Lung cancer is associated with decreased expression of perforin, granzyme B and interferon (IFN)-γ by infiltrating lung tissue T cells, natural killer (NK) T-like and NK cells

Abstract

There is a limited understanding how of lung cancer cells evade cytotoxic attack. Previously, we have shown reduced production of the cytotoxic mediator granzyme B by CD8(+) T cells in lung cancer tissue. We hypothesized that lung cancer would be further associated with decreased production of granzyme B, perforin and proinflammatory cytokines by other cytotoxic lymphocytes, natural killer (NK) T-like and NK cells, and that this would result from soluble mediators released by the cancer cells. Lung cancer and non-cancer tissue from five patients was identified by experienced pathologists. Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granzyme B and perforin were measured in CD4 and CD8(+) T, NK T-like cells and NK cells by flow cytometry. Correlation between cancer stage and granzyme B was analysed retrospectively for 21 patients. The effects of soluble factors released by lung cancer cells on production of cytotoxic mediators and cytokines was assessed, and the role of prostaglandin E2 (PGE)2 /COX investigated using indomethacin inhibition. There were significantly decreased percentages of T, NK T-like and NK cells expressing perforin, TNF-α and IFN-γ in cancer versus non-cancer tissue, and of CD8(+) T cells and CD8(+) NK T-like cells expressing granzyme B (e.g. NK T-like cells: non-cancer 30% ± 7 versus cancer 6% ± 2·5). Cancer cells released soluble factors that inhibited granzyme B, perforin and IFN-γ production that was partially associated with the PGE2 /COX2 pathway. Thus, lung cancer is associated with decreased expression of granzyme B, perforin and IFN-γ by infiltrating T cells, NK T-like and NK cells, possibly as a result of soluble factors produced by the cancer cells including PGE2 . This may be an important immune evasion mechanism.

Keywords: NK and NK T-like cells; cytotoxicity; granzyme B; interferon gamma; lung cancer.

Fig 1

Intracellular granzyme B expression in CD4⁺ and CD8⁺ T cell and natural killer (NK) T-like subsets and NK cells from normal (clear bars) and cancer tissue (grey bars). There was a significant decrease in the percentage of CD8⁺ T cells expressing granzyme B from cancer tissue compared with normal tissue. There was also a decrease in the percentage of CD8⁺ NK T-like cells and NK cells expressing granzyme B compared with normal tissue. Box-plots present median ± 25th and 75th percentiles (solid box) with the 10th and 90th percentiles shown by whiskers outside the box (*P < 0·05).

Fig 2

Perforin expression in CD4⁺ and CD8⁺ T cell and natural killer (NK) T-like subsets and NK cells from normal (clear bars) and cancer tissue (grey bars). There was a decrease in the percentage of CD4⁺ and CD8⁺ T cells expressing perforin from cancer tissue compared with normal tissue. There was also a decrease in the percentage of CD8⁺ NK T-like cells and NK cells expressing perforin compared with normal tissue. Data presented in box-plots as described in Fig. 1 (*P < 0·05; **P < 0·01).

Fig 3

Interferon (IFN)-γ production in CD4⁺ and CD8⁺ T cell and natural killer (NK) T-like subsets and NK cells from normal (clear bars) and cancer tissue (grey bars). There was a decrease in the percentage of CD4⁺ and CD8⁺ T cells, CD4⁺ and CD8⁺ NK T-like cells and NK cells producing IFN-γ from cancer tissue compared with normal tissue. Data presented in box-plots as described in Fig. 1 (*P < 0·05; **P < 0·01).

Fig 4

Inhibition of (a) interferon (IFN)-γ production and (b) tumour necrosis factor (TNF)-α in CD4⁺ and CD8⁺ T cells, natural killer (NK) T-like and NK cells (clear bars) in the presence of conditioned media from small-cell carcinoma (SBC)-1 cells compared with supernatant from the 16HBE normal human bronchoepithelial cell line (control). Data for three separate experiments (n = 3) are presented as % change compared to control. Significant decrease (*P < 0·05) in the percentage of lymphocyte subsets producing cytokines in the presence of SBC-1 supernatants. Neutralizing PGE₂ with 10 μM indomethacin (grey bars) significantly (#P < 0·05; ##P < 0·01) diminished the inhibitory effect of SBC-1 cancer cell line supernatant on IFN-γ and TNF-α production (three separate experiments, performed in triplicate).