Antitumor Effector B Cells Directly Kill Tumor Cells Via The Fas/FasL Pathway And Are Regulated By IL-10and IL-2

Part Ⅰ IL-10regulates the anti-tumor immunity of4T1TDLN B cellsObjective: Regulatory B cells (Bregs) are a subpopulation of B cells that can regulate immunity. In cancer immunity Bregs produce IL-10to suppress anti-tumor effect. In order to identify the role of IL-10+TDLN B cells or IL-10in inhibiting4T1pulmonary metastasis mediated by WT4T1TDLN B cells, IL-10expression of WT and IL-10-/-4T1TDLN B cells were detected and anti-tumor immunity of them in vitro and in vivo were also tested.Method:Using spontaneous pulmonary metastatic4T1mammary carcinoma,1×1064T1tumor cells in0.1ml PBS were injected subcutaneously (s.c.) into the lower flanks of syngeneic WT and IL-10-/-BALB/c mice to induce TDLNs. Nine days after4T1cell inoculation, the draining inguinal lymph nodes were collected in sterile and designated as TDLNs. Healthy LNs were directly collected from WT healthy mice. And then with CD19+microbeads, WT TDLN B cells, IL-10-/-TDLN B cells and LN B cells were purified from WT, IL-10-/-TDLNs and healthy LNs. The three kinds of purified B cells were activated and expanded (A/E) with LPS (5μg/ml) and anti-CD40mAb (2μg/ml) for3-4days in vitro. After A/E TDLN B cells were effector cells. WT TDLN, IL-10-/-TDLN and healthy LN B cells before and after A/E were analysed with their phenotype and function. After A/E WT and IL-10-/-TDLN B cells were adoptively transferred to study their efficacy in cancer immunotherapy and assessed their cytotoxicity through detecting LDH release in vitro. As to gain4T1tumor-bearing mice, healthy BALB/c mice were inoculated with5x1044T1cells in their mammary fat pads. Fourteen days after tumor inoculation, the tumor-bearing mice were treated with tail vein injection of3or15×106activated effector WT or IL-10-/-TDLN B cells, or107activated effector WT TDLN B cells with IL-10antibody administration. About2weeks after B cell transfer, all mice were sacrificed and lungs were harvested for enumeration of spontaneous pulmonary metastatic nodules. Spleens and peripheral blood were collected from all groups of mice for detecting cytotoxicity of T and B cells from spleens and PBMCs.Results:Detecting IL-10expression of WT TDLN, IL-10-/-TDLN and healthy LN B cells by flow cytometry, the purities of B cells were>95%using CD19+microbeads sorting. Among these freshly purified B cells,2-3%of WT B cells were CD19+IL-10+, but these cells were not detectable in the IL-10-/-B cells as expected. After in vitro activation and expansion with LPS plus anti-CD40, CD19+IL-10+cells in WT B cells increased to～11%, while CD19+IL-10+cells in the IL-10-/-B cells remained undetectable. In addition, there were almost no IL-10producing B cells in normal LN (<1%before A/E,;<2%after A/E). The therapeutic efficacies of IL-10-/-vs. WT TDLN B cells were tested, and it showed that IL-2alone or WT4T1TDLN B cells (3million/mouse) had a modest, but not significant reduction in pulmonary metastases compared with PBS control; adoptively transferred WT4T1TDLN B cells (15million/mouse) significantly inhibited the metastasis of4T1tumor cells from the mammary fat pads to the lungs (p<0.05). In comparison, IL-10-/-4T1TDLN B cells (15million/mouse) demonstrated a similar antitumor activity as WT4T1TDLN B cells (15million/mouse)(p>0.5). But IL-10-/-B cells (3million/mouse) inhibited metastases significantly more effectively than WT B cells (3million/mouse)(p<0.01). While in vitro neither IL-10-/-nor WT4T1TDLN B cells killed Renca and TSA notably, WT and IL-10-/-4T1TDLN B cells killed4T1tumor cells in a dose-dependent manner; Importantly, while at the higher E:T ratios (10:1and30:1), there was no significance in4T1cell death between the IL-10-/-and WT4T1TDLN B cells, IL-10-/-4T1TDLN B cells mediated4T1cell lysis much more effectively (p<0.05) than WT4T1TDLN B cells at a low E.T ratio (3:1). Subsequently accompanied with IL-10or isotype control antibody administration, infusion of10’activated WT TDLN B cells resulted in significantly (p<0.01) reducing spontaneous4T1metastases compared with the results using the same number of B cells without IL-10antibody (B cells only), or with IL-10antibody (no B cells), IgG or IgG1. Then spleens and peripheral blood from all