Lung CD8+ T cells in COPD have increased expression of bacterial TLRs

Abstract

Background: Toll-like receptors (TLRs) on T cells can modulate their responses, however, the extent and significance of TLR expression by lung T cells, NK cells, or NKT cells in chronic obstructive pulmonary disease (COPD) is unknown.

Methods: Lung tissue collected from clinically-indicated resections (n = 34) was used either: (a) to compare the expression of TLR1, TLR2, TLR2/1, TLR3, TLR4, TLR5, TLR6 and TLR9 on lung CD8+ T cells, CD4+ T cells, NK cells and NKT cells from smokers with or without COPD; or (b) to isolate CD8+ T cells for culture with anti-CD3ε without or with various TLR ligands. We measured protein expression of IFN-γ, TNF-α, IL-13, perforin, granzyme A, granzyme B, soluble FasL, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL9 in supernatants.

Results: All the lung subsets analyzed demonstrated low levels of specific TLR expression, but the percentage of CD8+ T cells expressing TLR1, TLR2, TLR4, TLR6 and TLR2/1 was significantly increased in COPD subjects relative to those without COPD. In contrast, from the same subjects, only TLR2/1 and TLR2 on lung CD4+ T cells and CD8+ NKT cells, respectively, showed a significant increase in COPD and there was no difference in TLR expression on lung CD56+ NK cells. Production of the Tc1 cytokines IFN-γ and TNF-α by lung CD8+ T cells were significantly increased via co-stimulation by Pam3CSK4, a specific TLR2/1 ligand, but not by other agonists. Furthermore, this increase in cytokine production was specific to lung CD8+ T cells from patients with COPD as compared to lung CD8+ T cells from smokers without COPD.

Conclusions: These data suggest that as lung function worsens in COPD, the auto-aggressive behavior of lung CD8+ T cells could increase in response to microbial TLR ligands, specifically ligands against TLR2/1.

Trial registration: ClinicalTrials.gov NCT00281229.

Figures

Figure 1

Gating strategy used to identify T cells, NK cells, and NKT cells isolated from human lung tissue. Lung tissue was dispersed and stained with CD45, CD3, CD8, CD4, and CD56 monoclonal antibodies to identify various cell subsets. (A) Viable CD45+ cells with a low side scatter profile were selected; (B) cells were further divided into CD3- and CD3+ fractions; (C) CD56+ cells were selected from the CD3- fraction and (E) NK cells were selected by excluding CD4 and CD8. (D) CD8+ and CD4+ cells were identified and (F) CD56+ was used to separate CD8+ NKT cells from CD8+ T cells and (G) CD4+ NKT cells from CD4+ T cells. Appropriate isotype-matched controls for all antibodies were used to determine positive staining.

Figure 2

Representative histograms demonstrating an increase in TLR expression on lung CD8+ T cells in COPD. CD8+ CD56- cells from human lung tissue were identified as described in the legend to Figure  1 and examined for TLR expression. Left panels are representative histograms (A, C, E, G) or density plot (I) from smokers without COPD and right panels are representative histograms (B, D, F, H) or density plot (J) from subjects with COPD. Expression of (A, B) TLR1, (C, D) TLR2, (E, F) TLR4, (G, H) TLR6, and (I, J) TLR2/1 are shown. Shaded profile, isotype staining; open profiles, antibody-specific staining.

Figure 3

Percentage of CD8+ T cells expressing bacterial TLRs is increased in COPD subjects. Lung tissue was dispersed and stained with monoclonal antibodies to measure TLR expression on CD8+ T cells (left panels), CD4+ T cells (middle panels), or CD56+ NK cells (right panels) from smokers without COPD (S; blue circles; n = 10) and with COPD (COPD; red circles; n = 18). The vertical axis shows the percentage of cells expressing a given TLR. Open circles represent individual patients. Lines represent the mean ± SEM. The Mann Whitney t-test was used to determine significant differences between groups. *, p < 0.05, **, p < 0.01.

Figure 4

Expression of TLR5 and TLR2/1 on lung CD8+ T cells and NK cells increases with worsening emphysema. Lung tissue was stained with monoclonal antibodies to measure TLR expression on T cells, NK cells, and NKT cells. Expression (vertical axis) of (A) TLR5 on CD8+ T cells, (B) TLR2/1 on CD8+ T cells, (C) TLR5 on NK cells, (D) TLR6 on NK cells, (E) TLR2/1 on NK cells, stratified by percent emphysema (−950 HU threshold) (horizontal axis). Open circles represent individual patients, n = 13. Spearman non-parametric analysis was used to calculate the rS value.

Figure 5

Stimulation of TLR2/1 on lung CD8+ T cells increases production of IFN-γ TNF-α and CCL3. CD8+ T cells were isolated from lung tissue of COPD patients (n = 7) and cultured for 48 hours with anti-CD3ε alone or with addition of specific ligands for the following TLRs (in parentheses): Pam3CSK4 (TLR2/1), HKLM (TLR2), LPS (TLR4), and Flagellin (TLR5). Supernatants were measured using multiplex beads for (A, E) IFN-γ, (B, F) TNF-α, (C, G) IL-13, and (D, H) CCL3. (A-D) Results are expressed as the fold change in protein concentration over the control stimulated with anti-CD3ε alone (represented by dashed line) or (E-H) as absolute values of protein. Bars represent the mean ± SEM. Kruskal-Wallis One-way ANOVA was used to look for differences between groups. *, p < 0.05, compared to the control.

Figure 6

Lung CD8+ T cells from COPD subjects have increased production of IFN-γ and TNF-α than CD8+ T cells from subjects without COPD. A-D, CD8+ T cells were isolated from lung tissue of smokers without COPD (S, n = 5) for comparison to CD8+ T cells from subjects with COPD (n = 7) and cultured with anti-CD3ε plus Pam3CSK4 for 48 hours. Supernatants were measured using multiplex beads for (A, E) IFN-γ, (B, F) TNF-α, and (C, G) IL-13 and (D, H) CCL3. (A-D) Results are expressed as the fold change in protein concentration over the control stimulated with anti-CD3ε alone (represented by dashed line) or (E-H) as absolute values of protein. Bars represent the mean ± SEM. The Mann–Whitney t-test was used to compare control versus COPD subjects. *, p < 0.05, compared to the control.