Altered effector function of peripheral cytotoxic cells in COPD

Abstract

Background: There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3-) cells and NKT-like (CD56+CD3+) cells.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results: The proportion of peripheral blood NKT-like (CD56+CD3+) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56+CD3-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56+CD3+) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells from smokers and HNS.

Conclusion: In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

Figure 1

Proportion and type of peripheral blood mononuclear cells in HNS (n = 12), smokers (n = 15) and COPD subjects (n = 10). Results show a significant decrease in the proportion of NK (CD56⁺CD3^-) cells (*; p < 0.05) and NKT-like (CD56⁺CD3⁺) cells (***; p < 0.001) in COPD subjects compared to HNS. Smokers had a significantly lower proportion of B-cells (***; p < 0.001) compared to the other two groups and a greater proportion of CD4⁺ T helper cells. Cell types were determined by flow cytometric analysis of monoclonal antibodies. CD19, B cells; CD4, T helper cells; CD8, cytotoxic killer cells; CD56⁺CD3^-, NK cells; CD56⁺CD3⁺, NKT-like cells.

Figure 2

Proportion of NK (CD56⁺ CD3^-) subsets (Panel A), NKT-like (CD56⁺ CD3⁺) subsets (Panel B) and CD8+ T lymphocyte subsets (Panel C), from the peripheral blood of HNS (n = 12), smokers (n = 15) and COPD subjects (n = 10). Panel A shows the proportion of CD56^brightCD16^- NK cells was significantly increased in COPD subjects compared to HNS (**; p < 0.01) and smokers (***; p < 0.001). Panel B shows significantly more CD8⁺CD56⁺CD3⁺ cells (**; p < 0.01) in the peripheral blood of COPD subjects compared to the other two groups and a significant decrease in the proportion of CD4⁺CD56⁺CD3⁺ cells (***; p < 0.001). Panel C shows cells of the highly cytotoxic effector memory phenotype (T_EMRA; CD8⁺CD45RO⁺RA⁺CD62L^-) were significantly decreased in COPD subjects (*; p < 0.05).

Figure 3

Representative flow cytometry plot (Panel A) showing the expression of both granzyme B and perforin (Panel B) in CD88⁺ T lymphocytes, CD56^dim CD16⁺ NK cells, CD56^bright CD16^- NK cells and NKT-like (CD56⁺CD3⁺) cells from non-smoking healthy participants (n = 12), healthy smokers (n = 15) and smokers with COPD (n = 10). Double stained cells (Panel B) are deemed cytotoxic (**; p < 0.01).

Figure 4

Cytotoxic activity of NK (CD56⁺ CD3^-) cells (Panel A) and NKT-like (CD56⁺ CD3⁺) cells (Panel B). Immunomagnetically separated cells (25,000) were cultured with K562 cells (5,000) giving an effector:target ratio of 5:1, in an LDH release cytotoxicity assay (***; p < 0.001).

Figure 5

Correlation of cytotoxic activity and lung function in NK (CD56⁺CD3^-) cells (Panel A) and NKT-like (CD56⁺CD3⁺) cells (Panel B) in COPD subjects. Immunomagnetically separated cells (25,000) were cultured with K562 cells (5,000) giving an effector:target ratio of 5:1, in an LDH release cytotoxicity assay compared to FEV₁ (% pred).

Figure 6

Cytotoxic activity of CD56⁺ cells in the presence of different concentrations of an anti-perforin antibody (A), an anti-granzyme B antibody (B) and a combination of the two (C) from HNS (n = 3), smokers (n = 3) and COPD subjects (n = 3). Immunomagnetically selected CD56⁺ cells were incubated with the stated concentration of antibody and used at the effector:target ratio of 5:1 against K562 cells in the LDH release cytotoxicity assay.