Population alterations of L-arginase- and inducible nitric oxide synthase-expressed CD11b+/CD14⁻/CD15+/CD33+ myeloid-derived suppressor cells and CD8+ T lymphocytes in patients with advanced-stage non-small cell lung cancer

Abstract

Background: Immune aberrations have been demonstrated in tumorogenesis, and myeloid-derived suppressor cells (MDSC) have shown to play a pivotal role in mediating immune suppression in animal models of human tumors. In the present study, we explored the clinical relevance of CD11b+/CD14⁻/CD15+/CD33+ MDSCs and the association of MDSCs with CD8+ cytotoxic T lymphocytes in patients with non-small-cell lung cancer (NSCLC).

Patients and methods: The population of CD11b+/CD14⁻ cells in peripheral blood mononuclear cells (PBMNC) was determined in 173 patients with NSCLC and 42 control subjects. The expression of CD15, CD33, IL-4R, INF-γR, iNOS and L-arginase were analyzed. Cocultures with CD8+ T lymphocytes and Jurkat cells were developed to determine the impact of MDSCs on the expression of CD3ζ of CD8+ T lymphocytes.

Results: Patients with treatment-naïve, advanced-stage NSCLC (n = 87) had an increased subpopulation of CD11b+/CD14⁻/CD15+/CD33+ cells in the PBMNCs with characteristics of MDSCs (P < 0.0001). The CD11b+/CD14⁻ cells in PBMNC also express IL-4R and INF-γR and can suppress CD3ζ expression in CD8+ T lymphocytes. The subpopulation of CD11b+/CD14⁻ cells in PBMNC was decreased in the advanced-stage NSCLC patients who had responsiveness to chemotherapy (n = 41, P < 0.0001) and in the early-stage NSCLC patients after removal of tumor (n = 8, P = 0.0391). Notably, a negative association existed between the population of CD11b+/CD14⁻ cells in PBMNC and the frequency of CD8+ T lymphocytes (n = 48, r = -0.3141, P = 0.0297).

Conclusions: Our study provided evidence of an increased pool of CD11b+/CD14⁻/CD15+/CD33+ MDSCs in the peripheral blood of NSCLC patients. For the suppressive effect of the cells on CD8+ T lymphocytes, these findings suggest the important role of the CD11b+/CD14⁻/CD15+/CD33+ MDSCs in mediating immunosuppression in NSCLC.

Fig. 1

A CD11b⁺/CD14⁻/CD15⁺/CD33⁺ subpopulation with expression of iNOS and l-arginase 1 was identified in PBMNCs from patients with treatment-naïve NSCLC. a CD11b and CD14 dual staining and flow cytometry for PBMNCs of NSCLC patients. The CD11b⁺/CD14⁻ cell population is denoted in the red circle in the CD11b versus CD14 density plot and in a red rectangle in the FSC versus SSC density plot. b The morphology of the magnetic microbead-isolated CD11b⁺/CD14⁻ cells in PBMNCs (modified Wright’s stain, ×400). c The CD15 and CD33 staining for the CD11b⁺/CD14⁻ subpopulation in PBMNCs (values are presented as mean ± SEM, n = 10, *P = 0.002, compared with IgG control, Wilcoxon signed rank test). d RT-PCR and Western blotting on the magnetic microbead-isolated CD11b⁺/CD14⁻ cells in PBMNCs for the expression of l-arginase 1. e RT-PCR and Western blotting on the magnetic microbead-isolated CD11b⁺/CD14⁻ cells in PBMNCs for the expression of iNOS. Flow cytometric plots, histograms, quick staining and blots are the representative data for one treatment-naïve NSCLC patient

Fig. 2

The CD11b⁺/CD14⁻ subpopulation in PBMNCs expressed IL-4 and INF-γ receptors, and suppressed the CD3ζ expression of CD8⁺ T lymphocytes and Jurkat cells. a The magnetic bead-isolated CD11b⁺/CD14⁻ cells in PBMNC were stained for IL-4R and INF-γR (mean fluorescence intensity as MFI, n = 10, *P = 0.002, compared with IgG control, Wilcoxon signed rank test). b The isolated CD11b⁺/CD14⁻ cells in PBMNCs were further cocultured with the magnetic bead-isolated homologous, heterologous CD8⁺ T lymphocytes from healthy subjects and Jurkat cells for 24 h. The MFI of CD3ζ of CD8⁺ T lymphocytes and Jurkat cells was analyzed by flow cytometry (n = 6, *P < 0.05, compared with CD8⁺ T lymphocytes in the absence of CD11b⁺/CD14⁻ cells in PBMNCs, Wilcoxon signed rank test). The values are presented as mean ± SEM. The flow cytometric histograms are the representative data for one treatment-naïve NSCLC patient

Fig. 3

The CD11b⁺/CD14⁻ population in PBMNCs was significantly increased in patients with treatment-naïve, advanced-stage NSCLC. As determined by flow cytometry, the frequency of the CD11b⁺/CD14⁻ cells in non-lymphocytic PBMNCs was compared in (a) patients with treatment-naïve, advanced stage NSCLC (n = 87) versus control subjects (n = 42), (b) stage IIIB disease (n = 27) versus stage IV disease (n = 60), and (c) different types of histology (adenocarcinoma, n = 38; squamous cell carcinoma, n = 32; other NSCLC, n = 17). Each point corresponds to an individual patient or a control subject. The bars denote mean values. P values are determined by the Mann–Whitney test (a, b) and from the Kruskal–Wallis test (c)

Fig. 4

The frequency of the subpopulation of CD8⁺ T lymphocytes was decreased in patients with treatment-naïve, advanced-stage NSCLC and there was an inverse relationship between the frequency of CD8⁺ T lymphocytes and that of 11b⁺/CD14⁻ cells in non-lymphocytic MNCs. As determined by flow cytometry, the ratio of CD4⁺/CD8⁺ T lymphocytes (a), the frequency (%) of CD4⁺ T lymphocytes (b) and CD8⁺ T lymphocytes were investigated in 55 patients with treatment-naïve, advanced stage NSCLC and 36 control subjects. Each point corresponds to an individual patient or a control subject. The bars denote mean values. P values are from Mann–Whitney test. d The relationship between the frequency (%) of CD8⁺ T lymphocytes and that of CD11b⁺/CD14⁻ cells in peripheral blood non-lymphocytic MNCs (r = −0.3141, n = 48, P = 0.0297, Spearman ranking test)

Fig. 5

The subpopulation of CD11b⁺/CD14⁻ cells in PBMNCs was decreased in the advanced-stage NSCLC patients who were in the status of partial response (PR) or stable disease (SD) after chemotherapy, and in the early-stage patients after removal of tumors. a The frequency (%) of CD11b⁺/CD14⁻ cells in peripheral blood non-lymphocytic MNCs in the advanced-stage NSCLC patients, who were treatment-naïve (n = 87), who had PR or SD after chemotherapy (n = 41), and who had progressive disease (n = 37). Each point corresponds to an individual patient. The bars denote mean values. P values were determined from the Mann–Whitney test. b The frequency (%) of CD11b⁺/CD14⁻ cells in peripheral blood non-lymphocytic MNCs in early-stage NSCLC patients before and after removal of tumors (n = 8). P values were determined with the Wilcoxon signed rank test