T-regulatory cells and programmed death 1+ T cells contribute to effector T-cell dysfunction in patients with chronic obstructive pulmonary disease

Abstract

Rationale: Previous studies from our laboratory have shown that peripheral blood mononuclear cells (PBMCs) from patients with chronic obstructive pulmonary disease (COPD) prone to exacerbations with nontypeable Haemophilus influenzae have impaired responses to lipoprotein P6. We hypothesized that an underlying immunosuppressive network could be responsible for the defective antibacterial immunity observed in these patients. We evaluated T regulatory cells (Tregs), myeloid-derived suppressor cells (MDSC), and exhausted T effector cells (programmed death 1 [PD-1](+)) in patients with COPD, because these cells are known to play a pivotal role in suppressing immune responses.

Objectives: We performed an in-depth characterization of Tregs, T effector cells, and MDSC in COPD and correlated their levels and function with disease severity.

Methods: Treg, effector T cell, and MDSC frequency from patients with COPD and healthy subjects' PBMCs were analyzed by flow cytometry. Treg immunosuppressive capacity was measured by in vitro suppression assay. The frequency of interferon-γ producing T cells and T-cell proliferation were measured after blocking CTLA-4 and PD-1. Plasma proinflammatory and immunosuppressive cytokine levels were measured.

Measurements and main results: Significantly increased levels of Tregs, MDSC, and PD-1(+) exhausted effector T cells were present in patients with COPD compared with healthy subjects. Tregs from patients with COPD suppressed P6-specific T-cell proliferation to a greater extent than Tregs from healthy subjects. Plasma levels of Treg-generated cytokines, IL-10, and transforming growth factor-β were elevated. Blockade of CTLA-4 resulted in significant augmentation of T-cell IFN-γ production in patients with COPD.

Conclusions: Functionally suppressive Tregs, MDSCs, and exhausted PD-1(+) T cells contribute to effector T-cell dysfunction in COPD.

Keywords: Foxp3+ Tregs; T effector cells; cytokines; lung function; myeloid-derived suppressor cells.

Figure 1.

Accumulation of immunosuppressive cells in patients with chronic obstructive pulmonary disease (COPD). (A) Frequency and (B) absolute number of CD4⁺Foxp3⁺ T regulatory cells (Tregs) in the peripheral blood of patients with COPD (n = 24) and healthy control subjects (n = 31). (C) Quantitative polymerase chain reaction analysis of Foxp3 gene expression in peripheral blood mononuclear cells of patients with COPD (n = 10) and healthy donors (n = 10). Lines represent mean values of Foxp3 mRNA copies normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) control. (D) Correlation between Foxp3 mRNA copies and CD4⁺Foxp3⁺ Treg frequency. (E) Frequency and (F) absolute number of CD3⁺CD4⁺ CD25⁺CD127⁻Foxp3⁺ Tregs in the peripheral blood of patients with COPD (n = 24) and healthy control subjects (n = 31). (G) Frequency and (H) absolute number of CD3⁺CD4⁺CD127⁺Foxp3⁻ effector T cells in patients with COPD and healthy control subjects. (I) Frequency and (J) absolute number of cells per milliliter of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with COPD (n = 24) and healthy donors (n = 31). (K) Correlation of CD11b⁺CD33⁺ MDSC frequency and CD4⁺Foxp3⁺ Treg frequency. Each symbol represents an individual COPD patient (black dots) or healthy control subject (grey squares); lines represent mean values for the group. ***P < 0.001, permutation t test; ^†P < 0.05 Hochberg adjustment for multiple comparison.

Figure 2.