groups of mice were collected and purified T and B cells from them, to assess the cytotoxicity of splenic and PBMC T and B cells. The splenic T and B cells, or PBMC T and B cells harvested from the hosts subjected to4T1TDLN B cell+IL-10antibody treatment were significant (p<0.05) to lyse4T1tumor cells more efficiently than the splenic T and B cells, or PBMC T and B cells prepared from the hosts subjected to4T1TDLN B cell alone,4T1TDLN B cell+IgG1, IL-10antibody only, or no treatment.Conclusion：In this study, with comparison of WT4T1TDLN B cells, IL-10-/-4T1TDLN B cells and healthy LN B cells, it has been proved that only WT4T1TDLN B cells contain IL-10+cells before and after A/E. In vitro TDLN B cells directly kill tumor cells in a tumor antigen-specific manner and the IL-10-/-TDLN B cells are more potent than WT TDLN B cells in such direct killing. IL-10-/-4T1TDLN B cells are more effective than WT4T1TDLN B cells on a per cell basis in adoptive immunotherapy. Adoptive immunotherapy using effector TDLN B cells is more effective in the absence of IL-10. In adoptive immunotherapy, Bregs in WT TDLN B cells produce IL-10to suppress their anti-tumor immunity. Part Ⅱ4T1TDLN B cells directly kill4T1tumor cells via Fas/FasL pathway and are regulated by IL-2in anti-tumor immunityObjection:Based on previous results, effector TDLN B cells were adoptively transferred to inhibit growth and metastasis of tumor cells, but its mechanisms mediated tumor cell death are not identified. Detecting FasL and IL-2R expression of TDLN B cells, we investigated the potential mechanisms of effector TDLN B cells inducing tumor cell death.Method:Using spontaneous pulmonary metastatic4T1mammary carcinoma,1×1064T1 tumor cells were injected subcutaneously (s.c.) into the lower flanks of syngeneic WT and IL-10-/-BALB/c mice to induce TDLNs. Nine days after4T1cell inoculation, TDLNs were collected in sterile. And then B cells were purified from TDLNs with CD19+microbeads. The purified B cells were activated and expanded (A/E) with LPS (5μg/ml) and anti-CD40mAb (2μg/ml) for3-4days in vitro. Then B cells were effector cells. The effect of anti-FasL on cytotoxicity of effector B cells was assessed through detecting LDH release in vitro. Before and after A/E TDLN B cells were analysed with their phenotype and function, and after A/E TDLN B cells were adoptively transferred. The healthy BALB/c mice were inoculated with5×1044T1cells into their mammary fat pads to induce4T1tumor-bearing mice. Fourteen days after tumor inoculation, the tumor-bearing mice were treated with tail vein injection of107activated effector TDLN B cells, with or without1L-2administration. About2weeks after B cell transfer, all mice were sacrificed and lungs were harvested for enumeration of spontaneous pulmonary metastatic nodules.Results:In order to confirm that tumor cell death mediated by TDLN B cells contained Fas/FasL pathway, we assessed the cytotoxicity of effector TDLN B cells through detecting LDH release with blocking FasL by anti-FasL. After co-cultured with4T1cells for8-12h, compared with lOμg/ml anti-FasL,30μg/ml anti-FasL significantly decreased the killing efficacy of4T1TDLN B cells on4T1tumor cells at the E:T ratios of10:1and30:1. Detecting FasL expression of IL-10-/-and WT4T1TDLN B cells, the results showed that approximately5%of the purified and anti-CD40/LPS activated/expanded (A/E) WT TDLN B cells expressed FasL and there was a similar percentage (-8%) of the IL-10-/-TDLN B cells expressing FasL. After the effector WT TDLN B cells were cultured with the target4T1cells at the ratio of3:1and10:1overnight, the FasL expression on the B cells increased from5.1%to13.5%and18.0%, respectively. We observed similar increases of FasL expression on the IL-10-/-TBLN B cells after their co-culturing with4T1tumor cells (16.2%and14.3%). And then we tested Fas expression of4T1cells and found that almost all4T1cells expressed Fas. At last in order to investigate the role of IL-2in adoptive immunotherapy of effector B cells, we compared the therapeutic efficacy of adoptively transferred WT TDLN B cells with vs. without IL-2administration. WT4T1TDLN B cells alone showed no efficacy, but adoptively transferred B cells with IL-2administration i.p. significantly inhibited the metastasis of4T1 tumor cells from