Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade

Abstract

PD-1 is a receptor of the Ig superfamily that negatively regulates T cell antigen receptor signaling by interacting with the specific ligands (PD-L) and is suggested to play a role in the maintenance of self-tolerance. In the present study, we examined possible roles of the PD-1/PD-L system in tumor immunity. Transgenic expression of PD-L1, one of the PD-L, in P815 tumor cells rendered them less susceptible to the specific T cell antigen receptor-mediated lysis by cytotoxic T cells in vitro, and markedly enhanced their tumorigenesis and invasiveness in vivo in the syngeneic hosts as compared with the parental tumor cells that lacked endogenous PD-L. Both effects could be reversed by anti-PD-L1 Ab. Survey of murine tumor lines revealed that all of the myeloma cell lines examined naturally expressed PD-L1. Growth of the myeloma cells in normal syngeneic mice was inhibited significantly albeit transiently by the administration of anti-PD-L1 Ab in vivo and was suppressed completely in the syngeneic PD-1-deficient mice. These results suggest that the expression of PD-L1 can serve as a potent mechanism for potentially immunogenic tumors to escape from host immune responses and that blockade of interaction between PD-1 and PD-L may provide a promising strategy for specific tumor immunotherapy.

Fig 1.

Inhibition of the cytotoxic activity of CTL clone by engagement of the PD-1 receptor with PD-L1 on the specific target cells. (A and B) Expression of PD-1, H-2L^d, and PD-L1 on 2C CTL clone and P815 tumor cells as well as their stable transfectant clones of PD-L1(P815/PD-L1 nos. 1–3) were analyzed by using a flow cytometry. Although not shown, none of the cells expressed PD-L2. (C) 2C cells were incubated with ⁵¹Cr-labeled P815 (○) or three independent P815/PD-L1 clones (□, ◊, ▵) in the absence or presence (▴) of 10 μg/ml rat anti-PD-L1 mAb F(ab′)₂ at varying effector-to-target (E/T) ratios for 4 h, and the specific ⁵¹Cr release was determined. The means and SE of triplicate cultures are indicated.

Fig 2.

Enhanced tumorigenicity and invasiveness of P815/PD-L1 cells in the syngeneic DBA/2 mice. (A) P815 (○) or P815/PD-L1 (•) cells were inoculated s.c. at 1 × 10⁶ cells per mouse into DBA/2 (Left) or H-2-shared BALB/c nu-nu (Right) mice, and local tumor growth was monitored. The mean tumor volumes and SE of six mice in each group are indicated. *, P < 0.01 by Student's t test. (B) DBA/2 mice (six mice per group) were inoculated with P815 (○) or P815/PD-L1 (no. 1 • and no. 2 ▴) cells as above and the survival rates were monitored. (C) DBA/2 mice inoculated s.c. with P815/PD-L1 cells were autopsied on day 18. Local s.c. tumors showing invasion across the abdominal wall into the peritoneal cavity (a and b) (×40 and ×400), spleen showing massive invasion of tumor cells (c), and liver with focal metastatic region (d).

Fig 3.

Inhibition of the tumorigenesis of P815/PD-L1 cells by the injection with anti-PD-L1 mAb in vivo. (A Left) Syngeneic P815 tumor-specific T cells were generated in DBA/2 mice as described in Materials and Methods and analyzed for the expression of CD4, CD8, and PD-1. (Center) The CD8+ T cells were incubated with ⁵¹Cr-labeled P815 (○) or P815/PD-L1 (•) cells at varying effector-to-target ratios for 4 h, and the specific cytotoxicity was determined. The means and SE of triplicated cultures are indicated. (Right) The CD8+ T cells (2 × 10⁶) were cocultured with 5 × 10⁶ P815 (open bar) or P815/PD-L1 cells in the absence (solid bar) or presence (shaded bar) of anti-PD-L1 mAb F(ab′)₂ (10 μg/ml) for 24 h, and IFN-γ in the culture supernatants was determined by enzyme-linked immunosorbent assay. The means and SE of triplicated cultures are indicated. (B) DBA/2 mice (10 mice per group) were inoculated s.c. with 3 × 10⁶ P815/PD-L1 cells, and normal rat IgG (○) or anti-PD-L1 mAb (□) was injected on days 1, 3, 5, and 7 at 0.1 mg per mouse each time. The mean tumor volumes and SE of 10 mice (Left) as well as their survival rates (Right) are indicated. *, P < 0.01 by Student's t test.

Fig 4.

Inhibition of the tumorigenesis of myeloma cells endogenously expressing PD-L1 in the normal syngeneic mice treated with anti-PD-L1 Ab or in the PD-1-deficient mice. (A) Expression of endogenous PD-L1 in myeloma and B16 melanoma cells was examined with a flow cytometry. (B) J558L myeloma cells (2.5 × 10⁵) were inoculated s.c. into the syngeneic BALB/c mice (nine mice per group) followed by the injection with normal rat IgG (○) or anti-PD-L1 mAb (□) (0.1 mg per mouse) on days 1, 3, 5, and 7. The mean tumor volumes and SE of nine mice are indicated. **, P < 0.01; *, P < 0.1 (by Student's t test). (C) J558L (Left, 2.5 × 10⁵) or B16 (Right, 1 × 10⁶) cells were inoculated s.c. into PD-1+/+ (○) and −/− (•) mice of BALB/c or B6 background, respectively. The mean tumor volumes and SE of four (the former) and 10 (the latter) mice are indicated.