Cytotoxic potential of lung CD8(+) T cells increases with chronic obstructive pulmonary disease severity and with in vitro stimulation by IL-18 or IL-15

Abstract

Lung CD8(+) T cells might contribute to progression of chronic obstructive pulmonary disease (COPD) indirectly via IFN-gamma production or directly via cytolysis, but evidence for either mechanism is largely circumstantial. To gain insights into these potential mechanisms, we analyzed clinically indicated lung resections from three human cohorts, correlating findings with spirometrically defined disease severity. Expression by lung CD8(+) T cells of IL-18R and CD69 correlated with severity, as did mRNA transcripts for perforin and granzyme B, but not Fas ligand. These correlations persisted after correction for age, smoking history, presence of lung cancer, recent respiratory infection, or inhaled corticosteroid use. Analysis of transcripts for killer cell lectin-like receptor G1, IL-7R, and CD57 implied that lung CD8(+) T cells in COPD do not belong to the terminally differentiated effector populations associated with chronic infections or extreme age. In vitro stimulation of lung CD8(+) T cells with IL-18 plus IL-12 markedly increased production of IFN-gamma and TNF-alpha, whereas IL-15 stimulation induced increased intracellular perforin expression. Both IL-15 and IL-18 protein expression could be measured in whole lung tissue homogenates, but neither correlated in concentration with spirometric severity. Although lung CD8(+) T cell expression of mRNA for both T-box transcription factor expressed in T cells and GATA-binding protein 3 (but not retinoic acid receptor-related orphan receptor gamma or alpha) increased with spirometric severity, stimulation of lung CD8(+) T cells via CD3epsilon-induced secretion of IFN-gamma, TNF-alpha, and GM-CSF, but not IL-5, IL-13, and IL-17A. These findings suggest that the production of proinflammatory cytokines and cytotoxic molecules by lung-resident CD8(+) T cells contributes to COPD pathogenesis.

Figures

Figure 1. Lung CD8+ T cells in COPD are predominately TEM cells at all disease stages, but their expression of CD69 and T-bet increases as FEV1 decreases

CD8+ T cells from lung tissue were either (A-D) stained and analyzed by flow cytometry or (E-G) isolated using positive selection magnetic beads and processed for real time RT-PCR. (A) representative histograms showing staining for CD27 (top panel) and CD62L (bottom panel) on gated lung CD8+ T cells of a COPD patient. Shaded profiles, isotype control; open profiles, specific staining. (B) the percentage of CD8+ T cells that display an effector memory phenotype (CD62L- CD27-) stratified by subject group. NS, non-smoker, S smokers with normal lung function, 1–4, GOLD stages of COPD severity. Spearman nonparametric analysis was used to calculate rS. Circles represent individual patients, bars represent the mean ± SEM (Cohort A,n = 42). (C, D) CD69 expression. (C) representative histograms from a normal smoker without COPD (top panel) and GOLD stage 4 subject (bottom panel). (D) the percentage of CD69+ CD8+ lung T cells stratified by subject group (Cohort A,n = 42). (E-G) mRNA expression by lung CD8+ T cells stratified by FEV1 (% predicted). G, T-bet. H, GATA-3. I, RORγ. Results are expressed as arbitrary units. Circles represent individual patients (Cohort B,n = 22). Spearman nonparametric analysis was used to calculaterS. N.S., not significant.

Figure 2. Expression of KLRG1, CD57 & IL-7R by lung CD8+ T cells

Lung CD8+ T cells were isolated for RNA analysis by real-time RT-PCR. A. KLRG1; B. CD57; C. IL-7R. Transcripts are expressed on the vertical axis as arbitrary units versus FEV1 (% predicted) on the horizontal axis. Circles represent individual subjects (Cohort B, n = 22). Spearman nonparametric analysis was used to calculate rS. N.S., not significant.

Figure 3. Expression of perforin and granzyme B by lung CD8+ T cells increases with worsening COPD severity

Lung CD8+ T cells were isolated for RNA analysis by real-time RT-PCR. A, perforin, B, granzyme B, and C, FasL were measured and are expressed on the vertical axis as arbitrary units versus FEV1 (% predicted) on the horizontal axis. Circles represent individual patients (Cohort B, n = 22). Spearman nonparametric analysis was used to calculate rS. N.S., not significant.

Figure 4. IL-18R expression on lung CD8+ T cells increases with worsening COPD severity

CD8+ T cells from lung tissue were stained and analyzed by flow cytometry. (A) representative histograms showing IL-18R mAb staining of gated CD8+ T cells from a smoker with normal lung function (top panel) and from a subject with advanced COPD (GOLD stage 4) (bottom panel). Shaded profiles, isotype control; open profiles, specific staining. (B) the percentage of CD8+ T cells that express IL-18R stratified by subject group (Cohort A,n = 42) as defined in the legend to Figure 1. (C) IL-18 protein levels from human lung tissue, stratified by group (Cohort C, n = 95). Spearman nonparametric analysis was used to calculaterS and p values. Open circles represent individual patients, bars represent the mean ± SEM.

Figure 5. Lung CD8+ T cell production of IFN-γ and TNF-α is increased by stimulation with IL-18 plus IL-12

Isolated lung CD8+ T cells were cultured for 48 h either with no stimulation (no stim), IL-18 alone, IL-12 alone, combined IL-18 and IL-12, or IL-15 alone. Supernatants were collected for protein measurement by Luminex assay. (A) IFN-γ; (B) TNF-α; (C) GM-CSF; (D) IL-5; (E) IL-13, and (F), IL-17; note the difference in scales between panel A and other panels. Bars are means ± SEM from five independent experiments. * p < 0.05 as compared to no stimulation (one-way ANOVA with Dunn’s post-hoc testing).

Figure 6. Lung CD8+ T cell production of Tc2 cytokines is not increased by TCR stimulation

Isolated lung CD8+ T cells were cultured for 48 h without (white columns) or with (gray columns) plate-bound anti-CD3ε. Supernatants were collected for protein measurement by Luminex assay. Bars are means ± SEM from five independent experiments. * p < 0.05 as compared to no anti-CD3ε stimulation (paired t-test).

Figure 7. IL-15 stimulation induces increased perforin production by lung CD8+ T cells

A, isolated lung CD8+ T cells were cultured for 48 h with either no stimulation, IL-18 alone, IL-12 alone, combined IL-18 plus IL-12, or IL-15 alone. Intracellular perforin expression by CD8+ T cells was measured by flow cytometry. Results are expressed as the fold-increase over non-stimulated controls. Bars represent means ± SEM from five individual experiments, * p < 0.05, compared to all other conditions (One-way ANOVA with Dunn’s post-hoc testing). (B) Whole lung IL-15 protein levels were measured by Luminex assay and stratified by subject group (Cohort C,n = 95). Circles represent individual patients, bars represent means ± SEM. (C) Immunohistochemical staining for IL-15 on frozen lung tissue from a representative subject (smoker with normal pulmonary function). Left panels, isotype control staining; right panels, IL-15 staining. Top panels, 20× magnification; bottom panels, 40× magnification.