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. 2020 Jan 17;10:3002.
doi: 10.3389/fimmu.2019.03002. eCollection 2019.

CD8 + T Cells Form the Predominant Subset of NKG2A + Cells in Human Lung Cancer

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Free PMC article

CD8 + T Cells Form the Predominant Subset of NKG2A + Cells in Human Lung Cancer

Yongyuan Chen et al. Front Immunol. .
Free PMC article

Abstract

Background: NKG2A is an inhibitory receptor of both T cells and natural killer (NK) cells. Persistent activation promotes T cells and NK cells to express NKG2A and results in the progression of chronic infection and cancer. However, the characteristics and subsets of NKG2A+ lymphocytes in human lung cancer are still unclear. Methods: Here, we used the Tumor Immune Estimation Resource database and immune profiling of paired biospecimens to uncover the correlation between NKG2A expression and immune infiltration levels in human cancer as well as the characteristics of NKG2A+ lymphocytes in human lung cancer. Results: We found that KLRC1 expression was especially correlated with CD8+ T-cell infiltration levels in 34 types of human cancer through the Tumor Immune Estimation Resource database. Moreover, NKG2A+ CD8+ T cells were the predominant subset of NKG2A+ lymphocytes in human lung cancer. In contrast, the NKG2A+ NK cells were decreased in tumors compared with the paired normal lung tissue. Tumor-infiltrating NKG2A+ CD8+ T cells expressed tissue-resident memory T cell (TRM cell) and exhausted T-cell markers. Cytokines and cytotoxic molecules secreted by tumor-infiltrating NKG2A+ CD8+ T cells were significantly lower than those secreted by NKG2A- CD8+ T cells in vitro. When stimulated with T-cell receptor activator, tumor-infiltrating NKG2A+ CD8+ T cells could secrete large amounts of granzyme B. Conclusions: Our findings demonstrate that tumor-infiltrating NKG2A+ CD8+ T cells form the predominant subset of NKG2A+ cells in human lung cancer and suggest that targeting NKG2A+ CD8+ T cells is a promising approach for future anti-lung cancer immunotherapy.

Keywords: CD8+ T cells; NKG2A; T-cell dysfunction; immune checkpoints; non-small cell lung cancer; tumor microenvironment.

