B7-H1 maintains the polyclonal T cell response by protecting dendritic cells from cytotoxic T lymphocyte destruction

Abstract

Induced B7-H1 expression in the tumor microenvironment initiates adaptive resistance, which impairs immune functions and leads to tumor escape from immune destruction. Antibody blockade of the B7-H1/PD-1 interaction overcomes adaptive resistance, leading to regression of advanced human cancers and survival benefits in a significant fraction of patients. In addition to cancer cells, B7-H1 is expressed on dendritic cells (DCs), but its role in DC functions is less understood. DCs can present multiple antigens (Ags) to stimulate dominant or subdominant T cell responses. Here, we show that immunization with multiple tumor Ag-loaded DCs, in the absence of B7-H1, vastly enhances cytotoxic T lymphocyte (CTL) responses to dominant Ag. In sharp contrast, CTL responses to subdominant Ag were paradoxically suppressed, facilitating outgrowth of tumor variants carrying only subdominant Ag. Suppressed CTL responses to subdominant Ag are largely due to the loss of B7-H1-mediated protection of DCs from the lysis of CTL against dominant Ag. Therefore, B7-H1 expression on DCs may help maintain the diversity of CTL responses to multiple tumor Ags. Interestingly, a split immunization approach, which presents dominant and subdominant Ags with different DCs, promoted CTL responses to all Ags and prevented tumor escape in murine tumor models. These findings have implications for the design of future combination cancer immunotherapies.

Keywords: B7-H1; PD-1; cytolytic T cells; dominant antigen; subdominant antigen.

Fig. 1.

B7-H1 on DCs has an opposite effect on T cell responses to dominant and subdominant Ags. (A and B) Naive OT-1 and H-Y T cells were transferred into mice and immunized with DCs purified from WT or B7-H1–KO male/OVA-Tg mice. After 5 d, spleen cells were analyzed by flow cytometry. (A and B) Representative plots (A) and absolute number (B) of OT-1 and H-Y T cells in spleens. (C and D) B6 mice were immunized s.c. with gp100 and TRP2 coloaded WT or B7-H1–KO DCs. At day 8, spleen cells were restimulated with the peptide in vitro. (C) IFN-γ⁺CD8⁺ T cells were counted. (D) Concentrations of IFN-γ in cultured supernatants detected by ELISA. Results represent at least three experiments unless otherwise stated. *P < 0.05 and **P < 0.01, determined by Student’s t test. Error bars indicate SD.

Fig. 2.

B7-H1–KO DCs are more susceptible to CTL lysis. (A) WT or B7-H1–KO DCs were loaded with 3 μM OVA peptides, CFSE-labeled, and cocultured with activated OT-1 cells at the indicated E/T ratio for 4 h. Cells were stained with DAPI and analyzed by flow cytometry. (B) WT DCs were cocultured with OT-1 cells in 10 µg/mL 10B5, G4, 43H12, or control Ig. (C and D) Ratios of WT/B7-H1–KO DCs (C) and representative plots (at E/T = 1:2) (D) after OT-1 cells were incubated with an equal mixture of OVA peptide-pulsed WT DCs (with CFSE) or B7-H1–KO DCs (without CFSE) with 10B5 or control Ig for 4 h in vitro. (E and F) activated OT-1 cells were transferred into mice with equal OVA peptide-pulsed WT and B7-H1–KO DCs. Twenty-four hours later, cells were collected, and DCs were counted by staining with mAbs to CD11c and B7-H1. Results are shown as ratios of WT/B7-H1–KO DCs (E) and representative plots of DCs with the transfer of 1 × 10⁶ OT-1 cells (F) in ascetic fluid.

Fig. 3.

DC immunization in the absence of B7-H1 eliminated tumors carrying dominant Ag but encouraged the emergence of tumors carrying subdominant Ag. (A) Schematic representation of vector design for pEGFP-N1 with the insertion of a minigene encoding OVA peptide (Left) and pDSRed-N1 with the insertion of a minigene encoding H-Y peptide (Right). (B) B6 mice were immunized twice at days −14 and −7 with BM-derived DCs from WT or B7-H1–KO male/OVA-Tg mice. Mice were challenged i.d. with an equal mixture of 5 × 10⁶ EL4-OVA/EGFP and EL4-HY/DSRed. Tumor growth is presented as a percentage of incidence (Left) and as mean tumor diameter (Right). (C) Tumors were surgically excised at day 50, and cells were analyzed by flow cytometry for EGFP and DSRed. (D) B6 mice were immunized s.c. with WT or B7-H1–KO DCs coloaded with gp100 and TRP2 and were inoculated i.d. with B16 cells. Mean tumor diameters (Left) and the mortality (Right) were recorded.

Fig. 4.

Split immunization with B7-H1 blockade prevents the loss of CTL responses to subdominant T cells and induces better tumor immunity. (A and B) Female B6 mice injected with purified naive OT-1 and H-Y cells were immunized with an equal mix of DCs from OVA Tg and WT male mice or with a mix of DCs from these strains on a B7-H1–KO background. Absolute number (A) and representative plots (B) of OT-1 and H-Y in spleens were analyzed at day 5. (C) Mice were immunized with an equal mix of WT BM DCs with gp100 or TRP2 peptides. Mice received 10B5 or control Ig i.p. concurrently with immunization. Spleen cells were obtained 8 d later and were restimulated with gp100 or TRP2 peptides for 72 h. Cell proliferation was shown by CCK8 dye staining. (D) Mice were immunized with B7-H1–KO DCs at day −7 and inoculated i.d. with B16 cells at day 0. DCs were pulsed with peptides in two ways: (i) DCs were loaded with a mix of 3 µM of gp100 and 3 µM of TRP2 peptides; (ii) DCs were divided into two tubes and pulsed separately with gp100 or TRP2 peptides (split DCs). After washing, DCs were mixed together for immunization. Tumor size is presented as the mean of tumor diameters.*P < 0.05 and **P < 0.01, determined by Student’s t test. Error bars indicate SD.