the injection site (mammary fat pad) to the lung (p>0.05); IL-2alone resulted in no significant reduction in pulmonary metastases compared with PBS control. Exogenous IL-2administration enhanced the antitumor reactivity of adoptively transferred effector B cells. Only IL-2or4T1TDLN B cells could’t inhibit pulmonary metastasis, but both of them together could. We tested IL-2R (CD25) expression on4T1TDLN B cells, and it showed that expressions of IL-2R on freshly purified TDLN B cells from WT and IL-10"’ mice were similar (about10%). After A/E in vitro, expressions of IL-2R were increased both on WT and IL-10-/-TDLN B cells to16.7%and17.9%.Conclusion:Both of IL-10-/-and WT4T1TDLN B cell contain similar number of FasL+cells, and even after co-cultured with4T1cells. FasL on the surface of4T1TDLN B cells interact with Fas on4T1cells and mediate4T1cell to die, so TDLN B cells kill tumor cells via Fas/FasL pathway. TDLN B cells expressed IL-2R which is necessary for effector TDLN B cells to suppress pulmonary metastasis in vivo. These suggest that IL-2may act on TDLN B cells directly. The mechanisms of effector TDLN B cells in adoptive immunotherapy contain Fas/FasL pathway and IL-2regulation. Part Ⅲ Tracking adoptively transferred B cells in vivoObjection:We have proved that effector B cells can produce IL-10to suppress anti-tumor therapeutic effect, mediated tumor cell death via Fas/FasL pathway and IL-2administration regulated their anti-tumor efficacy because of their IL-2R expression. But it is not clear that how and where effector TDLN B cells migrate and locate in vivo after adoptive transfer. In order to find the result, adoptively transferred TDLN B cells were labeled with CMTMR and tracked in vivo.Method:Using spontaneous pulmonary metastatic4T1mammary carcinoma,4T1tumor cells were injected subcutaneously (s.c.) into the lower flanks of BALB/c mice to induce TDLNs. Nine days after4T1cell inoculation, TDLNs were collected in sterile. And then B cells were purified from TDLNs with CD19+microbeads. The purified B cells were activated and expanded (A/E) with LPS (5μg/ml) and anti-CD40mAb (2μg/ml) for3-4days in vitro. Then B cells were effector cells and labeled with10μM CMTMR at37℃for45min in the dark. The healthy BALB/c mice were inoculated with4T1cells in their mammary fat pad to induce4T1tumor-bearing mice. Fourteen days after tumor inoculation, the tumor-bearing mice or healthy mice were treated with tail vein injection of107labeled effector TDLN B cells with IL-2administration. At Day1/5/9/14after B cell transfer, the adoptively transferred mice were sacrificed and their tumors, lungs spleens and TDLNs were harvested for detecting labeled B cells in these tissues and organs by flow cytometry.Results: In order to acknowledge migration and the role of effector TDLN B cells in the hosts, they were labeled with CMTMR to tracking them in vivo. Firstly we observed that all of B cells were labeled successfully. At Day1/5/9/14after B cell transfer, we detected live labeled CD19+B cells in tumors, lungs spleens and TDLNs by flow cytometry. Adoptively transferred B cells represented a high percentage of the total CD19+B cells in the tumor site and lung peaking at Day9post-transfer. Transferred B cells remained high in the tumor and lung at Day14when lung metastases were examined, but transferred B cells in TDLNs and spleens were at low percentages all through adoptive immunotherapy. In addition, transferred TDLN B cells in lungs of tumor-bearing mice were clearly more than that of healthy mice. According to data acquired by flow cytometry to calculate the actual numbers of transferred B cells, a smaller fraction of the B cells found in the spleens and TDLNs but that the total numbers of transferred B cells in the spleen and TDLN were higher in comparison to those in the tumors and lungs.Conclusion: These data suggest that transferred B cells do not preferentially home to the tumor sites, but that they are more prone to entering the sites of tumor than endogenous B cells. Adoptive TDLN B cells can directly act on primary tumor and metastatic tumor cells in lung to inhibit lung metastases, and the systemic localization and/or survival of the adoptively transferred B cells are/is critically dependent on interaction with the4T1tumor cells in vivo.