Experimental study on in vitro preparation of adoptive immunotherapy cells induced by CD40 in combination with cytokines / 中国免疫学杂志

Objective:To explore a new method of the cultivation of adoptive immunotherapy cells.Methods: Mononuclear cells was isoplated by density gradient centrifugation and then proliferated by using CD 40-agonist monoclonal antibody 5C11、cytokine of IFN-αand IL-7(CD40 group)in vitro.During the culturing procedure ,the cell morphology was obersved by optical microscope.The percentage of T-lymphocytes, NK-T cells, Treg cells and the cell proliferation, which were compared with CIK group CD3mAb activated,was detected on the 9th day.Results:There was no significant difference of CD 4+/CD8+T cells percentage between the two groups.But the Treg cells percentage of CIK group was far higher than that of CD 40 group,while the percentage of CD3+CD56+NK-T cells was lower than that of CD 40 group.And a group of Mo-NK-DC cells were observed in the CD 40 group.Conclusion: The new method of adoptive immunity therapy has been established in this study could increase the percentage of NK -T cells which had the ability to kill tumor cells.Simultaneously ,it is reduced the amount of Treg cells significantly.

The research of antitumor activities in vitro of DCs loading antigen prouced by radiofrequency ablation of tumor combined with CIK cells / 国际肿瘤学杂志

Objective To study the in vitro anti-tumor activity of dendritic cells (DCs) loading with antigen produced by radiofrequency ablation of tumor lysate in situ combined with cytokine-induced killer cells (CIK).Methods CIK ceils derived from BALB/C mouse spleen and DCs derived from bone marrow were prepared,and experimental model of murine colon carcinoma were established for radiofrequency ablation.The supernatant of tumor tissue in situ lysis after repeated freezing and thawing were tested by lowry protein quantitative statutory,amounting to a final concentration of 5 μg/ml,then load to the first 5 days of culture DCs (Ag-DC),2 days later,co-cultured with CIK cells after the first seven days of culture 48 h (Ag-DC-CIK).Flow cytometry was used to analyze costimulatory molecules on the surface of the cells,and CCK-8 assay to detect in vitro cytotoxic activity.Results The DCs loading with antigen resulted in an increase in the proportion of CD86 + CD11 c +,MHC Ⅱ + CD11 c + and MHC Ⅱ + CD80 + cells.The main effector cells of CIK cells were CD3 + NK1.1 + cells.The percentage of CD3 + NK1.1 + cells was 1.45％ on the first day of the culture ; while when they had been cultured for 7 days,the percentage CD3 + NK1.1 + significantly increased to 36.9％.The cytotoxicity of Ag-DC-CIK cells toward C26 cells was much more efficient than that of DC-CIK,CIK cells.The cytotoxic activity of the former was significantly lower than the latter and the same target ratio.When the ratios of effector cells to target cells were 5 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (74.9 ± 3.5) ％,; while the DC-CIK was (71.2 ± 2.1) ％ and the CIK cells was (68.7 ± 2.9) ％.The difference was statistically significant(F =7.007,P =0.007).When the ratios of effector cells to target cells were 10 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (82.3 ± 4.5) ％,while the DC-CIK cells was (77.1 ± 5.1) ％,and the CIK cells was (72.7 ± 2.8) ％.The difference was statistically significant (F =7.727,P =0.005).When the ratios of effector cells to target cells were 20 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (83.2 ± 1.9) ％,while the DC-CIK cells was (77.2 ± 4.2) ％,and the CIK cells was (73.0 ± 2.6) ％.The difference was statistically significant (F =16.594,P =0.000).Conclusion DCs loading with antigen produced by radiofrequency ablation of tumor in situ pyrolysis products can improve in vitro cytotoxic activity combined with CIK cells,which can provide a new comprehensive cancer treatment strategy.

