Fig 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.

Fig 3: 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.

Fig 4: ICOS is upregulated in Melan-A-specific CD8+ T cells isolated after combined therapy and is involved in chemoimmunotherapy-induced AKT activation. (A) Top panel shows representative staining with anti-ICOS (open histogram) or isotype (filled histogram) mAbs, in two T-cell clones isolated from Arm1 (Pt08.90) and Arm2 (Pt15.26) patients after the treatments (POST). Bottom panel shows pooled data of ICOS expression in Melan-A-specific T-cell clones (n = 15 from two patients of Arm1, n = 18 from three patients of Arm2), isolated before (PRE) and after therapy (POST). Cells were stained after 48 h stimulation with plate-bound anti-CD3 mAb (2 µg/mL) plus rIL-2 and shown in both scatter-plot graph and box-and-whisker diagram, with 5–95 percentile. Each dot represents the mean value out of three–five independent experiments. The mean ± SEM of each group is shown. (B) Correlation between p-AKT and ICOS expression. T-cell clones isolated after both treatments were grouped according to pSerAKT expression levels and the percentage of ICOS+ cells (bottom panel) and shows higher expression of ICOS in AKThigh T cells. Top panel, percentage of clones with ICOS levels > 30% cut-off in p-AKTneg/low versus p-AKThigh groups. p value was calculated with one-tail Fisher’s exact test. */?p = 0.05, **/??p = 0.01, ***/???p = 0.001, Mann–Whitney two-sample test (*) and two-tail Student’s test (?), respectively. (C) Left panel: effect of ICOS blockade on AKT activation (p-Ser473-AKT) tested in a representative Melan-A specific T-cell clone (Arm2, Pt15.26, POST), following activation by irradiated PBMC-derived APCs plus rIL-2 (-). Blockade of ICOS engagement with anti-ICOS-L (ICOSLi) mAb (10 µg/mL) induced a marked downregulation of p-AKT. Right panel: effect of ICOS engagement on AKT activation (p-Ser473-AKT) tested in a representative Melan-A specific T-cell clone (Arm2, Pt15.111, POST), following activation by anti-CD3 mAb (-). The anti-ICOS-agonist (ICOSa) mAb (12.5 µg/mL) induced upregulation of p-AKT. Gel loading control is represented by total AKT expression. Densitometric quantification of protein bands was determined by NIH ImageJ software. Columns show the mean ± SD of p-AKT/AKT ratios (OD) from two independent experiments. (D) Top panel: bars represent the mean % (± SD) of TNF-a, IFN? and GrB inhibition by ICOS blockade from two experiments performed on Pt15.26 T cell clone (representative of three different T-cell clones tested). Bottom panel: bars represent the mean % (± SD) of the increased polyfunctional activity induced by ICOS co-stimulation, as calculated with respect to the isotype control mAb.

Fig 5: 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.