Inducible Costimulator-C-X-C Motif Chemokine Receptor 3 Signaling is Involved in Chronic Obstructive Pulmonary Disease Pathogenesis

Abstract

Background: The role of inducible costimulator (ICOS) signaling in chronic obstructive pulmonary disease (COPD) has not been fully elucidated.

Methods: We compared the percentages of ICOS⁺ T cells and ICOS⁺ regulatory T (Treg) cells in CD4⁺ T cells and CD4⁺CD25⁺FOXP3⁺ Tregs, respectively, in the peripheral blood of smokers with or without COPD to those in healthy controls. We further characterized their phenotypes using flow cytometry. To investigate the influence of ICOS signaling on C-X-C motif chemokine receptor 3 (CXCR3) expression in COPD, we evaluated the expression levels of ICOS and CXCR3 in vivo and in vitro.

Results: ICOS expression was elevated on peripheral CD4⁺ T cells and CD4⁺ Tregs of COPD patients, which positively correlated with the severity of lung function impairment in patients with stable COPD (SCOPD), but not in patients with acute exacerbation of COPD (AECOPD). ICOS⁺CD4⁺ Tregs in patients with SCOPD expressed higher levels of coinhibitors, programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with Ig and ITIM domains (TIGIT), than ICOS^-CD4⁺ Tregs, whereas ICOS⁺CD4⁺ T cells mostly exhibited a central memory (CD45RA^-CCR7⁺) or effector memory (CD45RA^-CCR7^-) phenotype, ensuring their superior potential to respond potently and quickly to pathogen invasion. Furthermore, increased percentages of CXCR3⁺CD4⁺ T cells and CXCR3⁺CD4⁺ Tregs were observed in the peripheral blood of patients with SCOPD, and the expression level of CXCR3 was higher in ICOS⁺CD4⁺ T cells than in ICOS^-CD4⁺ T cells. The percentage of CXCR3⁺CD4⁺ T cells was even higher in the bronchoalveolar lavage fluid than in matched peripheral blood in SCOPD group. Lastly, in vitro experiments showed that ICOS induced CXCR3 expression on CD4⁺ T cells.

Conclusions: ICOS signaling is upregulated in COPD, which induces CXCR3 expression. This may contribute to increased numbers of CXCR3⁺ Th1 cells in the lungs of patients with COPD, causing inflammation and tissue damage.

Keywords: CXCR3; ICOS; T cell; Treg; chronic obstructive pulmonary disease.

Figure 1

Expression of inducible costimulator (ICOS) on CD4⁺ T cells and CD4⁺ regulatory T (Treg) cells in the peripheral blood of four groups of people. (A) Representative flow cytometric dot plots of ICOS expression on CD4⁺ T cells and CD4⁺CD25⁺FOXP3⁺ Tregs of patients with stable chronic obstructive pulmonary disease (SCOPD). Comparisons of the percentage of ICOS⁺ T cells within CD4⁺ T cells (B) and CD4⁺CD25⁺FOXP3⁺ Tregs (C) in the peripheral blood from 15 healthy controls (HC), 11 healthy smokers (HS), 22 patients with SCOPD, and 14 patients with acute exacerbation of COPD (AECOPD). Correlations of the frequencies of ICOS⁺CD4⁺ T cells (D and E) and ICOS⁺CD4⁺CD25⁺FOXP3⁺ Tregs (F and G) with forced expiratory volume in one second/forced vital capacity (FEV1/FVC) and FEV1 (% predicted), respectively (n = 22). *p<0.05, **p<0.01, ****p<0.0001.

Figure 2

Characteristics of ICOS⁺ or ICOS⁻ Tregs and the relationship between inducible costimulator (ICOS) and forkhead box protein 3 (FOXP3). (A) Comparisons of the expression levels of Ki-67, IKAROS family zinc finger 2 (Helios), programmed cell death protein 1 (PD-1), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) in ICOS⁺CD4⁺CD25⁺FOXP3⁺ Tregs and ICOS⁻CD4⁺CD25⁺FOXP3⁺ Tregs in the peripheral blood from patients with stable chronic obstructive pulmonary disease (SCOPD, n = 6). Representative flow cytometric plots of the expression level of FOXP3 in four subsets of CD4⁺ T cells gated by CD25 and ICOS in healthy controls (HC, n = 15), healthy smokers (HS, n = 11), and patients with SCOPD (n = 22) (B) and the corresponding histograms (C). (D) Quantification of FOXP3 expressed by ICOS⁺CD4⁺CD25⁺FOXP3⁺ Tregs or ICOS⁻CD4⁺CD25⁺FOXP3⁺ Tregs of HC (n = 15), HS (n = 11) and patients with SCOPD (n = 22). Significance was determined by two-way ANOVA analysis followed by Tukey’s multiple comparisons test (D). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

Comparisons of the characteristics of ICOS⁺CD4⁺ T cells and ICOS⁻CD4⁺ T cells in the peripheral blood of patients with stable chronic obstructive pulmonary disease (SCOPD). (A) The proportions of naïve T cells (CD45RA⁺CCR7⁺), T_CM cells (CD45RA⁻CCR7⁺), T_EM cells (CD45RA⁻CCR7⁻) and T_EMRA cells (CD45RA⁺CCR7⁻) in ICOS⁺CD4⁺ T cells and ICOS⁻CD4⁺ T cells of 6 patients with SCOPD. (B) Representative flow cytometric dot plots of the expression of CD45RA, CD62L, CCR7, CD27, CD28, CD69, CD57, killer cell lectin-like receptor subfamily G member 1 (KLRG-1), programmed cell death protein 1 (PD-1), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) on ICOS⁺CD4⁺ T cells and ICOS⁻CD4⁺ T cells and the corresponding histograms (n = 6). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

Inducible costimulator (ICOS) signaling induces C-X-C motif chemokine receptor 3 (CXCR3) expression in vivo and in vitro. (A) Representative flow cytometric dot plots of the frequencies of CXCR3⁺CD4⁺ T cells and CXCR3⁺CD4⁺CD25⁺FOXP3⁺ Tregs within patients with stable chronic obstructive pulmonary disease (SCOPD). Comparisons of the percentage of CXCR3⁺ T cells within CD4⁺ T cells (B) and CD4⁺CD25⁺FOXP3⁺ Tregs (C) in the peripheral blood from 15 healthy controls (HC), 11 healthy smokers (HS), 22 patients with SCOPD (SCOPD), and 14 patients with acute exacerbation of COPD (AECOPD). (D) Representative gating of CXCR3⁺ICOS⁺CD4⁺ T cells and CXCR3⁺ICOS⁻CD4⁺ T cells and the corresponding histograms (n = 22). (E) Naïve CD4⁺ T cells obtained from the peripheral blood of healthy people were cultured with plate-bound anti-CD3/28 mAbs in the presence of IL-2 with or without anti-ICOS mAb (ISA-3) for four days. The frequency of CXCR3⁺CD4⁺ T cells was analyzed with flow cytometry. Comparable results were obtained from six independent experiments. (F) The percentage of CXCR3⁺CD4⁺ T cells in the peripheral blood and corresponding BALF of four patients with SCOPD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.