Preparation and characterization of a novel functional anti-human CD83 monoclonal antibody / 细胞与分子免疫学杂志

AIM: To prepare and characterize a novel anti-human CD83 monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD83 transfectant (L929/CD83) as immunogen. The spleen B cells of the mice were fused with Sp2/0 myeloma cells. The hybridoma cells were screened with CD83 transfectant (L929/CD83 and 293/CD83) by FCM. The biological characteristics of antibody were investigated by rapid isotyping analysis, karyotype analysis, competitive inhibition test and Western blot. RESULTS: One hybridoma cell line named 9D8 was obtained, which had the property of secreting antihuman CD83 monoclonal antibody steadily, This mAb specifically recognized CD83 molecule expressed on human mature DC, activated T cells, and tumor cell line Daudi, myeloma cell line 8226. This mAb can recognize a distinct epitope from comercial mAb(HB15e). CONCLUSION: One hybndoma cell line has been developed successfully, which may lay a fundation for further study on the function of this molecule.

Significance of gp130 molecule activation in the differentiation and maturation of dendritic cells / 中国免疫学杂志

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.

Biological characteristics of tumor apoptotic cells-plused-dendritic cells induced by tumor lysates supernatants / 中国病理生理杂志

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

Establishment and identification of humanized SCID mouse model / 中国免疫学杂志

Objective:To establish and identify humanized-SCID mouse model(hu-SCID).Methods:SCID mouse was treated by CTX to inhibit the hemocytopoiesis. With successive 4-day injection, human peripheral blood mononuclear cells(PBMC) were engrafted into SCID mouse through intraperitoneal injection. After 4, 8 and 12 weeks of engraftment, peripheral blood, spleen and liver tissues of engrafted SCID mouse were harvested. Human CD3~+, CD19~+ cells in peripheral blood were analyzed by inflorescence microscopy and FCM, human CD3~+, CD19~+ cells in spleen and liver tissues were observed by immune histochemistry, and human IgG level in SCID mouse serum was measured by ELISA.Results:After engraftment of 4, 8 and 12 weeks, human CD3~+, CD19~+ cells in SCID peripheral blood were identified by inflorescence microscopy and the percents were 31% and 10% respectively by FCM analysis. And these cells could be evidenced after 12 weeks later. Through immune histochemistry human CD3~+、CD19~+ cells were detected in mouse spleen but not in liver tissue. Furthermore the titer of human IgG in mouse serum was 390,1 100 and 1 040 ?g/ml at each time point respectively.Conclusion:Our experimental results demonstrated that a bona fide humanized SCID model was established.

The inhibitory repressive effect of IL-10 on the monocyte-derived DC maturation,differation and biological function in vitro / 中国免疫学杂志

Objective:To investigate the negative effect of the IL-10 on monocyte-derived DC maturation and differation iv vitro,and the potentiation of the TNF-? or sCD40L to inhibit or reverse the IL-10′s inhibitory effect on monocyte-derived DC.Methods:The expression of the surface molecules on DC was detected by FACS analysis.The potentiation to stimulate T cell proliferation was assayed by 3H-TdR incorporation,and IL-12 secretion in the DC supernatant measured by ELISA.Results:In vitro DC-inducing system IL-10 had an obviously negative effect on the maturation as well as the potentiation to stimulate the T cell proliferation and IL-12 secretion of the immature monocyte-derived DC,and IL-10 could drive monocyte-derived DC differentiate into the macrophages.The negative effect was also correlative to the concentration of the added IL-10;The results also showed that IL-10 hadn′t any negative effect on mature DC induced by sCD40L,but to some extent could reduce the mature DC induced by TNF-? to produce IL-12;Furthermore the inhibitory effect of IL-10 can′t be reversed by adding TNF-? or sCD40L after IL-10 was added to the DC-inducing culture system for three days.Interesting by adding sCD40L not TNF-? to the DC-inducing culture system with IL-10 at the same time can inhibit the negative effect of IL-10 completely.Conclusion:IL-10 is an important biological factor produced in tumor microenvironment for escaping the attack of the immune system by repressing maturation,potentiation to costimulate the T cells and IL-12 secretion of the immature monocyte-derived DC.The reverse effect of TNF-? and sCD40L on IL-10 negative effect on monocyte-derived was different.All together suggested that CD40 signal has important values to obtain the therapeutic DC for the tumor immune intervention.