B Cells Loaded With Tumor-Derived Autophagasomes (DRibbles) Can Prime Antigen-Specific Naive T Cells And Elicit Anti-tumor Immunity

Autophagy is an important process of cell life, which cytopasmic organelles or long-lived proteins are wrapped in double-membrane structures to form autophagosomes. Then they are fused with lysosomes to form autolysosomes, and degrade the contents. It is demonstrated that autophagy plays an improtant role in cell energy cycle, metabolism, cell updating and maintaining the steady state. Based on previous work, proteasome inhibitor was added to block the degradation of short-lived proteins and impel them to enter the degradation pathway of autophagy. Meanwhile, the autophagy inducer was used to promote the formation and release of autophagosomes and NH4Cl was added to inhibit the function of lysosome, so the proteins in autophagosomes wouldn’t be degraded. Based on this, the natural and induced autophagosomes (DRips in Blebs, DRibbles) were prepared. DRibbles contain antigens of tumor.cells and various danger-associated molecular patterns(DAMPs) such as HSPs, calreticulin and HMGB1.Cancer is one of the major diseases that threaten human health. In tumor immune response, T cells are involved in killing tumor cells and controlling tumor growth. But tuomr antigens failing to effectively transmit to T cells is an important reason leading to immune escape. Therefore, tumor cells must rely on professional antigen presenting cells(APCs) to present tumor antigens.B cells mediate humoral immunity response and are antigen presenting cells as well. Compared with DCs, B cells have larger quantity and good uniformity, and it is reported that B cells play an important role in the body’s anti-tumor immune response. However, naive B cells cannot active naive T cells. Instead, they may inactivate naive T cells. Although LPS are used to stimulate B cells and up-regulate their MHC Ⅰ, MHCⅡ and CD80/86, naive T cells were not activated. For several years, people focus on CD40. Studies showed that CD40 activated B cells loaded with varions antigens (peptides, tumor cell lysate or retroviral RNA transfections antigens, etc) can induce antigen-specific CD4+ and CD8+ T cells response. CpG ODN enhanced expression of class I MHC and the costimulatory molecule CD86 and increased endocytic activity of B cells. Our previous studies showed that DRibbles can induce B cells activation in a TLR2-MYD88 dependent manner and the activated B cells are able to cross-present antigens in DRibbles and then elicit effector T cells response. In this work, we further study whether B cells loaded with DRibbles can prime antigen-specific naive T cells and elicit anti-tumor immunity, and whether CpG and anti-CD40 antibody can enhance the anti-tumor immunity.Objective:1. Study whether DRibbles can stimulate B cells to induce naive T cells immune response and B cells loaded with DRibbles can elicit antitumor immunity.2. Study whether CpG and anti-CD40 antibody can further enhance the antitumor effect of B/DRibbles.Method:1. Whether B cells loaded with DRibbles can prime antigen-specific naive T cells and elicit anti-tumor immunity.1.1 Purified CD8+OT-1 naive T cells stimulated with DRibbles(OVA+) alone, B cells alone, purified B-cells loaded with DRibbles(OVA+) or whole tumor cell lysate. Medium alone and DC/DRibbles were included as the negative and positive controls. Cross-presentation of OVA containing DRibble antigens by B cells was measured by flow cytometry analysis of CFSE labeled naive OT-1 CD8+ T cells.1.2 EG7 cells were used to establish subcutaneous lymphoma model in C57BL/6 mouse. These tumor-bearing mice were inject with B/DRibbles through lymph nodes and tail vein, and received adoptive transfer of OT-I spleen cells. Tumor volume and percentage of survival were determined over time.1.3 On the 10th day after the intranodal immunization, lymphocytes were harvested from lymph nodes and spleen of the vaccinated mice. The frequency of Vβ5.1/5.2+CD8+OT-I T cells in the spleen was measured by flow cytometry analysis. Lymphocytes were re-stimulated by DRibbles. After 72 hours stimulation, IFN-y production in the supernatantd was determined by ELISA.2. Whether CpG and anti-CD40 antibody can further enhance the anti-tumor immunity of B/DRibbles.2.1 Purified B cells are co-cultured with DRibbles (OVA+)、DRibbles(OVA+) combined with CpG and anti-CD40 antibody for 3 days. The expression of MHC Ⅰ、MHC Ⅱ and costimulatory molecule CD86 on B cells are measured by flow cytometry.2.2 EG7 cells were used to establish subcutaneous lymphoma model in C57BL/6 mouse. These tumor-bearing mice were inject with B/DRibbles which is pretreated with CpG and anti-CD40 antibody through lymph nodes and tail vein. Tumor volume and percentage of survival were determined over time.2.3 On the 10th day after the intranodal immunization, lymphocytes were harvested from lymph nodes and spleen of the vaccinated mice. Lymphocytes were re-stimulated by different antigens. After 72 hours stimulation, IFN-γ production in the supernatantd was determined by ELISA.Results:1. Whether B cells loaded with DRibbles can prime antigen-specific naive T cells and elicit anti-tumor immunity.1.1 The proliferation of OT-1 naive CD8+ T cells induced by OVA+ DRibbles loaded B cells was significantly greater than that by DRibbles alone (32.2% vs 1.1%), B cells alone (32.2% vs 7.6%) and tumor lysate-loaded B cells (32.2% vs 10.3%) in vitro.1.2 After adaptive transferring OT-I cells, compared with mice injected with PBS, B cells loaded with DRibbles could lead to complete eradication of established tumors and median survival days are significantly prolonged (>84days vs 28days). Without adaptive transferring OT-I cells, B cells loaded with DRibbles could suppressed tumor growth and prolong the survival time of tumor-bearing mice (43 days vs 21days) compared with mice injected with PBS.1.3 B/DRibbles could induce the immune responses of T cells. After adaptive transferring OT-I cells, the percentages of Vβ5.1/5.2+CD8+ T cells among total splenocytes of mice in B/DRibbles group were markedly increased and had a significantly higher level of IFN-γ after in comparison to other groups. On wildtype mouse model, lymphocytes harvested from mice of group B/DRibbles produced a significantly higher amount of IFN-y compared with lymphocytes from.other groups of mice.2. Whether CpG and anti-CD40 antibody can further enhance the anti-tumor immunity of B/DRibbles.2.1 DRibbles(OVA+) with combined of CpG and anti-CD40 antibody are able to stimulate B cells and further up-regulate the expression of MHC Ⅰ (98.6% vs 12,7%), MHC Ⅱ molecules(84.8%/290 vs 92.3%/1210), costimulatory molecule CD86 (16% vs 54.8%) on B cells, significantly higher than the control.2.2 B/DRibbles pretreated with CpG and anti-CD40 antibody could further enhance the antitumor effect, and tumor growth wad suppressed and further prolong the survival time of tumor-bearing mice(45 days vs 42 days) compared with mice injected with B/DRibbles.2.3 B/DRibbles pretreated with CpG and anti-CD40 antibody could further enhance the immune response, mice produced a significantly higher level of IFN-y than lymphocytes from mice of other treatment groups when re-stimulated with EG7-DRibbles.Conclusion:1. B cells loaded with DRibbles could induce naive T cell activation in vitro, and DRibbles-loaded B cells efficiently enhanced immune response and inhibited tumor growth when it is vaccinated to mice.2. DRibbles-loaded B cells stimulated by CpG and anti-CD40 antibody could further enhance their antitumor effect.