Basal Gene Expression by Lung CD4+ T Cells in Chronic Obstructive Pulmonary Disease Identifies Independent Molecular Correlates of Airflow Obstruction and Emphysema Extent

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 NCT0028122

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)

Background Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. Methods The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Results Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Conclusions Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. Trial registration This study was registered with ClinicalTrials.gov as NCT01969344

Human CD56+ cytotoxic lung lymphocytes kill autologous lung cells in chronic obstructive pulmonary disease.

CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3-) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly "cytotoxic" CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.ClinicalTrials.gov NCT00281229

Identification and characterization of human lung NK cells and CD56+ T cells.

<p>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). (<b>A, C, E</b>) Representative staining: isotype controls on left, specific staining on right; (<b>B, D, F</b>) 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. (<b>A</b>) 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>B</b>) NK cells (blue bars) versus CD56+ T cells (orange bars). (<b>C, D</b>) 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). (<b>E, F</b>) 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).</p

Summary of subject demographics, smoking history, and spirometry.

<p>Data are presented as average (SD), except for sex, smoking status,</p><p>ICS, and resection for cancer ratios.</p><p>ICS, inhaled corticosteroids;</p><p>M, male;</p><p>F, female.</p

Increased cytotoxicity by lung CD56+ cells correlates with decreased pulmonary function.

<p>Human lung CD56+ cells were co-cultured with autologous lung target cells and % cytotoxicity was determined as described in the Methods. (<b>A</b>) 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>B</b>) FEV₁ % predicted (x-axis) versus % cytotoxicity (y-axis). Spearman non-parametric correlation was used to determine the p-value. (<b>C, D</b>) 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 (<b>C</b>) and DLCO % predicted (<b>D</b>) 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 (<i>n</i> = 6); □, mild COPD (<i>n</i> = 12); Δ, severe COPD (<i>n</i> = 10). Lines represent the mean ± SEM.</p

Human lung CD56+ cells spontaneously kill autologous lung CD45− cells in vitro.

<p>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. (<b>A</b>) 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>B</b>) % Cytotoxicity (y-axis) for target cells cultured with CD56+ cells (blue circles), CD8+ cells (orange circles), or CD4+ cells (green circles); <i>n</i> = 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.</p

The percentage of epithelial cells expressing MICA/MICB is increased with COPD severity.

<p>Human lung tissue was dispersed and stained with monoclonal antibodies against CD45, CD56, NKG2D, CD326, and MICA/MICB. (<b>A</b>) 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>B</b>) The percentage of CD56+ cells that express NKG2D (y-axis) versus FEV₁ % predicted (x-axis). x, smokers without COPD (<i>n</i> = 10); □, mild COPD (<i>n</i> = 5); Δ, severe COPD (<i>n</i> = 10). N.S., not significant. (<b>C</b>) 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. (<b>D</b>) The percentage of CD326+ epithelial cells that express MICA/MICB (y-axis) versus FEV₁ % predicted (x-axis). x, smokers without COPD (<i>n</i> = 10); □, mild COPD (<i>n</i> = 5); Δ, severe COPD (<i>n</i> = 10). Spearman correlation was used to determine the p value.</p

Linear regression model to evaluate ability of variables to predict cytotoxicity of CD56+ cells.

<p>Linear regression model to evaluate ability of variables to predict cytotoxicity of CD56+ cells.</p