Objective:To observe the expression of BTLA on T cells during activation and further analyze its inhibitory effects on T cell activation in different phases.Methods: T cells from PBMC were enriched by negative selection using magnetic beads.Expression of BTLA,CTLA-4 and PD-1 on freshly isolated human T cells and kinetics expression of BTLA,CILA-4 and PD-1 on CD3 mAb stimulated T cells were examined by flow cytometry.T cells were stimulated by anti-CD3 mAb combined with anti-CD28 mAb in the presence of anti-BTLA mAb 8H9,then T cell proliferation was tested by MTT assay in the different culture time.Immature DCs were generated from monocytes cultured in the medium containing GM-CSF and IL-4, and further driven to maturation by anti-CD40 mAb.Expression of HVEM on DCs was measured by flow cytometry.T cells were co-cultured with DCs in the presence of soluble 8H9 or anti-HVEM antibody to block HVEM-BTLA interaction,T cell proliferation was measured by MTT assay.Results:Freshly isolated T cells exhibited high levels of BTLA expression, but not CTLA-4 and PD-1.After T cell activation, BTLA expression decreased on first 2 days, with rapidly increasing to high levels.Unlike BTLA, expression of CTLA-4 and PD-1 was gradually increased during T cell activation.8H9 significantly inhibited the proliferation of T cell stimulated by CD3 mAb and CD28 mAb.8H9 could still exhibit inhibitory effect on T cell proliferation after 24 h or 48 h of preactivation by CD3 mAb plus CD28 mAb stimulation.HVEM was highly expressed on immature DCs, and down-regulated on mature DCs.Blockade of BTLA by soluble 8H9 or anti-HVEM antibody enhanced DC-mediated T cell proliferation within 48 h.Conclusion: BTLA signal enhances the threshold of T cell activation and plays importantly negative regulatory role in the initiation and early phase of T cell activation.

Objective:To study the effects of agonistic anti gp130 monoclonal antibody B S12 on the differentiation, maturation and function of dendritic cells (DC).Methods:Monocytes isolated from human peripheral blood were cultured with GM CSF plus IL 4, and differentiated into immature DC. The phenotype of DC was analyzed by cytometry after the addition of B S12 antibody to the culture of immature DC. In addition, the abilities for DC to uptake antigen, secrete IL 12, initiate the mixed lymphocyte reaction and chemoattract T cells were tested. The effects of agonistic anti CD40 monoclonal antibody 5C11 on the differentiation, maturation and function of DC were simutaneously compared with those of B S12 antibody.Results:Agonistic anti gp130 monoclonal antibody B S12 had DC to up regulate the expression of CD1a, costimulatory molecules CD80 and CD86 as well as CD83, which is special marker for mature DC, down regulate the expression of CD14. Moreover, B S12 antibody decrease the up take of antigen by DC, enhance the abilities for DC to secrete IL 12, initiate the mixed lymphocyte reaction and chemoattract T cells. The comparison of roles of B S12 and 5C11 antibodies in DC showed that 5C11 was more effective than B S12.Conclusion:The direct stimulation of gp130 on immature DC by B S12 antibody could induce immature DC to differentiate into mature DC.

Objective To research the mechanism of the changes of T lymphocyte subtypes and provide reference for clinically prevention,diagnosis and treatment for NSCLC through analysis of the expression of Th1 ,Th2 in Non-small-cell carcinoma (NSCLC)patients.Methods Whole blood (EDTA anticoagulant treatment)from 60 NSCLC patients and 60 healthy sub-j ects were collected to detect of the expression of CD3 +T cells,CD4+T cells and CD8+T cells on T lymphocytes and the lev-els of Th1 and Th2 cells by flow cytometer (FCM),and the absolute value of T lymphocyte by hematology analyzer.Results Compared with normal control group,after surgery 1~3 days NSCLC groups,the percent of CD3 +T,CD4+T,CD8+T cells and the CD4+/CD8+ ratio in the NSCLC patients before surgery were significantly reduced 58.40±10.27 vs 66.58± 6.84,31.32±8.65 vs 39.40±6.43,34.23±8.00 vs 24.31±8.16,0.96±0.23 vs 1.58±0.23 (t=-6.726~14.916,P<0.05).The percent of CD3 +T,CD4+T,CD8+T cells and the CD4+/CD8+ ratio in the NSCLC patients after surgery 1~3 days were also significantly decreased 56.31±8.00 vs 66.58±6.84,27.72±7.55 vs 39.40±6.43,33.69±7.10 vs 24.31± 8.16,0.87±0.31 vs 1.58±0.23 (t=-6.720~14.367,P<0.05).The percent of CD4+T cells in the NSCLC patients af-ter surgery 4~7 days was increased 33.23±4.13 vs 39.40±6.43(t=6.257,P<0.05).Compared with the control group, within the helper T cell subsets,the cell content of Th1,Th2 cells (× 10/μl)and the Th1/Th2 ratio were significantly changed in different extent in the NSCLC group before surgery 6.79±1.34 vs 12.52±3.56,4.82±0.51 vs 2.32±0.82, 1.39±0.84 vs 5.36±1.42 (t=-20.087~18.630,P<0.05).The content of Th1 cells was lower in the NSCLC patients after 1~3 days and 4~7 days 8.86±1.52 vs 12.52±3.56,7.02±1.27 vs 12.52±3.56 (t=7.339~11.275,P<0.05). Conclusion The NSCLC patients presented immune dysfunction,like T lymphocytes and helper T cells decreased and Th2 cells were clearly in the ascendant.Also,the cytotoxic T cells increased by the stimulation of cancer cells,but they began to decrease after the surgery.

