TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells

Abstract

The combination of immunotherapy and chemotherapy is regarded as a promising approach for the treatment of certain types of cancer. However, the underlying mechanisms need to be fully investigated to guide the design of more efficient protocols for cancer chemoimmunotherapy. It is well known that danger-associated molecular patterns (DAMPs) can activate immune cells, including dendritic cells (DCs), via Toll-like receptors (TLRs); however, the role of DAMPs released from chemical drug-treated tumor cells in the activation of the immune response needs to be further elucidated. Here, we found that colorectal cancer (CRC) cells treated with oxaliplatin (OXA) and/or 5-fluorouracil (5-Fu) released high levels of high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). After OXA/5-Fu therapy, the sera of CRC patients also exhibited increased levels of HMGB1 and HSP70, both of which are well-known DAMPs. The supernatants of dying CRC cells treated with OXA/5-Fu promoted mouse and human DC maturation, with upregulation of HLA-DR, CD80 and CD86 expression and enhancement of IL-1β, TNF-α, MIP-1α, MIP-1β, RANTES and IP-10 production. Vaccines composed of DCs pulsed with the supernatants of chemically stressed CRC cells induced a more significant IFN-γ-producing Th1 response both in vitro and in vivo. However, the supernatants of chemically stressed CRC cells failed to induce phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating an essential role of TLR4 in DAMP-induced DC maturation and activation. Furthermore, pulsing with the supernatants of chemically stressed CRC cells did not efficiently induce an IFN-γ-producing Th1 response in TLR4-deficient DCs. Collectively, these results demonstrate that DAMPs released from chemically stressed cancer cells can activate DCs via TLR4 and enhance the induction of an anti-tumor T-cell immune response, delineating a clinically relevant immuno-adjuvant pathway triggered by DAMPs.

Figure 1

HMGB1 and HSP70 release from chemically stressed CRC cells and in the sera of CRC patients after chemotherapy. (a) CT26 cells or (b) SW480 cells (3×10⁵/ml) were treated with 0.5 mg/ml OXA, 10 mg/ml 5-Fu or a combination of 0.5 mg/ml OXA and 10 mg/ml 5-Fu. Supernatants were collected after 0, 6, 12, 18, 24, 30, 36 and 42 h of culture. HMGB1 and HSP70 levels in the supernatants were measured by ELISA. (c) HMGB1 and HSP70 levels in the sera of patients with advanced CRC were measured by ELISA before and after FOLFOX chemotherapy. The data are presented as the mean±s.e.m. of three independent experiments. CRC, colorectal cancer; 5-Fu, 5-fluorouracil; HMGB1, high-mobility group box 1; HSP, heat shock protein; OXA, oxaliplatin.

Figure 2

Phenotypic and functional maturation of human DCs induced by the supernatants of chemically stressed SW480 cells in a TLR4-dependent manner. (a) Human DCs (5×10⁵/ml) cultured on day 5 were treated with 100 µl/ml untreated CTR-sup, OXA-sup, 5-Fu-sup or OXA+5-Fu-sup or 100 ng/ml LPS for 24 h and then collected for flow cytometry analysis of HLA-DR, CD80 and CD86 expression. (b) Cytokine production by DCs stimulated with the supernatants of chemically stressed SW480 cells. Normal human DCs (normal group) and DCs blocked with the TLR4 antagonist HTA125 (TLR4 antagonist group) were cultured for 5 days and then stimulated with 100 µl/ml untreated CTR-sup, OXA-sup, 5-Fu-sup or OXA+5-Fu-sup or 100 ng/ml LPS for 24 h. The levels of IL-6, IL-1β, TNF-α, MIP-1α, MIP-1β, RANTES and IP-10 in the supernatants were measured by ELISA. The results are presented as the mean±s.d. of triplicate samples. **P<0.01; *P<0.05. DC, dendritic cell; 5-Fu, 5-fluorouracil; 5-Fu-sup, supernatants from SW480 cells treated with 5-Fu; LPS, lipopolysaccharide; NS, no significant difference; OXA, oxaliplatin; OXA+5-Fu-sup, supernatants from SW480 cells treated with OXA and 5-Fu; OXA-sup, supernatants from SW480 cells treated with OXA; TLR, Toll-like receptor; untreated sup-CTR, supernatants from untreated SW480 cells.

Figure 3

Phenotypic and functional maturation of mouse DCs induced by the supernatants of chemically stressed CT-26 cells in a TLR4-dependent manner. (a) DCs from wild-type C57BL/6 mice (WT) and TLR4-deficient C57BL/6 mice (TLR4^−/−) were cultured for 5 days and then stimulated with 100 µl/ml untreated CTR-sup, OXA-sup, 5-Fu-sup or OXA+5-Fu-sup or 100 ng/ml LPS for 24 h. The cells were then collected for flow cytometry analysis of I-a^b, CD80 and CD86 expression. (b) Cytokine production by DCs stimulated with the supernatants of chemically stressed CT-26 cells. The levels of IL-6, IL-1β, TNF-α, MIP-1α, MIP-1β, RANTES and IP-10 in the supernatants were measured by ELISA. The results are presented as the mean ± SD of triplicate samples. **P<0.01; *P<0.05. DC, dendritic cell; 5-Fu, 5-fluorouracil; LPS, lipopolysaccharide; NS, no significant difference; OXA, oxaliplatin; TLR, Toll-like receptor.

Figure 4

DCs pulsed with the supernatants of chemically stressed CRC cells induced an IFN-γ-producing Th1 cell response in vitro in a TLR4-dependent manner. (a) Human peripheral blood lymphocytes from health donors were stimulated with autologous DCs (normal group) pulsed with 100 µl/ml untreated CTR-sup, OXA-sup, 5-Fu-sup or OXA+5-Fu-sup or with autologous DCs treated with the TLR4 antagonist HTA125 (TLR4 antagonist group) before pulsing. An IFN-γ ELISPOT was used to detect the specific Th1 immune response. (b) Mouse splenocytes from wild-type (WT) and TLR4-deficient (TLR4^−/−) C57BL/6 mice were stimulated with syngeneic BMDCs pulsed with 100 µl/ml untreated CTR-sup, OXA-sup, 5-Fu-sup or OXA+5-Fu-sup. IFN-γ ELISPOT analysis of the splenocytes was then used to assay specific Th1 immune responses. The results are expressed as the number of positive SFCs/2×10⁵ lymphocytes or spleen cells. The columns are the mean±s.e.m. of three independent experiments. **P<0.01. BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; 5-Fu, 5-fluorouracil; OXA, oxaliplatin; SFCs, spot forming cells; TLR, Toll-like receptor.

Figure 5

Immunization with DCs pulsed with the supernatants of chemically stressed CT-26 cells induced more potent anti-tumor immunity. Balb/c mice were subcutaneously immunized in the thigh with DCs that had been pulsed with untreated CTR-sup, OXA-sup, 5-Fu-sup or OXA+5-Fu-sup. Five days after the final immunization, the mice were subcutaneously challenged with 2×10⁵ CT-26 tumor cells in the flank area. (a) Tumor growth curves. Following CT-26 tumor challenge, tumor growth was monitored by measuring the diameter of the tumor every 2 days and was recorded as the average tumor diameter. (b) Survival of the immunized mice after CT-26 tumor challenge. Each group contained 10 mice. DC, dendritic cell; 5-Fu, 5-fluorouracil; OXA, oxaliplatin.