| Our previouse study showed that the autophagosomes isolated from tumor-cells could be up-taken by B cells, resulting in activation, antibody secretion and cytokine production of B cells, especially the high levels of IL-10 production. IL-10 has been considered as a key trait of Bregs, thus promote us to wonder whether tumor-released autophagosomes represent the factors responsible for the generation of Bregs.Purpose:To investigate the potential role of tumor cells-released autophagosomes (TRAP) in regulating IL-10-producing B cell differentiation and function; to verify whether TRAP-induced IL-10-producing B cells possess a regulatory function; the mechanisms of inducing IL-10+B cells by TRAP; to verify whether autophagosomes were exist in cancer patient and could reproduce the effects on human B cells.Methods:1. Murine splenocytes were cultured with TRAP or LPS for 72h, the frequencies of IL-10+B cells in splenocytes were analyzed by FACS, and levels of IL-10 in culture supernatant were confirmed by a standard ELISA assay. Then the putified B cells were stimulated with TRAP or equivalent amount of cell homogenate, 50% tumor cell culture supernatant,50% TRAP-depleted tumor cell culture supernatant or equivalent amount of TRAP, and levels of IL-10 in culture supernatant were assessd by ELISA. The surface marker (CD5, CD1d, CD21, CD23, CD43, CD86, MHCⅡ) expressed on TRAP-induced IL-10+B cells were analyzed by FACS. TRAP was i.v. iniected to WT mice for 3 times, the frequency of IL-10+B cells in the splenocytes and frequencies of CD5+CD1dhigh B cells in TRAP-induced IL-10+ B cells was analysed by FACS.2. For proliferation experiments, CD4+ or CD8+ T cells purified from wild-type C57BL/6 mice by magnetic microbeads were labeled with CFSE, and then cultured in a 24-well plate pre-coated anti-CD3 and anti-CD28. B cells or TRAP-induced B cells from wild-type C57BL/6 or IL-10 deficient mice were co-cultured with those T cells at a ratio of 1:1. Four days later, proliferation of CD4+ or CD8+ T cells was evaluated with CFSE dilution by flow cytometry. In transwell culture, TRAP-induced B cells were added in the upper chamber whereas T cell in the lower chamber. To assess whether the inhibitory effect of induced B cells was antigen specific, CFSE labeled OT-1 splenocytes were stimulated with OVA257-264 (1 μg/ml), followed by culturing with TRAP-induced B cells. Mice bearing 6-day established E.G7-OVA tumors (s.c.) were vaccinated with OVA-pulsed DCs, and then adoptively transferred (i.v.) with OVA+ or OVA-TRAP-induced B cells. At day 7 after last vaccination, three mice from each group were killed and the frequency of IFN-γ+ CD4+ T and IFN-γ+ CD8+ T cells in spleens was determined by intracellular cytokine staining after in vitro stimulation with the OVA protein for 24 h. The growth of E.G7-OVA tumors in the remaining five mice were continuously monitored and measured.3. Splenic B cells from WT mice and mice deficient in TLR2, TLR4, or their adaptor proteins, MyD88, were examined for IL-10 production and phospho-P65 after stimulating with TRAP. Moreover, the suppressive capacity of TLR2, TLR4 and MyD88 deficient B cells after stimulated with TRAP were evaluated by inhibiting T cell prolifieration. In some experiments, B cells were pretreated with the inhibitors Bayl 1-7082 for 60 minutes, and then stimulated subsequently with TRAP for 72. Level of IL-10 in culture supernatant were confirmed by ELISA. To understand how TLRs signaling induces B cell-derived IL-10 production, we assessed phosphorylation of NF-κB p65 in WT, TLR2-/-,TLR4-/-,and MyD88-/-B cells after culturing with TRAP for 1h.4. TRAP were incubated with anti-HMGB1 to block the HMGB1 antigen epitopes in TRAP, and then incubated with purified B cells. The effect of HMGB1 on IL-10+B cells differentiation induced by TRAP was determined. TRAP was isolated from B16-F10 cells which transfected with HMGB1-shRNA, and cultured with purified B cells for 72h. The level of IL-10 in the supernatant was determind by ELISA. B cells were stimulated with TRAP or equal amount of sonicated TRAP for 72h, levels of IL-10 in the supernatant was determind by ELISA.5. Malignant effusions were collected from 14 clinical cancers patients, and the pellet fractions were obtained after ultracentrifugation of the effusions. An extensive biochemical characterization was performed by FACS, Western blot and electron microscopy. PBMCs were prepared from healthy blood donors and then stimulated with malignant effusions or ascitis derived autophagosomes with different intensities of HMGB1 expression. The percentage of IL-10+B cells in PBMC was analyzed by FACS, and the correlations between generated IL-10+B cells and expression levels of HMGB1 was also evaluated. Then CFSE-labeled CD8+T and CD4+T cells were cultured with autophagosomes-induced B cells in a 24-well plate pre-coated anti-CD3. The proliferation of T cell was determined by FACS.Results:1. FACS analysis revealed that TRAP treatment significantly increased the frequency of IL-10-producing B cells. Moreover, the elevated levels of IL-10 in culture supernatant were confirmed by a standard ELISA assay. Interestingly, an equivalent amount of cell homogenate from E.G7-OVA cells did not induce IL-10 secretion at significant levels. More importantly, depletion of TRAP by ultracentrifugation impaired the ability of tumor cell culture supernatant to induce IL-10 production. We found that TRAP-induced IL-10+ B cells were positively stained for CD5 with high levels of CD1d expression, and with slightly up-regulated MHC class II. FACS analysis showed α ～2.7-fold increase in the frequencies of IL-10-producing B cells as the result of TRAPs injections. Notably, frequencies of CD5+CDldhigh B cells in TRAP-induced