Altered CD94/NKG2A and perforin expression reduce the cytotoxic activity in malignant pleural effusions

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Abstract

CD94/NKG2A is an inhibitory receptor expressed by NK cells and cytotoxic lymphocytes and, upon activation by HLA-E, downregulates the cytolytic activities of these cells thus representing a tumour immune escape mechanism.

This study was aimed at assessing whether cytotoxic lymphocytes (CD8+) and NK cells from malignant pleural effusions have a deregulated expression of CD94/NKG2A.

The expression of membrane CD94/NKG2A and perforin was evaluated by flow-cytometry in CD8+ and NK cells from pleural effusions and autologous peripheral blood of cancer (n = 19) and congestive heart failure (CHF) (n = 11) patients. Intracellular CD94/NKG2A expression was evaluated by flow-cytometry in pleural effusion CD8+ and NK cells from cancer patients (n = 10). Cytotoxic activity against cancer cells exerted by pleural and autologous peripheral blood T lymphocytes from cancer patients was assessed by flow-cytometry assay.

Pleural CD8+ from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood and CHF pleural effusions. Reduced numbers of NK cells were present in pleural effusions from both cancer and CHF patients. Pleural NK from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood. Pleural T lymphocytes from cancer patients exhibited a reduced cytotoxic activity against cancer cells when compared to autologous peripheral blood T lymphocytes. The intracellular expression of CD94/NKG2A in CD8+ and NK cells from cancer patients was higher than membrane expression.

In conclusion, this study provides compelling evidences of new mechanisms underlying the reduced host defence against cancer within the pleural space.

Introduction

Malignant pleural effusions are mostly due to metastatic involvement of the pleural compartment by heterogeneous primaries and recur in 15% of patients who die with malignancies and account for up to 50% of the exudates in many clinical series.1 Exudative malignant pleural effusions are characterised by the accumulation of fluid enriched in proteins and by the recruitment of elevated numbers of mononuclear cells into the pleural space.2 Host defence mechanisms against tumour cells are mainly related to the activity of CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells.3 CD8+ CTL exert their cytotoxic activity on tumour cells by releasing toxic mediators, such as perforin and granulysin4 or by inducing cell apoptosis via FAS/FAS ligand pathway.5 Like CD8+ CTL, NK cells kill tumour target cells without prior sensitisation, relying on directed exocytosis of secretory lysosomes and on activation of FAS/FAS ligand-induced cell death. The magnitude and diversity of CD8 positive CTL- and NK-mediated responses against tumour cells reflect the outcome of a balance between signals of activation and inhibition. An accurate and precise control of this balance is essential for mounting protective anti-tumour responses and for concurrently limiting potential immunopathologic complications.6 The CD94/NKG2A heterodimer is an inhibitory receptor expressed by CD8+ CTL and NK cells that, upon activation by HLA-E, downregulates their cytolytic activity against tumour cells.7 Although, some studies have shown that pleural effusion mononuclear cells exhibit several functional defects,8 limited information is available regarding the expression and the activity of the CD94/NKG2A complex in CD8+ CTL and NK cells isolated from malignant pleural effusions.

The aims of the present study were to determine whether CD8+ CTL and NK cells from malignant pleural effusions have a deregulated expression/activation of CD94/NKG2A and whether this phenomenon may contribute to a reduced cytotoxic activity against cancer cells in the pleural compartment.

Section snippets

Pleural fluid collection

Peripheral blood (PB) and pleural fluid (PF) were obtained from patients with congestive heart failure (CHF) (n = 11; age range 50–80 years) and cancer (n = 19, age range 45–78 years). Pleural fluids were collected by thoracentesis, before the initiation of chemotherapy. All subjects gave informed written consent and the study was approved by the institutional review board for human studies and was consistent with Helsinki Declaration. Patients were recruited at the Dipartimento di Medicina Interna e

CD8+ CTL and NK cells in cancer and in CHF patients

Altered numbers of NK and CD8+ cells or functional defects of these cell types may contribute to reduce the efficiency of the immune responses against cancer cells.3, 4 The first step of this study was to assess whether the altered numbers of CD8+ and NK cells were specifically present in malignant PF comparing malignant PF and PB from cancer patients with PF and PB from CHF patients.

Whilst no significant differences were observed in the percentage of CD8+ CTL between cancer and CHF patients in

Discussion

CD8+ CTL and NK cells display effector functions, including the recognition and lysis of infected, stressed, or transformed cells and the production of immunoregulatory cytokines and chemokines.12 Tumour microenvironment may be unfavourable for CD8+ CTL and NK cell activity and may contribute to tumour immune escape. Malignant effusions are a relatively easily accessible source of tumour-associated effector cells and represent a suitable model for the study of interactions between tumour cells

Conflict of interest statement

None declared.

Acknowledgements

This work was supported by the Italian National Research Council (C.N.R.), the University of Parma, and the University of Palermo.

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