Therapeutic antitumor efficacy of B cells loaded with tumor-derived autophagasomes vaccine (DRibbles)

Abstract

Tumor-derived autophagosomes (DRibble) selectively capture tumor-specific antigens and induce a dramatic T-cell activation and expansion when injected into lymph nodes of naive mice. Both dendritic and B cells can efficiently cross-prime antigen-specific T cells. In this report, we demonstrated that a booster vaccination with naive B cells loaded with DRibbles eradicated E.G7-OVA tumors in mice that were previously treated with adoptive transfer naive OT-I T cells and intranodal immunization with DRibbles derived from E.G7 tumors. The antitumor efficacy was accompanied by a heighten number of tumor-specific interferon-γ-producing T cells and antibodies. However, the same treatment in the absence of adoptive T-cell transfer exhibited a limited efficacy. In contrast, when DRibble-loaded B cells were activated with CpG and anti-CD40 antibody before use as booster vaccines, established E.G7 tumors were completely eradicated in the absence of T-cell transfer. Therefore, our results document that B cells could efficiently cross-present tumor-specific antigens captured by DRibbles and suggest that naive B cells can be deployed as an effective and readily accessible source of antigen-presenting cells for cancer immunotherapy clinical trials.

Figures

FIGURE 1

B cells loaded with DRibbles were efficient antigen-presenting cells (APCs) at restimulating primed CD8+ T cells. A, B cells purified from the C57/BL6 mice were analyzed by flow cytometry for CD19 and CD11c expression. B, Histogram and (C) bar graph were shown. DRibbles were collected from EG7-OVA tumor cells expressed OVA protein. B cells were stimulated with or without DRibbles [or whole tumor cell lysate (10 μg/mL total proteins, or 0.1 μg/mL OT-I SIINFEKL peptide)], or DRibbles alone (10 μg/mL) were then coincubated with CFSE-labeled effector OT-I CD8+ T cells. Activation of T cells was assessed by CFSE dilution on day 5. Percentage of divided OT-I T cells is shown as the mean±SEM. Data are representative of results from 2 to 4 independent experiments.

FIGURE 2

Vaccination with B cells pulsed with EG7.OVA cell-derived DRibbles and adoptively transfer of OT-I T cells eradicated E.G7-OVA tumors. A, Experimental scheme illustrates the immunotherapy protocol. C57BL/6 mice (n=5) with preestablished EG7 tumors (previously injected with 5×105 EG7 tumor cells) were treated with DRibbles injected directly into both inguinal lymph nodes and received adoptive transfer of OT-I spleen cells (1×107). Two intravenous injections of 5×106 DRibbles-loaded B cells or unloaded B cells or PBS were given 3 and 6 days after the first injection of DRibbles. Untreated mice served as the controls. Tumor volume (B) and percentage of survival (C) in mice bearing subcutaneous tumors were monitored over time. Lymphocytes were collected from lymph nodes and spleens of vaccinated mice on the 10th day after the first immunization. (D) The spleen cells were stained with antibodies against OT-1 clonal T-cell receptor Vβ antibodies and CD8. The expression of Vβ5.1/5.2+ and CD8+ on T cells was analyzed by flow cytometry. The percentage of divided OT-I T cells is shown as mean±SEM derived from 3 mice per group (n=3). E and F, The lymphocytes were restimulated with DRibbles in vitro. Supernatants were harvested for detection of secretory interferon (IFN)-γ by ELISA after 72 hours (E) or the intracellular IFN-γ staining were performed to determine the frequency of antigen-specific T cells after 12 hours (F). *P<0.05, ***P<0.001. Data are representative of 3 independent experiments with similar results.

FIGURE 3

Boosting with DRibble-loaded B-cell vaccine suppressed the tumor growth but generated no cure without adoptive transfer of OT-I T cells in an established E.G7 murine thymoma model. A, Experimental scheme. C57BL/6 mice (n=5) with preestablished EG7 tumors (previously injected with 5×105 EG7 tumor cells) were first treated with DRibbles injected directly into both inguinal lymph nodes. Two intravenous injections of 5×106 DRibbles-loaded B cells or dendritic cells (DCs), unloaded B cells or PBS were given 3 and 6 days after the first injection of DRibbles. Untreated mice served as controls. Tumor volume (B) and percentage of survival (C) in mice bearing subcutaneous tumors were monitored over time. D and E, The tumor cells were stained with antibodies against CD4 and CD8. The frequency of CD8+ and CD4+ T-cell infiltrating tumors was analyzed by flow cytometry. Percentages of CD8+ (D) and CD4+ (E) T cells are shown as mean±SEM derived from 3 mice per group (n=3). *P<0.05, **P<0.01, ***P<0.001. The typical result from 3 independent experiments was shown.

FIGURE 4

Detection of interferon-γ-producing T cells in vaccinated E.G7 tumor-bearing mice after restimulation with DRibbles. Lymphocytes were collected from lymph nodes and spleens of vaccinated mice on the 10th day after the first immunization. A, Representative spleens from each group mice were shown. B, Total number of splenocytes of each group mice was counted. The lymphocytes were coincubated with DRibbles for 72 hours. C, Images were taken after 72 hours of incubation (×200). D, Supernatants were harvested for detection of secretory interferon (IFN)-γ by ELISA. **P<0.01, ***P<0.001. Data presented were obtained as a result of triplicates.

FIGURE 5

Booster vaccination with DRibble-loaded B cells delayed the tumor growth in an established BNL murine hepatocellular carcinoma model. A, B-cell therapy scheme in an established BNL murine hepatocellular carcinoma model. Immunocompetent BALB/c mice (n=5) with preestablished BNL tumors (previously injected with 2×106 BNL tumor cells) were treated with DRibbles injected directly into both inguinal lymph nodes. Two intravenous injections of 5×106 DRibbles-loaded B cells or unloaded B cells were given 3 and 6 days after the first injection of DRibbles. Untreated mice served as controls. Tumor volume (B) and percentage of survival (C) in mice bearing subcutaneous tumors were monitored over time. D and E, The frequency of CD8+ and CD4+ T cells infiltrated the tumors was analyzed by flow cytometry. Percentages of CD8+ (D) and CD4+ (E) T cells are shown as mean±SEM derived from 3 mice per group (n=3). F and G, Lymphocytes were collected from lymph nodes and spleens of vaccinated mice were restimulated with DRibbles. The frequency of interferon (IFN)-γ-producing T cells and secreted IFN-γ were detected by intracellular cytokine staining 12 hours later (F) and ELISA 72 hours later (G), respectively. *P<0.05, **P<0.01. Data are representative of 3 independent experiments with similar results.

FIGURE 6

TLR9 and CD40 stimulation of DRibble-loaded B cells completely regressed established tumors in an E.G7 murine thymoma model. A, Experimental scheme. The E.G7 tumor-bearing mice were established as described in Figure 2 with without adoptive transfer of OT-I T and B cells loaded with DRibbles in the presence of CpG and anti-CD40 antibodies. Tumor volume (B) and percentage of survived mice (C) were recorded. D, Lymphocytes collected from lymph nodes and spleens of vaccinated mice were coincubated with EG7-DRibbles, B16F10-Dribbles, and anti-CD3 for 72 hours. The supernatants were harvested for the detection of secretory interferon (IFN)-γ by ELISA. ***P<0.001. A representative result from 3 independent experiments is shown.