Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

Abstract

Background: Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3zeta expression. However, the hypothesis of tumor counterattack is still controversial.

Methods: We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG(1) peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3zeta, CD3epsilon, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3zeta and CD3epsilon in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied.

Results: Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3epsilon but not CD3zeta chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3epsilon, but not CD3zeta, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors.

Conclusions: Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3epsilon in T-cells induced by NSCLC cells might lead to T-cell dysfunction.

Fig. 1

FasL expression on NSCLC. a Histograms obtained by flow cytometry from HeLa cells (positive control) and five representative lung carcinoma cell lines. Cells were stained with 100413 clone anti-FasL mAb (dark area) or with an isotype-matched IgG (solid line). Similar results were obtained using the NOK-1 and Alf-2.1 mAbs (not shown). b Immunocytochemistry of FasL in tonsillar tissue and HeLa cells (positive controls). A granular pattern was observed in HeLa cells. Most of NSCLC cell lines were negative for FasL expression. RCC1-20 (not shown) and Calu-1 cell lines showed a low expression of FasL with a homogeneous pattern. Representative cell lines are shown (original magnification, ×1,000). c A representative result from tumor cell-enriched fraction obtained from pleural effusion is shown; tumor cells were gated from the CD45-negative population to exclude leukocytes, which were FasL-positive cells. Cells were stained with 100413 clone anti-FasL mAb (dark area) or with an isotype-matched IgG (solid line). d FasL mRNA expression in NSCLC cell lines. HeLa cells and stimulated-Jurkat T-cells (+) were used as positive controls. From a panel of 12 NSCLC cell lines, only RCC1-20 and Calu-1 cells expressed FasL mRNA

Fig. 2

Absence of apoptosis in Fas-sensitive Jurkat T-cells after co-culture with NSCLC cell lines. a Cellular distribution of Jurkat T-cells in a PKH-26 dye versus side scatter dot plot graph. b Annexin V-FITC histograms showing non-apoptotic Jurkat T-cells gated from the PKH-26-negative cells. c Absence of apoptosis in Jurkat T-cells detected by propidium iodide staining and further subG₁ peak detection. rhFasL (positive control) induced apoptosis in Jurkat T-cells

Fig. 3

Lack of apoptosis in Jurkat T-cells after treatment with plasma or pleural effusion samples from lung adenocarcinoma patients. a Viability of Jurkat T-cells co-cultured with plasma or pleural effusion samples (diluted 1:3 with complete RPMI-1640 medium) from adenocarcinoma patients after 96 h of incubation, determined by Annexin V-FITC/propidium iodide staining. A small reduction in viability was observed in Jurkat T-cells treated with malignant pleural effusion (PE), *P < 0.05 with respect to control. b Kinetic of apoptosis in Jurkat T-cells treated with pleural effusion sample from a representative lung adenocarcinoma patient

Fig. 4

Absence of apoptosis in Jurkat T-cells co-cultured with microvesicles (1–2 μg/ml) from eight NSCLC cell lines and detected by Annexin V-FITC/propidium iodide staining. As positive controls microvesicles (MV) from PHA-stimulated Jurkat T-cells and from HeLa cell lines were used. Microvesicle-free supernatant (SN) from Jurkat T-cells did not induced apoptosis

Fig. 5

Expression of CD3ε, CD3ζ, and CD28 in Jurkat T-cells and in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients. a Down-regulation of CD3ε and CD28 but not CD3ζ expressions in Jurkat T-cells after direct or indirect co-culture with representative NSCLC cell lines: A-549, A-427, and OSJV-1. Control: gray area; direct co-culture: solid line; indirect co-culture: discontinuous line; isotype-matched control: gray line. Results from three independent experiments. b Microvesicles released by NSCLC cell lines did not reduce CD3ε, CD3ζ, and CD28 expressions. In contrast, microvesicles-free supernatants down-regulated CD3ε and CD28 but not CD3ζ expressions in Jurkat T-cells. Results from two representative NSCLC cell lines (AJG-1 and OSJV-1) are shown. Microvesicles from PHA-stimulated Jurkat T-cells were employed as positive control for CD3ε and CD3ζ down-regulation. Control: gray area; microvesicle treatment: solid line; treatment with microvesicle-free supernatant: discontinuous line; isotype-matched control: gray line. Results obtained from three independent experiments. c MFI values for CD3ζ and CD3ε expression in CD4+T and CD8+T cells from peripheral blood and pleural effusion (PE) of healthy donors (HD) and lung adenocarcinoma patients (Adeno)