Basal gene expression by lung CD4+ T cells in chronic obstructive pulmonary disease identifies independent molecular correlates of airflow obstruction and emphysema extent

Abstract

Lung CD4+ T cells accumulate as chronic obstructive pulmonary disease (COPD) progresses, but their role in pathogenesis remains controversial. To address this controversy, we studied lung tissue from 53 subjects undergoing clinically-indicated resections, lung volume reduction, or transplant. Viable single-cell suspensions were analyzed by flow cytometry or underwent CD4+ T cell isolation, followed either by stimulation with anti-CD3 and cytokine/chemokine measurement, or by real-time PCR analysis. In lung CD4+ T cells of most COPD subjects, relative to lung CD4+ T cells in smokers with normal spirometry: (a) stimulation induced minimal IFN-γ or other inflammatory mediators, but many subjects produced more CCL2; (b) the T effector memory subset was less uniformly predominant, without correlation with decreased IFN-γ production. Analysis of unstimulated lung CD4+ T cells of all subjects identified a molecular phenotype, mainly in COPD, characterized by markedly reduced mRNA transcripts for the transcription factors controlling TH1, TH2, TH17 and FOXP3+ T regulatory subsets and their signature cytokines. This mRNA-defined CD4+ T cell phenotype did not result from global inability to elaborate mRNA; increased transcripts for inhibitory CD28 family members or markers of anergy; or reduced telomerase length. As a group, these subjects had significantly worse spirometry, but not DLCO, relative to subjects whose lung CD4+ T cells expressed a variety of transcripts. Analysis of mRNA transcripts of unstimulated lung CD4+ T cell among all subjects identified two distinct molecular correlates of classical COPD clinical phenotypes: basal IL-10 transcripts correlated independently and inversely with emphysema extent (but not spirometry); by contrast, unstimulated IFN-γ transcripts correlated independently and inversely with reduced spirometry (but not reduced DLCO or emphysema extent). Aberrant lung CD4+ T cells polarization appears to be common in advanced COPD, but also exists in some smokers with normal spirometry, and may contribute to development and progression of specific COPD phenotypes.

Trial registration: ClinicalTrials.gov as NCT00281229.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. TCR-stimulated lung CD4+ T cells produce less IFN-γ protein but more CCL2 in COPD.

Isolated lung CD4+ T cells were stimulated with plate-bound anti-CD3ε for 48 hours. Supernatants were collected and Luminex assays were used to measure (A) IFN-γ, (B) CCL2, and (C) CCL3 for smokers with normal spirometry (orange diamonds; n = 6) and COPD subjects (blue diamonds; n = 23 for panel A; n = 18 for panels B & C). Each symbol represents an individual patient; solid lines represent the mean ± SEM. The dashed line represents the minimum detectable concentration. The Mann Whitney t-test was used to determine significant differences between groups.

Figure 2. The TEM subset of lung CD4+ T cells is significantly decreased in COPD patients.

Single cell suspensions of mechanically disaggregated human lung tissue were stained and analyzed by flow cytometry. A, B. Representative flow plots showing staining for CD62L (horizontal axis) versus CD27 (vertical axis) on gated CD4+ T cells from (A) a smoker normal spirometry and (B) a subject with COPD. Small blue numbers are the percentage of CD4+ T cells in each quadrant. C–E. Aggregated percentages of lung CD4+ T cells from smokers with normal spirometry (orange; n = 7) and COPD subjects (blue; n = 15) that were (C) negative for both CD62L & CD27 (TEM), (D) positive for both CD62L & CD27, or (E) negative for CD62L but positive for CD27. Box-&-whisker plots indicating the first & third quartiles (box), median (band) and range (whiskers). The Mann Whitney t-test was used to determine significance.

Figure 3. Production of IFN-γ protein by stimulated lung CD4+ T cells correlates with CD103+ expression.

