Increased tumor-infiltrating CD8(+)Foxp3(+) T lymphocytes are associated with tumor progression in human gastric cancer

Abstract

Background: CD8(+)Foxp3(+) T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown.

Methods: The levels of CD8(+)Foxp3(+) T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8(+)Foxp3(-) T cells was investigated in vitro. The suppressive function of CD8(+)Foxp3(+) T lymphocytes was analyzed by their effect on CD4(+) T-cell proliferation and IFN-γ production. The percentages of CD8(+)Foxp3(+) T lymphocytes were evaluated for the association with tumor stage.

Results: The frequency of CD8(+)Foxp3(+) T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8(+)Foxp3(+) T lymphocytes were activated effector cells (CD45RA(-)CD27(-)). TGF-β1 levels were positively correlated with the frequency of CD8(+)Foxp3(+) T lymphocytes in tumor tissues, and in vitro TGF-β1 could induce the generation of CD8(+)Foxp3(+) T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8(+)Foxp3(+) T lymphocytes suppressed the proliferation and IFN-γ production of CD4(+) T cells. Finally, intratumoral CD8(+)Foxp3(+) T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage.

Conclusions: Our data have shown that increased intratumoral CD8(+)Foxp3(+) T lymphocytes are associated with tumor stage and potentially influence CD4(+) T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.

Fig. 1

Distribution of CD3⁺CD8⁺Foxp3⁺ T lymphocytes in TIL, NIL, TDLN, and PBMC of patients with GC. a A representative flow cytometry analysis of lymphocytes in TIL and NIL of the same patients for Foxp3 versus CD25 expression after gating on CD3⁺CD4⁺ or CD3⁺CD8⁺. b The frequencies of CD3⁺CD4⁺Foxp3⁺ and CD3⁺CD8⁺Foxp3⁺ T cells in TIL and NIL from 40 GC patients (horizontal bars, mean ± SEM; each circle, single donor). c The representative dot plot of CD25⁺Foxp3⁺ cells among CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in TDLN and PBMC of the same patient. d The frequency of CD3⁺CD8⁺Foxp3⁺ T cells in matched pairs of TDLN and PBL from 19 patients. Statistical analysis in b and d was shown by Student’s paired t test. Data were the mean ± SEM

Fig. 2

Phenotypic characteristics of CD8⁺Foxp3⁺ T lymphocytes in tumors. a Surface expression of CD27 and CD45RA on CD8⁺Foxp3⁺ T lymphocytes from TIL of two patients (Pt). b Freshly isolated TIL from tumor tissues were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-Foxp3, anti-CTLA-4, anti-GITR, anti-HLA-DR, anti-CD122, anti-CCR4, anti-CXCR4, and anti-CD127 antibodies. Cells were gated on CD3⁺CD8⁺Foxp3⁺ T cells and the expressions of CD25, CTLA-4, GITR, HLA-DR, CD122, CCR4, CXCR4, and CD127 were analyzed

Fig. 3

The differentiation of CD8⁺Foxp3⁺ T cells induced by TGF-β1. a TGF-β1 levels in tumor tissues (GCT, black bars) and non-tumor tissues (NT, white bars) of GC patients. Data were the mean ± SEM. b Correlation between the percentages of CD8⁺Foxp3⁺ T cells and the concentrations of TGF-β1 in tumor tissues of the patients. c Puried CD8⁺ T cells were cultured for 5 days with indicated different concentrations of rhTGF-β1 in the presence of rhIL-2, anti-CD3, and anti-CD28 antibodies. A representative dot plot is shown Foxp3 induction in CD8⁺ cells by flow cytometry. d The mean ± SEM of three independent experiments for the induction of CD8⁺Foxp3⁺ T cells

Fig. 4

Suppressive function of CD8⁺Foxp3⁺ T cells in vitro. a CFSE-labeled CD4⁺ T cells were cultured alone or with sorted CD8⁺CD25⁺ T cells from TIL of GC patients at 1:1 for 5 days in the presence of anti-CD3 and anti-CD28 antibodies. The suppressive effect of CD8⁺CD25⁺ T cells on the proliferation and IFN-γ production of CFSE-labeled CD4⁺ T cells was detected by flow cytometry. A representative flow cytometry analysis is shown from one of two patients. b CD8⁺Foxp3⁺ T cells were induced by TGF-β1 (10 ng/ml) for 5 days and then isolated by sorting CD8⁺CD25⁺ T cells. CFSE-labeled CD4⁺ T cells were cultured alone or with sorted TGF-β1-induced CD8⁺CD25⁺ T cells at 1:1 for 5 days in the presence of anti-CD3 and anti-CD28 antibodies. A representative flow cytometry analysis for the proliferation and IFN-γ production of CFSE-labeled CD4⁺ T cells is shown from one of three independent experiments

Fig. 5

Association of intratumoral CD8⁺Foxp3⁺ and CD4⁺Foxp3⁺ T lymphocytes with GC stage. Percentages of CD8⁺Foxp3⁺ and CD4⁺Foxp3⁺ T lymphocytes in TIL (a), TDLN (b), and NIL (c) between stage I–II and stage III–IV GC patients. P values of < 0.05 are considered significant