Figures

Figure 1
Figure 1
Data mining of the Tumor Immune Estimation Resource (TIMER) database. (A) Human KLRC1 expression levels in different tumor types from the TCGA database were determined by TIMER (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Prognostic roles of KLRC1 and immune-related factors in lung adenocarcinoma (LUAD) from The Cancer Genome Atlas (TCGA) database were determined by TIMER. (C) Prognostic roles of KLRC1 and immune-related factors in lung squamous cell carcinoma (LUSC) from the TCGA database were determined by TIMER. (D) KLRC1 expression is significantly negatively related to tumor purity and has significant positive correlations with infiltrating levels of CD8+ T cells, macrophages, neutrophils, and dendritic cells in LUAD, but no significant correlations with infiltrating levels of B cells and CD4+ T cells. (E) KLRC1 expression is significantly positively correlated with tumor purity and infiltrating levels of B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells in LUSC, other than CD4+ T cells. (F) Correlation between KLRC1 and CD8A expression in LUAD. (G) Correlation between KLRC1 and CD8A expression in LUSC.
Figure 2
Figure 2
NKG2A expression on CD8+ T cells and natural killer (NK) cells in lung cancer. (A) Representative gating strategy for the flow cytometric analysis of CD8+ T cells and NK cells in NSCLC. (B) Representative flow cytometric analysis of CD8+ T cells (upper panels) in CD3+ leukocytes and NK cells (lower panels) in the CD45+ leukocytes in non-small cell lung carcinoma (NSCLC). Cells from peripheral blood (PB; n = 6), N (healthy normal tissue adjacent to the tumor, n = 6), and T (tumor, n = 6) were analyzed by flow cytometry. Numbers in plots indicate the percent of cells in respective gates. (C) Bar diagram shows the percentages of CD8+ T cells (upper panels) in CD3+ leukocytes and NK cells (lower panels) in the CD45+ leukocytes and absolute numbers in all the cells in NSCLC. Data are shown as the mean ± SEM; n = 6; *p < 0.05; **p < 0.01. (D) Representative flow cytometric analysis of NKG2A expression on CD8+ T cells (upper panels) and NK cells (lower panels) in NSCLC. (E) Bar diagram shows the NKG2A expression on CD8+ T cells (upper panels) and NK cells (lower panels) and its absolute numbers in all the cells in NSCLC. Data are shown as the mean ± SEM; n = 6; *p < 0.05; **p < 0.01. (F) Bar diagram shows the absolute numbers of CD8+ T cells and NK cells in NKG2A+ CD45+ leukocytes. Data are shown as the mean ± SEM; n = 6; *p < 0.05; **p < 0.01; ***p < 0.001. (G) Paraffin sections from lung cancer patients (scale bars represent 50 μm for right inserts) were stained with anti-human NKG2A (red) and anti-human CD8 (green) for immunofluorescent (IF) staining. One of six independent experiments is shown. N (healthy normal tissue adjacent to the tumor, n = 6) and T (tumor, n = 6).
Figure 3
Figure 3
Tumor-infiltrating NKG2A+ CD8+ T cells express CD103 and PD-1. (A) Representative flow cytometric analysis of the expression of NKG2A and CD103 on CD8+ T cells in NSCLC. Cells from peripheral blood (PB; n = 6), N (healthy normal tissue adjacent to the tumor, n = 6), and T (tumor, n = 6) were analyzed by flow cytometry. (B) Bar diagram summarizes the expression of NKG2A and CD103 on CD8+ T cells in NSCLC. Data are shown as the mean ± SEM; n = 6; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C) Representative flow cytometric analysis of the expression of NKG2A and CD103 on NK cells in NSCLC. (D) Bar diagram summarizes the expression of NKG2A and CD103 on NK cells in NSCLC. Data are shown as the mean ± SEM; n = 6; *p < 0.05; **p < 0.01; ***p < 0.001. (E) Representative flow cytometric analysis of the expression of NKG2A and PD-1 on CD8+ T cells in NSCLC. (F) Bar diagram summarizes the expression of NKG2A and PD-1 on CD8+ T cells in NSCLC and its absolute numbers. Data are shown as the mean ± SEM; n = 6; NS, no statistical significance; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 4
Figure 4
Anti-tumor cytokine profile of the NKG2A+ CD8+ T cells. (A) Representative flow cytometric analysis of the expression of NKG2A and intracellular IFN-γ produced by CD8+ T cells with BFA and Monensin (blue) or with BFA, Monensin, PMA, and Ionomycin (red) in NSCLC. (B) Cells from N (healthy normal tissue adjacent to the tumor, n = 6) and T (tumor, n = 6) were analyzed by flow cytometry. Bar diagram summarizes the levels of intracellular IFN-γ produced by CD8+ NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; *p < 0.05. (C) Representative flow cytometric analysis of the expression of NKG2A and intracellular TNF-α produced by CD8+ T cells. (D) Bar diagram summarizes the levels of intracellular TNF-α produced by CD8+ NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; NS, no statistical significance. (E) Representative flow cytometric analysis of the expression of NKG2A and intracellular granzyme B produced by CD8+ T cells. (F) Bar diagram summarizes the levels of intracellular granzyme B produced by CD8+ NKG2A+ T cells. Data are shown as mean ± SEM; n = 6; NS, no statistical significance; **p < 0.01.
Figure 5
Figure 5
T-cell receptor (TCR) signal influences the expression of NGK2A and the secretion of IFN-γ and granzyme B. The single-cell suspension of paired normal tissue and tumor stimulated for 72 h with the recommended dose concentration of CD3 and CD28 or nothing. (A) Bar diagram shows the NKG2A expression change. N (healthy normal tissue adjacent to the tumor, n = 6), and T (tumor, n = 6). Data are shown as the mean ± SEM; n = 6; NS, no statistical significance; **p < 0.01. (B) Representative flow cytometric analysis of the expression of NKG2A and intracellular IFN-γ produced by CD8+ T cells. (C) Bar diagram summarizes the levels of intracellular IFN-γ produced by CD8+ NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; *p < 0.05. (D) Representative flow cytometric analysis of the expression of NKG2A and intracellular granzyme B produced by CD8+ T cells. (E) Bar diagram summarizes the levels of intracellular granzyme B produced by CD8+ NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; **p < 0.01; ***p < 0.001. (F) Representative flow cytometric analysis of the expression of NKG2A and PD-1 on CD8+ T cells. (G) Bar diagram summarizes the levels of PD-1 on CD8+ NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; *p < 0.05.
Figure 6
Figure 6
The expression of NGK2A on tumor-infiltrating CD8+ T cells is TCR strength-dependent. The single-cell suspension of paired normal tissue and tumor stimulated for 72 h with nothing or 1/4 or 1/2 or recommended dose concentration of CD3 and CD28. (A) Bar diagram shows the NKG2A expression change. N (healthy normal tissue adjacent to the tumor, n = 7) and T (tumor, n = 7). Data are shown as the mean ± SEM; n = 6; *p < 0.05; **p < 0.01. (B) Representative flow cytometric analysis of the expression of NKG2A and intracellular IFN-γ produced by CD8+ T cells. (C) Bar diagram summarizes the levels of intracellular IFN-γ produced by CD8+ T cells and CD8+NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; *p < 0.05. (D) Representative flow cytometric analysis of the expression of NKG2A and intracellular granzyme B produced by CD8+ T cells. (E) Bar diagram summarizes the levels of intracellular granzyme B produced by CD8+ T cells and CD8+NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; **p < 0.01; ***p < 0.001. (F) Representative flow cytometric analysis of the expression of NKG2A and PD-1in CD8+ T cells. (G) Bar diagram summarizes the levels of PD-1 in CD8+ T cells and CD8+NKG2A+ T cells. Data are shown as the mean ± SEM; n = 6; *p < 0.05.

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