Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer

Abstract

T-cell tolerance is an important mechanism for tumor escape, but the molecular pathways involved in T-cell tolerance remain poorly understood. It remains unknown whether the inhibitory immunoreceptor programmed death-1 (PD-1) plays a role in conditions of human non-small cell lung cancer (NSCLC). In this study, we detected PD-1 expression on CD8+ T cells from healthy control peripheral blood mononuclear cells (PBMCs) and the PBMCs of NSCLC patients as well as NSCLC tissues. Results showed that tumor-infiltrating CD8+ T cells had increased PD-1 expression and impaired immune function, including reducing cytokine production capability and impairing capacity to proliferate. Blockade of the PD-1/PD-L1 pathway by the PD-L1-specific antibody partially restored cytokine production and cell proliferation. These data provide direct evidence that the PD-1/PD-L1 pathway is involved in CD8+ T-cell dysfunction in NSCLC patients. Moreover, blocking this pathway provides a potential therapy target in lung cancer.

Figure 1

PD-1 is highly upregulated by tumor-infiltrating CD8⁺ T cells in patients with NSCLC. (a) Frequencies of PD-1-expressing CD8⁺ T cells in 58 blood samples, 16 lung tissue samples for LC patients and 23 blood samples for healthy controls. Each dot represents one individual. (b) PD-1 staining representative dot plots for one blood sample from a healthy person, as well as one blood sample and one tumor tissue sample from LC. Values in the upper right quadrant indicate the percentage of cells that express PD-1. (c) MFI of PD-1 expression on CD8⁺ T cells in PBMC and lung tissue samples for LC patients and healthy controls. Each dot represents one individual. (d) Positive rate of PD-1 in PBMCs and lung tissues from the same individual for 16 LC patients with. LC, lung cancer; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; PD-1, programmed death-1.

Figure 2

Preterminally differentiated phenotype and functional impairment of PD-1⁺ tumor-infiltrating CD8⁺ T cells. (a) Expression levels in PD-1⁺CD8⁺ T cells of CD45RA, CD127 (IL-7-receptor-α), CD27 and CCR7. (b) Representative dot plots of IFN-γ and IL-2 intracellular staining gated on CD8-positive cells for one blood sample from a healthy person, as well as one blood sample and one tumor tissue sample from LC. PBMCs and tumor tissues were stimulated with PMA and ionomycin for 6 h in the presence of 3 µg/ml of Brefeldin A and were stained with anti-CD8, fixed and permeabilized, followed by intracellular staining with antihuman IFN-γ or anti-IL-2 antibody. (c and d) Cytokine production in CD8⁺ T cells: IFN-γ (c) and IL-2 (d). (e) Proliferating fold of CD8-positive cells stimulated by anti-CD3 and anti-CD28. IFN, interferon; LC, lung cancer; PBMC, peripheral blood mononuclear cell; PD-1, programmed death-1; PMA, phorbol 12-myristate 13-acetate.

Figure 3

Blocking the PD-1/PD-L1 pathway increases production of effector molecules and proliferation of tumor-infiltrating CD8⁺ T cells. A purified CD8⁺ T-cell stimulation assay was performed in the presence of anti-CD3 and anti-CD28 alone and alternately with an antibody blockade of PD-L1 or PD-L2. (a) A fold of proliferating CD8-positive cells in the presence or absence of anti-PD-L1 or anti-PD-L2 antibodies. (b) Production of IFN-γ in the supernatants of 3-day cultures measured by ELISA in the presence or absence of anti-PD-L1 or anti-PD-L2. IFN, interferon; LC, lung cancer; PBMC, peripheral blood mononuclear cell; PD, programmed death.

Figure 4

Increased tumor-infiltrating CD8⁺ T-cell apoptosis and blocking the PD-1/PD-L1 pathway cannot abrogate apoptosis. (a) Increased apoptosis of tumor- infiltrating CD8⁺ T cells upon incubation with anti-CD3 for 3 days. Cells were harvested and examined for apoptosis by double staining with annexin V⁺ and an antibody against CD8. Apoptosis was calculated as the percentage of annexin V⁺ cells in CD8⁺ fraction. (b) Apoptosis of tumor-infiltrating CD8⁺ T cells in the presence of anti-PD-L1 or anti-PD-L2. LC, lung cancer; PBMC, peripheral blood mononuclear cell; PD, programmed death.