PD-L1/B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor (TCR) Transgenic CD8⁺ T Cells

Markedly improved 2C effector function in the absence of PD-1. A, cytolytic activity. Primed 2C/RAG2^−/− or 2C/RAG2^−/−/PD-1^−/− T cells were cocultured with B16.C-IFN or B16-SIY-IFN (48 h IFN-γ pretreatment) for 4 h at the indicated E:T cell ratios, and lytic function was determined by ⁵¹Cr release. The difference between wild-type and PD-1^−/− 2C cells was significant using an unpaired t test (P < 0.001). B and C, cytokine production. Primed 2C/RAG2^−/− or 2C/RAG2^−/−/PD-1^−/− T cells were stimulated with B16.SIY or B16.SIY-IFN cells, and supernatants were collected after 18 h. Levels of IL-2 (B) and IFN-γ (C) were determined by ELISA. Tumors alone indicates cytokine levels detected without T cells. Medium indicates cytokine levels detected with T cells alone. Similar results were observed in at least three experiments.

Improved tumor cell elimination in vitro by PD-1-deficient 2C cells. Equal numbers of primed 2C/RAG2^−/− T cells (A and B) or 2C/RAG^−/−/PD-1^−/− cells (C) were cocultured with B16.SIY (A) or IFN-γ-pretreated B16-SIY (B and C) in 24-well plates. Areas representative of the cell density were photographed at day 3. Similar results were found on days 4 and 5 and in at least three experiments (data not shown).

A, PD-1 expression on 2C T cells during priming. Purified 2C/RAG2^−/− (top row) and 2C/RAG^−/−/PD-1^−/− (bottom row) T cells were primed with mitomycin C-treated P815.B71, stained with FITC anti-PD-1, and analyzed by flow cytometry at the indicated time points. At day 4, the cells were collected, purified by Ficoll-Hypaque centrifugation, reprimed for another 4 days, and analyzed similarly. B, PD-L1 expression by 2C T cells. Purified 2C/RAG2^−/− (top row) and 2C/RAG^−/−/PD-1^−/− (bottom row) T cells were primed with mitomycin C-treated P815.B71 and stained for PE PD-L1 on day 0 (left panels) and day 2 (right panels). C, augmentation of T-cell activation with anti-PD-L1 antibody during the effector phase but not the priming phase. Purified 2C/RAG2^−/− cells were primed for 4 days and reprimed for 4 days with mitomycin C-treated P815.B71 in the absence or presence of anti-PD-L1 mAb or control immunoglobulin. At day 8, cells were collected, purified by Focoll-Hypaque centrifugation, and stimulated with B16.SIY-IFN (left panel). Control primed 2C cells were also stimulated in the presence of anti-PD-L1 mAb or control immunoglobulin (right panel). After 18 h, supernatants were collected, and levels of interleukin 2 were determined by ELISA. Similar results were seen for IFN-γ (data not shown) and in two independent experiments.

A, PD-L1 expression on murine tumor cell lines. The indicated tumor cell lines were cultured in complete medium with 10% FCS in the absence (solid gray) or presence (solid black) of 20 ng/ml murine IFN-γ for 48 h, stained with PE-anti-PD-L1, and analyzed by flow cytometry. Isotype control antibody staining was performed in parallel (black line). No expression of PD-L2 was observed on any tumor cell line (data not shown). Similar results were observed in at least two experiments. B and C, PD-L1 expression on human melanoma cell lines. The indicated tumor cell lines were cultured in complete medium with 10% FCS in the absence or presence of 200 ng/ml human IFN-γ for 48 h, stained with PE-antihuman PD-L1, and analyzed by flow cytometry. Isotype control antibody staining was performed in parallel. Representative flow cytometry histogram of SKMel28, showing control immunoglobulin staining (black line) and PE PD-L1 without (solid gray) or with (solid black) pretreatment with IFN-γ, is shown in B. Changes of mean fluorescence intensity of all tumor lines analyzed are shown in C. Similar results were observed in at least two experiments.

Increased proliferation of primed 2C/RAG2^−/−PD-1^−/− T cells in response to B16.SIY-IFN or HTR.C tumor cells. Purified primed 2C/RAG2^−/− ( ) and 2C/RAG^−/−/PD-1^−/− (▪) cells were stimulated with either mitomycin C-treated B16.SIY-IFN (A) or HTR.C (B) cells or a 5:1 ratio of anti-CD3:anti-CD28-coated beads for 48 h. [³H]Thymidine was added during the last 6 h, and incorporated radioactivity was measured. Similar results were observed in at least two experiments.