GARP and CTLA-4 expressing T regulatory cells (Tregs) in patients with chronic obstructive pulmonary disease (COPD). (A) Frequency of GARP⁺ Tregs and (B) GARP expression levels measured by mean fluorescent intensity (MFI) on Tregs from COPD (n = 24) and healthy subjects (n = 31). (C) Quantitative polymerase chain reaction analysis of GARP gene expression in peripheral blood mononuclear cells of patients with COPD (n = 10) and healthy donors (n = 10). Lines represent mean values of GARP mRNA copies normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) control. (D) Frequency of CTLA-4⁺ Tregs and (E) CTLA-4 expression levels. (F) Quantitative polymerase chain reaction analysis of CTLA-4 gene expression in peripheral blood mononuclear cells of patients with COPD (n = 10) and healthy donors (n = 10). Lines represent mean values of CTLA-4 mRNA copies normalized to GAPDH control. (G) Correlation of either GARP⁺ Treg frequency or (H) CTLA-4⁺ Treg frequency to CD25⁺CD127⁻Foxp3⁺ Treg frequency in patients with COPD (n = 24) and healthy control subjects (n = 31). Each symbol represents an individual COPD patient (black dots) or healthy control subject (grey squares); lines represent mean values for the group. ***P < 0.001, permutation t test; ^†P < 0.05 Hochberg adjustment for multiple comparison.

Figure 3.

High suppressive potential of T regulatory cells (Tregs) and functional exhaustion of effector T cells in patients with chronic obstructive pulmonary disease (COPD). (A and B) Frequency of CD4⁺PD-1⁺CD127⁺ T cells and PD-1 expression level on CD4⁺ T cells measured as mean fluorescent intensity (MFI). (C and D) Proliferative response of sorted CD4⁺CD25⁻CD127⁺ effector cells to P6 protein in the presence or absence of purified CD4⁺CD25⁺CD127⁻ autologous Tregs. Each symbol represents an individual COPD patient (black dots) or healthy control subject (grey squares); lines represent mean values for the group. *P < 0.05, ***P < 0.001, permutation t test; ^†P < 0.05 Hochberg adjustment for multiple comparison. PD-1 = programmed death 1.

Figure 4.

Elevated levels of immunosuppressive cytokines in patients with chronic obstructive pulmonary disease (COPD). Cytokine-specific sandwich ELISA of plasma from patients with COPD and healthy normal subjects were performed to measure the levels of circulating (A) IL-10, (B) transforming growth factor (TGF)-β1, (D) IFN-γ, and (E) IL-12. (C) Correlation of plasma TGF-β1 levels and frequency of CD4⁺Foxp3⁺ T regulatory cells (Tregs). Each symbol represents an individual patient with COPD (black dots) or healthy control subject (grey squares); lines represent mean values for the group. **P < 0.01, ***P < 0.001, permutation t test; ^†P < 0.05 Hochberg adjustment for multiple comparison.

Figure 5.

Correlation of immune parameters with lung functions of patients with chronic obstructive pulmonary disease (COPD). (A) Correlation of GARP⁺ Foxp3⁺ T regulatory cells (Tregs), (B) levels of plasma IFN-γ, and (C) levels of plasma IL-12 with lung functions (FEV₁) of patients with COPD. Each symbol represents an individual COPD patient (black dots).

Figure 6.

Enhanced T-cells functionality after blocking immune check-points in patients with chronic obstructive pulmonary disease. (A) Proliferation of CD4⁺ T cells (left), frequency of CD4⁺ T cells producing IFN-γ (middle) and expression levels of IFN-γ on CD4⁺ T cells (right) measured as mean fluorescence intensity (MFI). (B) Proliferation of CD8⁺ T cells (left), frequency of CD8⁺ T cells producing IFN-γ (middle), and expression levels of IFN-γ on CD8⁺ T cells (right) measured as MFI. Peripheral blood mononuclear cells from patients with chronic obstructive pulmonary disease (n = 3) were stimulated in vitro in the presence or absence of anti–CTLA-4 or programmed death 1 (PD-1) antibody. T-cell proliferation was measured based on dilution of carboxyfluorescein succinimidyl ester and proliferation index was calculated using the Flowjo Version 9 proliferation platform (TreeStar). Intracellular cytokine staining was performed to measure IFN-γ. (C) Cytokine-specific sandwich ELISA of the culture supernatants from proliferation assay was performed to measure the levels of secretory IFN-γ, IL-6, IL-10, IL-17-A, transforming growth factor (TGF)-β1, and tumor necrosis factor (TNF)-α. *P < 0.05, **P < 0.01, ***P < 0.001, permutation t test; ^†P < 0.05 Hochberg adjustment for multiple comparison.