Table 1.
Summary of subject demographics, smoking history, and spirometry.
Figure 1.
Identification and characterization of human lung NK cells and CD56+ T cells.
Lung tissue was dispersed, stained with monoclonal antibodies against CD45, CD3, CD56, and CD16 and analyzed by flow cytometry to select a viable population comprised predominately of lung lymphocytes (CD45+, low side-scatter cells). (A, C, E) Representative staining: isotype controls on left, specific staining on right; (B, D, F) Frequency of various lung lymphocyte populations in individual subjects as a percentage of the total viable lung lymphocyte population; note difference in scale of panel B. (A) Ungated staining for CD3 and CD56 identifies four distinct populations: NK cells (CD56+ CD3−); CD56+ T cells (CD56+ CD3+); conventional T cells (CD56− CD3+); and double-negative cells (predominately B cells). (B) NK cells (blue bars) versus CD56+ T cells (orange bars). (C, D) After gating on CD3− cells, staining for CD56 and CD16 identifies two lung NK populations: CD56+ CD16+ (light blue circle & columns) and CD56+ CD16− (dark blue circle & columns). (E, F) After gating on CD3+ cells, staining for CD56 and CD16 identifies two lung CD56+ T cell populations: CD56+ CD16+ (dark orange circle & columns) and CD56+ CD16− (light orange circle & columns). By Kruskal-Wallis one-way ANOVA, there are no significant differences between subject groups for any of these three lung cell populations (B, D, F).
Figure 2.
The frequency of lung NK cells, but not lung CD56+ T cells, co-expressing CD8 correlates with FEV1 % predicted.
(A–C) NK cells; (D–F) CD56+ T cells. (A) Representative staining for CD8 on CD56+ CD3− NK cells from a smoker without COPD (left panel) and a COPD subject (right panel); blue line, CD8+ staining; grey line, isotype control. (B) Subjects were categorized by pulmonary function (x-axis) versus the percentage of NK cells that co-express CD8 (y-axis); x, smokers without COPD (n = 5); □, mild COPD (n = 9); Δ, severe COPD (n = 9); Kruskal-Wallis one-way ANOVA testing was used to determine significance. (C) The percentage of NK cells that co-express CD8 (y-axis) versus FEV1 % predicted (x-axis). Spearman correlation was used to determine the p-value. (D) Representative staining for CD8 and CD4 on CD56+ CD3+ T cells from a smoker without COPD (left panel) and a COPD subject (right panel). The numbers in the quadrants are the % of each subset among all lung CD56+ T cells. (E) CD4 single-positive (brown bars), CD8 single-positive (orange bars), and CD8/CD4 double-negative (grey bars) are shown as the percentage of lung CD56+ T cells for individual subjects. No difference was seen between groups. (F) The percentage of lung CD56+ T cells (y-axis) that express CD4 (brown symbols) or CD8 (orange symbols) versus FEV1 % predicted (x-axis); x, smokers without COPD (n = 5); □, mild COPD (n = 9); Δ, severe COPD (n = 9).
Figure 3.
The percentage of epithelial cells expressing MICA/MICB is increased with COPD severity.
Human lung tissue was dispersed and stained with monoclonal antibodies against CD45, CD56, NKG2D, CD326, and MICA/MICB. (A) Representative staining showing the expression of NKG2D on CD45+ CD56+ cells from a smoker without COPD (left panel) and a subject with COPD (right panel). Blue line, NKG2D+ staining; grey line, isotype control. (B) The percentage of CD56+ cells that express NKG2D (y-axis) versus FEV1 % predicted (x-axis). x, smokers without COPD (n = 10); □, mild COPD (n = 5); Δ, severe COPD (n = 10). N.S., not significant. (C) Representative staining showing the expression of MICA/MICB on CD45−, CD326 (EpCAM)+ epithelial cells from a smoker without COPD (left panel) and a subject with COPD (right panel). Blue line, MICA/MICB+ staining; grey line, isotype control. (D) The percentage of CD326+ epithelial cells that express MICA/MICB (y-axis) versus FEV1 % predicted (x-axis). x, smokers without COPD (n = 10); □, mild COPD (n = 5); Δ, severe COPD (n = 10). Spearman correlation was used to determine the p value.
Figure 4.
Human lung CD56+ cells spontaneously kill autologous lung CD45− cells in vitro.
CD56+ cells were isolated from dispersed human lung tissue using magnetic beads. CD8+ cells were isolated from the CD56 depleted fraction and CD4+ cells were isolated from the CD56− and CD8− depleted fraction. The remaining cells were used as autologous target cells. Target cells were cultured either alone or with CD56+ cells, CD8+ cells, or CD4+ cells at a ratio of 1 target to 10 effectors. After 4 hours, all cells were collected and stained with CD45, Annexin-V, and 7-AAD for flow cytometry. Target cells were identified as CD45− with a high side scatter. (A) Representative staining of Annexin-V on target cells that were cultured with CD56+ cells (left panel), CD8+ cells (middle panel), and CD4+ cells (right panel). (B) % Cytotoxicity (y-axis) for target cells cultured with CD56+ cells (blue circles), CD8+ cells (orange circles), or CD4+ cells (green circles); n = 28. Lines represent the mean ± SEM. The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups.
Figure 5.
Increased cytotoxicity by lung CD56+ cells correlates with decreased pulmonary function.
Human lung CD56+ cells were co-cultured with autologous lung target cells and % cytotoxicity was determined as described in the Methods. (A) Subjects were categorized by pulmonary function (x-axis) versus % cytotoxicity (y-axis). The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups. (B) FEV1 % predicted (x-axis) versus % cytotoxicity (y-axis). Spearman non-parametric correlation was used to determine the p-value. (C, D) The same subjects were separated into two groups based on their CD56+ cell function: non-cytotoxic (<8.5% cytotoxicity) or cytotoxic (≥8.5% cytotoxicity) and then FEV1 % predicted (C) and DLCO % predicted (D) were analyzed. The parametric unpaired Student t-test was used to determine significant differences between the two groups. In all figures, x, smokers without COPD (n = 6); □, mild COPD (n = 12); Δ, severe COPD (n = 10). Lines represent the mean ± SEM.
Table 2.
Linear regression model to evaluate ability of variables to predict cytotoxicity of CD56+ cells.