Isolated lung CD4+ T cells were stimulated with plate-bound anti-CD3ε for 48 hours. Supernatants were collected and Luminex assays were used to measure IFN-γ. A. Aggregated percentages of CD103+ cells among lung CD4+ T cells of smokers with normal spirometry (maize; n = 7) and COPD subjects (blue; n = 17). The Mann Whitney t-test was used to determine significant differences between groups. B. Correlation between stimulated IFN-γ protein concentrations (vertical axis, log scale) and percentage of CD103+ lung CD4+ T cells (horizontal axis). Smokers with normal spirometry (orange diamond; n = 3) and COPD subjects (blue diamonds; n = 14) are a subset of those in panel A, as not all subjects had corresponding protein data. Statistics by Spearman correlation.

Figure 4. Unsupervised hierarchical clustering separates subjects based on unstimulated lung CD4+ T cell gene expression profiles.

Transcripts for 27 genes were measured by real-time RT-PCR from isolated lung CD4+ T cells and results were displayed as a heat map. Columns represent individual subjects; gene identities are shown on the right. Red = down-regulated, white = unchanged, blue = up-regulated, grey = missing data. Based on spirometry plus clinical diagnosis, subjects were classified (top) either as smokers without COPD (green; n = 8) or COPD (purple; n = 23). Unsupervised hierarchical clustering divided the subjects into two distinct groups, as indicated by the dendrogram. Group A contained 14 of 23 COPD subjects and two of eight smokers without COPD, while Group B consisted of the remaining six smokers without COPD and the other nine COPD subjects. The Row Z scores were calculated from log-transformed arbitrary units.

Figure 5. Group A subjects are characterized by worse spirometry than Group B subjects.

Pulmonary function was assessed for all subjects and is presented based on the groups defined in Figure 4. (A) FEV1% predicted, (B) FEV1/FVC ratio, and (C) DLCO % predicted are shown for Group A subjects (Xs; n = 16) and Group B subjects (circles; n = 15). Orange symbols represent smokers without COPD (n = 8) and blue symbols represent subjects with COPD (n = 23). Symbols represent individual patients, lines represent the mean ± SEM. The Mann Whitney t-test was used to determine significant differences between groups. N.S., not significant.

Figure 6. Group A subjects have significantly reduced transcripts for multiple polarizing transcription factors & signature cytokines.

Lung CD4+ T cells were isolated for RNA analysis by real-time RT-PCR and mRNA transcripts are displayed as arbitrary units according to the subject groups defined by the heat map analysis in Figure 4. Group A (Xs; n = 16) and Group B (circles; n = 15). (A) T-bet, (B) GATA-3, (C) ROR-γ, (D), FOXP3, (E) IFN-γ, (F) IL-5, (G) IL-17A, (H) IL-22, (I) CCL2, (J) CHOP, (K) CTLA-4 and (L) ICOS. In both groups, orange symbols represent smokers without COPD (n = 8) and blue symbols represent subjects with COPD (n = 23). Symbols represent individual patients, lines represent the mean ± SEM. The Mann Whitney t-test was used to determine significant differences between groups. N.S., not significant.

Figure 7. Decreased IL-10 mRNA transcripts in lung CD4+ T cells correlate with worsening emphysema.

Lung CD4+ T cells were isolated for analysis of IL-10 mRNA by real-time RT-PCR. (A) mRNA transcripts presented in the Group A and Group B subject groups defined in Figure 4. Symbols, which represent individual subjects, are as described in the Legend to Figure 6; lines represent the mean ± SEM. The Mann Whitney t-test was used to determine significant differences between groups; N.S., not significant. (B) Relationship of IL-10 mRNA transcripts in lung CD4+ T cells, expressed as arbitrary units (vertical axis), to percent emphysema (−950 HU threshold) (horizontal axis) (n = 17). Spearman non-parametric analysis was used to calculate rS and p values.

Figure 8. Group A subjects have expansion of an unusual population (CD62L−, CD27+), but not of TEM.

Available flow cytometry data on subjects in Group A and Group B were analyzed to determine the fraction of lung CD4+ T cells in each of the quadrants defined by CD62L and CD27 expression. (A) CD62L− CD27− TEM; (B) CD62L+ CD27+; (C) CD62L− CD27+. Symbols represent individual patients, lines represent the mean ± SEM. Group A subjects (Xs; n = 8) and Group B subjects (circles; n = 6); orange symbols represent smokers without COPD (n = 3) and blue symbols represent subjects with COPD (n = 10). The Mann Whitney t-test was used to determine significant differences between groups. N.S., not significant.