with scoring (P < 0.05). Fas+ islet cells increased (P < 0.0005), correlated positively with scoring (P < 0.01). In insulitis lesions, islet cells expressed FasL that increased gradually (P < 0.0005) and correlated positively with scoring (P < 0.01). The infiltrating cells were all Fas negative. But these mononucleated cells showed the expression of FasL and CD8, both increasing gradually (P < 0.0005). Furthermore, there was certain correlation between the expression of some antigens: in islet cells, between Fas and insulin (negative, P < 0.01), insulin and FasL (negative, P < 0.01), and Fas and FasL (positive, P < 0.01). In the infiltrating cells, the expression of CD8 was correlated with FasL (positively, P < 0.01); it was also found that there was a negative correlation between Fas+ islet cells and CD8+ mononucleated cells (P < 0.05).

CONCLUSIONTo sum up, there may be some important and complicated effects by Fas-FasL on the damage of beta cells and the regulation of autoreactive T cells in NOD insulitis, which will facilitate further studies in human type 1 diabetes.

Animals ; Autoimmune Diseases ; etiology ; immunology ; metabolism ; Diabetes Mellitus, Experimental ; etiology ; immunology ; Fas Ligand Protein ; Female ; Inflammation ; immunology ; metabolism ; Insulin ; blood ; Islets of Langerhans ; immunology ; metabolism ; Membrane Glycoproteins ; physiology ; Mice ; Mice, Inbred NOD ; fas Receptor ; physiology

OBJECTIVETo investigate apoptosis of tumor infiltrating dendritic cells (TIDC) and their expression of Fas/FasL (CD95/CD95L) in human endometrioid adenocarcinoma.

METHODSThe apoptotic rate of TIDC was measured in 45 cases of endometrioid adenocarcinoma and 20 cases of normal endometrium tissues (control) by double-label immunohistochemistry using the monoclonal antibody S-100 protein and TUNEL technique. The expressions of Fas and FasL in TIDCs were detected using double-label immunohistochemistry and imaging analysis.

RESULTSThe apoptotic rate of TIDCs in endometrioid adenocarcinoma were significantly higher than that in normal endormetrium [(13.02∓0.64)% vs (6.82∓0.53)%, P<0.05]. The expression levels of Fas in the TIDCs were significantly lower, whereas FasL expression significantly higher in endometrioid adenocarcinoma than in normal endormetrium (7.88∓1.05 vs 19.25∓3.03, P<0.05; 12.95∓2.25 vs 7.51∓1.14, P<0.05).

CONCLUSIONIncreased apoptosis of the TIDCs and abnormal expression of Fas/FasL in TIDCs in endometrioid adenocarcinoma may lead to tumor immune escape.

Apoptosis ; physiology ; Carcinoma, Endometrioid ; immunology ; metabolism ; pathology ; Case-Control Studies ; Dendritic Cells ; immunology ; Endometrial Neoplasms ; immunology ; metabolism ; pathology ; Fas Ligand Protein ; genetics ; metabolism ; Female ; Humans ; Lymphocytes, Tumor-Infiltrating ; immunology ; Tumor Escape ; fas Receptor ; genetics ; metabolism

OBJECTIVETo investigate the expression of FasL in rat cryptorchidism and its significance.

METHODSTwenty-four male SD rats (22-day old) were randomly divided into two groups: unilateral cryptorchid group (n = 12) and pseudo-operation group (n = 12). When the rats were 110-day old, blood samples were taken and the rats were killed for analysis. Immunohistochemical method (SP) was used to detect FasL expression in testes and ELISA method to detect serum antisperm antibody (AsAb).

RESULTSThe positive FasL expression rates in cryptorchid and contralateral testes were significantly higher than those in pseudo-operation group (P < 0.001). The serum AsAb positive rates in the cryptorchid group and the pseudo-operation group were 41.7% and 0, respectively, with significant difference(P < 0.01).

CONCLUSIONSFasL expression upregulating in both testes of the unilateral cryptorchid rat may be a protective response of the testis to autoimmunity.

Animals ; Autoantibodies ; blood ; Cryptorchidism ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Fas Ligand Protein ; biosynthesis ; Immunohistochemistry ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatozoa ; immunology ; Testis ; metabolism ; Up-Regulation

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.
Transplantation, Homologous/immunology ; Transforming Growth Factor beta1/*immunology ; Sertoli Cells/*immunology/*transplantation ; Mice, Inbred C57BL ; Mice ; Male ; Graft Survival/*immunology ; Female ; Fas Ligand Protein/*immunology ; Complement System Proteins/*immunology ; Clusterin/*immunology ; Cells, Cultured ; Cell Survival ; Animals