Increased expression of immunosuppressive molecules on intratumoral and circulating regulatory T cells in non-small-cell lung cancer patients

Abstract

The expression patterns of immunosuppressive molecules on regulatory T (Treg) cells have not been elucidated in non-small-cell lung cancer (NSCLC) patients. In this study, a total of 88 patients including 53 patients with NSCLC, 17 patients with lung non-malignant diseases, and 18 healthy volunteers were enrolled. Increased number of total CD4(+)CD25(+)FoxP3(+) Treg cells and elevated expressions on the surface of several inhibitory molecules including CTLA-4, LAG-3 and PD-1 have been observed in the peripheral blood of NSCLC patients. We found that intratumoral Treg cells from NSCLC patients express the highest levels of co-inhibitory molecules compared to Treg cells isolated from tumor adjacent tissues or from peripheral blood of cancer patients, which is in consistent with the enhanced immunosuppressive function of these co-inhibitory molecules. Moreover, the number of Treg cells and their functional surface molecules increased during the progression of lung cancer. Elevated plasma levels of TGF-β and IL-10 in NSCLC patients were also observed in NSCLC patients compared to that in healthy volunteers. Our findings further support the role of Treg cells in the tumor microenvironments in NSCLC patients.

Keywords: Regulatory T cells; immunosuppressive molecules; non-small-cell lung cancer; tumor-infiltrating lymphocytes.

Figure 1

CTLA-4, LAG-3, PD-1 and CD39 expressions in peripheral blood Treg cells of NSCLC patients. Cells in different groups were labeled as described in the Materials and Methods, and detected by FCM. A. Representative flow cytometric analysis for detecting CD4⁺CD25⁺Fxop3+ Treg cells, or CTLA-4, LAG-3, PD-1 and CD39 on Treg cells. B. Treg cells and CTLA-4 expressions on Treg cells are elevated in peripheral blood of late stage NSCLC patients (*, P<0.05).

Figure 2

Comparison of Treg cells and expressions of inhibitory molecules in tumor tissues and corresponding adjacent tissues by flow cytometry. A. Representative flow cytometric analysis of a NSCLC patient; B. analysis of paired tumor and tumor-adjacent tissues obtained from 13 NSCLC patients (*P<0.05, **P<0.01).

Figure 3

Treg cells and expressions of inhibitory molecules on Treg cells isolated from tumor tissues and peripheral blood. Local tumor-infiltrating or circulating Treg cells, as well as surface expressions of LAG-3, PD-1, and CTLA-4, were determined by flow cytometry. TT: tumor tissues; PB: peripheral blood.

Figure 4

Tumor-infiltrating Treg cells have increased suppressive properties. A. CFSE-labeled CD4⁺CD25^- T cells were co-cultured with unlabelled CD4⁺CD25⁺ Treg cells from different sources at indicated ratios. After four days of treatment with anti-CD3 and anti-CD28 antibodies for stimulation of T cell proliferation, CD4⁺CFSE⁺ T cells were analyzed by flow cytometry. B. Inhibitory activity of Treg from different sources on T cells in Patient #1 and Patient #8. Treg: CD4⁺CD25⁺ regulatory T cells; Teff: CD4⁺CD25^- effective T cells; Treg-TT: Treg cells in tumor tissues; Treg-TAT: Treg cells in tumor adjacent tissues; Treg-PB: Treg cells in peripheral blood.

Figure 5

Plasma levels of TGF-β and IL-10 are elevated in NSCLC patients. All plasma samples of the 88 cases were collected and detected by ELISA (*P<0.05, **P<0.01).