Dual Control of Antitumor CD8 T Cells through the Programmed Death-1/Programmed Death-Ligand 1 Pathway and Immunosuppressive CD4 T Cells: Regulation and Counterregulation

PD-1 expression on CD4 and CD8 T cells in the tumor and the local lymph nodes. A, PD-1 expression on CD8⁺ T cells from the tumor-draining lymph nodes (DLN) was significantly increased compared with non-DLN (NDLN); unpaired Student’s t test. Representative dot plots gated on lymphocytes show PD-1 expression on CD8⁺ T cells from the non-DLN (left panel) and DLN (right panel). Percentage of PD-1^high CD8⁺ T cells is indicated. B, TCR-transgenic HA-specific CD8⁺ T cells from the NDLN (left panel), DLN (center panel), and tumor (right panel) were analyzed for expression of PD-1 and LFA-1. Percentages of PD-1⁺CD8⁺ T cells are indicated. Cells shown are gated on CD8⁺ cells. C, PD-1 expression on CD4⁺ T cells from the tumor nondraining lymph nodes (left panel) and draining lymph nodes (right panel). Percentages of PD-1⁺CD4⁺ T cells are indicated.

Impact of in vivo PD-L1 blockade on tumor growth and T cell activation. A, Tumor growth in AB1-HA tumor-bearing BALB/c mice treated with 200 μg of anti-PD-L1 Ab (days 8, 11, and 14 posttumor inoculation). Data shown are mean ± SEM (n = 3) from one experiment. ∗∗, p < 0.05 when untreated is compared with anti-PD-L1. B, PD-1 and ICOS expression on CD4⁺ T cells from the nondraining lymph nodes (NDLN; left panels) and draining lymph nodes (DLN; right panels) from untreated (top panels) or anti-PD-L1-treated (bottom panels) mice. Tumor-bearing mice were left untreated or treated with anti-PD-L1 (days 11 and 14), and lymph nodes were harvested at day 16. C, PD-1 and ICOS expression on CD8⁺ cells, as per B.

Increased activation of CD8 T cells by PD-L1 blockade after CD4 depletion. Representative depletion data are shown for PBL samples at day 3 after GK1.5 Ab injection (A) and for lymph nodes at day 16 after GK1.5 Ab injection (B). C, Effect of CD4 depletion on the activation of T cells in the tumor-draining lymph nodes. Tumor-bearing mice were left untreated or depleted of CD4⁺ T cells (days 8, 11, and 14) with or without PD-L1 blockade (days 8, 11, and 14). Draining lymph node cells were harvested on day 16 and were gated on CD8⁺ (upper row) or CD4⁺ (lower row) cells and analyzed for PD-1 and ICOS expression. D, As per C, but showing expression of PD-1 and Ki-67.

A, CD4 depletion with PD-L1 blockade promotes CTL activity. Mice were either left untreated or treated with anti-PD-L1, CD4 depletion, or a combination of the two (days 7, 10, and 13). Two days after the last dose of treatment (day 15), 2 × 10⁷ HA-coated CFSE-labeled target splenocytes were injected i.v., and their recovery in the draining lymph nodes (DLN), nondraining lymph nodes (NDLN), spleen, and tumor was measured 18 h later. Responses of individual mice are shown with group mean (horizontal scale bar; n = 3 per group). ∗, p < 0.05; ∗∗∗, p < 0.005 (unpaired Student’s t test). B, MHC I pentamer staining on tumor-draining lymph nodes in CD4-depleted and/or PD-L1-blocked mice. Tumor-bearing mice were treated with Abs from day 7 onward (anti-CD4 on day 7; anti-PD-L1 on days 8 and 11), and lymph nodes were harvested on day 14 and used for pentamer analysis. C, Expression of ICOS on pentamer⁺ CD8 T cells, as shown in B.

Effect of CD4 depletion on effect of PD-L1 blockade. Tumor-bearing mice were left untreated or depleted of CD4⁺ T cells using GK1.5-depleting Ab (days 6, 9, and 12) with or without PD-L1 blockade (days 10, 13, and 16). Data shown are mean (n = 5) from one experiment. ∗∗, p < 0.005, anti-PD-L1 vs anti-PD-L1 + anti-CD4, day 13–28; ∗, p < 0.05, anti-PD-L1 vs anti-PD-L1 + anti-CD4, day 28–41.

PD-L1 blockade increases CD4⁺ regulatory T cell numbers. A, Percentage of foxp3⁺CD4⁺ T cells present in the draining lymph nodes of untreated mice or mice treated with poly(I:C), anti-PD-L1, or both (days 7, 10, and 13; top panel). Lymph nodes were harvested at day 14. ∗, p < 0.05; ∗∗, p < 0.01; unpaired Student’s t test. Representative plots of the same data (bottom panel). Numbers indicated represent the percentage of CD4⁺ T cells positive for foxp3. B, ICOS expression on CD4⁺ foxp3⁺ T regulatory cells from the draining lymph nodes of mice treated, as for A. C, ICOS-L expression on tumor-infiltrating CD11c⁺ DCs. CD11c⁺ DCs were gated (left panel) and analyzed for ICOS-L expression (green graph). Isotype control staining is shown in red.