AIM: To prepare the efficient tumor-DC vaccines, dendritic cells(DC) derived from 6-8 weeks Balb/c mice bone marrow progenitor cells were pulsed by apoptotic SP2/0 tumor cells and induced maturation by SP2/0 tumor lysates supernatants. Then SP2/0 tumor burdening Balb/c mice were immunized by the tumor-DC vaccines to observe the therapeutic effects in vivo .METHODS: Immature DC were derived by recombinant murine GM-CSF and IL-4, then were pulsed by SP2/0 apoptotic cells. Tumor-DC vaccines were stimulated by LPS and SP2/0 tumor lysates supernatants prepared by four cycles repetitive freezing and thawing, respectively. -thymidine incorporation test and standard 4h [ 51 Cr] release assay were used to detect the proliferation and activation of cytotoxic T lymphocytes (CTL) stimulated by DC in vitro . (4-5)?10 5 DC were immunized in the right inguen of SP2/0 tumor burdening Balb/c mice and most mice received three cycles immunization every two weeks. Changes of the tumor and mice life-spans were recorded. RESULTS: In vitro proliferation and activation of CTL induced by the tumor-DC vaccines of tumor lysates supernatants or LPS stimulation group were more powerful than other groups ( P

Objective: To prepare anti-VSIG4 monoclonal antibodies and characterize their biological functions.Methods: BALB/c mice were immunized with transfected cell line (L929/VSIG4L) as immunogen.The spleen B cells of the mice were fused with SP2/0 and hybridoma cells were screened with transfected cell line (L929/VSIG4) by FCM.After acquisition of the hybridomas secreting anti-VSIG4 mAb,their biological activities were investigated by indirect immunofluorescence,Western blot,competitive inhibition test,and MTT assay.Results:Two stable hybridomas,9A7 and 9D5 were obtained,which could continuously secrete specific anti-VSIG4 monoclonal antibodies.The following biological activity studies showed that these monoclonal antibodies could recognize the natural VSIG4 expressed on the macrophages and several cancer cell lines,such as Jurkat,THP-1 and H446.Furthermore,they could block the inhibitory effects of VSIG4 on proliferation of T cells in vitro.Conclusion: Two hybridomas secreting anti-VSIG4 monoclonal antibodies have been established.These monoclonal antibodies provide useful tools for further studying VSIG4's biological function and its unknown receptor.

OBJECTIVETo explore the effect of recombinant human soluble CD(40) ligand (rhsCD(40)L) and CD(40)L cDNA transfected cell (CD(40)L-TC) on the behavior of malignant B lymphocytes, and investigate the possibility of using rhsCD(40)L as a new bio-factor in tumor immunotherapy.

METHODrhsCD(40)L and CD(40)L-TC were obtained by gene recombinant techniques. Multiple myeloma cell lines, XG2, XG7, U266 and 8226, B-lymphoma cell lines, Raji and Daudi were selected to detect responses to rhsCD(40)L and CD(40)L-TC stimulation. Cell growth curve, cell cycle, early apoptosis as well as membrane surface molecules on these cell lines were analyzed.

RESULTS(1) The expression levels of CD(40) molecule on malignant B lymphocytes showed heterogeneity. High level of CD(40) on XG2, moderate on 8266, Raji, and Daudi, and no expression on U266 and XG7 were detected. The rhsCD(40)L stimulation gave rise to a typical homo-type cell aggregation of XG2 and Daudi. Meanwhile, at least 10 to 20 of CD(40)(+) XG2 or CD(40)(+) Daudi cells were found adherent to one pre-treat ed CD(40)L-TC. (2) Co-incubation with rhsCD(40)L (5 micro g/ml), or CD(40)L-TC (tumor cell: CD(40) = 5:1) resulted in a significant inhibition of in vitro cell growth of XG2, Raji and Daudi, with G(1)-phase arrest for XG2 and G(2)-phase for Raji and Daudi. These two kinds of CD(40) stimulators induced XG2, Raji and Daudi cells to apoptosis in vitro. The apoptotic rate for XG2 was 23.3% (rhsCD(40)L) and 18.8% (CD(40)L-TC), for Daudi 14.2% and 15.9%, and for Raji 11.6% and 8.9% respectively. (3) Phenotype analysis showed that CD(95) expression levels were significantly up-regulated on XG2, Raji and Daudi after stimulation with rhsCD(40)L or CD(40)L-TC, and CD(80) and CD(18) expression levels on Raji were respectively enhanced and decreased.

CONCLUSIONThe abilities to directly inhibit XG2, Daudi and Raji cell proliferation, to induce themapoptosis, as well as to up-regulate immune co-stimulator molecule CD(80) expression on Raji cells would make rhsCD(40)L a potential bio-factor for tumor immuno-therapy.

B-Lymphocytes ; drug effects ; metabolism ; pathology ; CD40 Antigens ; metabolism ; CD40 Ligand ; genetics ; metabolism ; pharmacology ; Cell Division ; drug effects ; Coculture Techniques ; DNA, Complementary ; genetics ; Humans ; Lymphoma, B-Cell ; metabolism ; pathology ; Recombinant Proteins ; pharmacology ; Time Factors ; Transfection ; Tumor Cells, Cultured ; drug effects