IL-10+ B cells were significantly increased comparing with IL-10- B cells (36.7% vs 11%), but these increased CD5+CDldhigh B cells represented a small part of TRAP induced IL-10 producing B cells.2. The proliferation of CD4+ T and CD8+ T cells were strongly suppressed in the presence of TRAP-induced B cells, but not in the presence of control B cells. More importantly, we found that TRAP-induced B cells, even separated from T cells by a porous membrane, still had an intact suppressive activity on T cell proliferation. While B cells from IL-10-/- mice, after pre-stimulated by TRAP, were incapable of inhibiting the proliferation of CD4+ T and CD8+ T cells. Similar with the OVA+ -TRAP, OVA--TRAP induced B cells still had inhibitory effect on T-cell proliferation. OVA-pulsed DC vaccinations induced high frequencies of IFN-γ+ CD8+ T and IFN-γ+ CD4+ T cells, whereas transfer of OVA+ or OVA- TRAP-induced B cells, but not control B cells, resulted in a significant decrease in the frequencies of both IFN-γ+ CD8+ T and CD4+ T cell. Consistently, the decreased frequencies of IFN-γ+ T cells in the TRAP-induced B cells injected mice led to a decline of IFN-γ secreted by T cells when re-stimulated in vitro with OVA. Furthermore, we found that injection with TRAP-induced B cells, but not control B cells, obviously abrogated the antitumor efficacy and improved survival induced by DCOVA vaccination.3. Intracellular staining for IL-10 and FACS analysis showed that TLR2 and MyD88-deficient, but not TLR4-deficient B cells failed to produce IL-10 in response to TRAP compared with WT B cells. TRAP can triggers P65 phosphorylation in WT and TLR4-/-B cells, but not TLR2-/- and MyD88-/- B cells. Moreover, B cells from TLR2 or MyD88-deficient mice, after pre-stimulated with TRAP, were incapable of inhibiting CD8+ and CD4+ T cell proliferation stimulated by anti-CD3/CD28 antibodies, while B cells from TLR4-deficient mice still retained the intact suppressive capacity in response to TRAP. FACS analysis and ELISA detection showed that pre-incubation with the NF-κB inhibitor Bay11-7082 completely abrogated IL-10 production of B cells stimulated with TRAP. TRAP stimulation induced the phosphorylation of NF-κB p65 in B cells from WT and TLR4-, but not TLR2- and MyD88-deficient mice.4. FACS analysis showed that more than 93% of LC3B+ TRAP carried abundant levels of HMGB1. Subsequently, we found that pre-treatment of TRAP with a functional anti-HMGB1 antibody led to a nearly complete loss of effect of TRAP on inducing IL-10-producing B cells compared pre-treatment with isotype antibody. ELISA detection showed that TRAP derived from HMGB1 shRNA transfected B16-F10 cells had a reduced capacity to induce IL-10 production in B cells compared with TRAPs from WT cells or cells transfecting negative control shRNA. Interestingly, we also found that the sonicated TRAPs significantly reduced their potential effects on inducing IL-10 production of B cells compared with the intact TRAP.5. The transmission electron microscopy observation indicated that high-speed pellet fractions from ascitisor malignant effusions of cancer patients contained an abundant of double-membrane vesicles with structural properties similar to autophagosomes. Moreover,95% of these membrane vesicles from cancer patients were positively stained with the typical autophagosomal marker LC3-II. These criterias, including their morphological characteristics and typical autophagosome marker, indicated that the vesicles we isolated from cancer patients were extracellular autophagosome (MedAP). Interestingly, abundant expression of the HMGB1 was also found to be presented in these MedAP. Subsequently, PBMCs were prepared from healthy blood donors and then stimulated with MedAP with different intensities of HMGB1 expression. Similarly, a significant increase in the percentage of IL-10-producing B cells was observed when stimulated with MedAP, and the frequency of induced IL-10-producing B cells showed dynamic changes, with a close correlation with the expression levels of HMGB1. In anti-CD3 mAb-induced T cell proliferation system, both the proliferation of CD4+ T cells and CD8+ T cell could be inhibited by culturing with MedAP-induced B cells.Conclusions:1. Autophagosomes could be passively released into the media by the tumor cells; these tumor cell-released autophagosomes were able to induce B cells to differentiate into IL-10 producing B cells in vitro and in vivo. TRAP induced IL-10-producing B cells were hybrid regulatory B cell subsets which contained B10 cells, but not splenic marginal-zone B cells.2. TRAP induced B cells could inhibit T-cell proliferation in response to nonspecific stimuli (anti-CD3/CD28 antibody) and specific stimuli (OVA257-264). Adoptive transfer of TRAP induced B cells to the DCOVA vaccinated tumor bearing mice leads to suppression of IFN-y production by effector CD4+ and CD8+ T cells and thus favorite tumor progression.3. TLR2 and its adaptor MyD88, were involved in the induction of IL-10-producing B cells by TRAP, thus might be responsible for the immunosuppressive functions of induced B cells. Membrane-associated HMGB1 expressed on TRAP was critical in inducing IL-10 producing B cells, which was detrimental for the antitumor response. Most importantly, membrane integrity of TRAP was required for the stimulatory effect.4. Cancerous malignant effusions contained a large numbers of LC3B positive autophagosomes which had abundant expression of HMGB1. MedAP could induce the differentiation of IL-10+B cells, and the frequency of induced IL-10-producing B cells was positively correlated with the intensity of HMGB1 expression. MedAP-induced B cells have the suppressive capacity. |
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