ABSTRACT
Title
Frequency and phenotype of circulating invariant NKT cells in malignant pleural mesothelioma patients
Authors
E. Altomare1, S. Fallarini1, M. Botta2, G. Lombardi1.
1DISCAFF – University of “Piemonte Orientale A. Avogadro” Novara, Italy
2SOC Oncology -Hospital of “Santo Spirito” Casale Monferrato (AL), Italy
1DISCAFF – University of “Piemonte Orientale A. Avogadro” Novara, Italy
2SOC Oncology -Hospital of “Santo Spirito” Casale Monferrato (AL), Italy
Abstract
Malignant pleural mesothelioma (MPM) is a rare tumor of the mesothelium, strongly related to asbestos exposure, with a very poor prognosis. Multimodality treatments for MPM, including surgery, radiotherapy, or chemotherapy, are of limited efficacy and several novel approaches are under development. The combination of pemetrexed with cisplatin demonstrated a positive benefit on multiple outcomes and, recently, became a major component of standard treatments. Active or passive immunotherapy can represent another highly promising approach, but the achievement of therapeutic benefits on MPM remains to be clarified (Grégoire 2010).
Invariant natural killer (iNKT) cells are a subset of T cells that recognize glycolipid antigens presented by the CD1d molecules. Upon activation, iNKT cells rapidly produce both Th1- and Th2-specific cytokines, such as interferon (IFN)-γ and interleukin (IL)-4, and exhibit both antigen-specific and NK-like cytolytic activities. Their peculiar capacity to produce cytokines provides iNKT cells with the ability to link the innate and adaptive immune responses.iNKT cells promote antimicrobial and anti-tumor immunity, actively induce T cell tolerance and protect against autoimmune diseases. Various iNKT cell alterations (frequency, phenotype, activity) have been reported in cancer patients (Molling et al., 2008), but no data are available on the iNKT cells in MPM patients.
The aims of the present work were to analyze the frequency and/or the subset ratio of iNKT cells in MPM patients, compared to healthy volunteers, and to evaluate possible correlations between these cells and the state and/or severity of MPM. We analyzed the cell surface expressions of CD3, CD4, CD8 and Vα24Vβ11 (specific iNKT cell markers) on peripheral blood mononuclear cells (PBMC), isolated from patients with MPM and matched healthy volunteers. We then evaluated the intracellular expression of IFN-γ and IL-4 cytokines by flow cytometry (FACSVantage-SE) after 2.5 ng/ml PMA and 1mg/ml ionomycin stimulation for 4 h at 37°C in the presence of 5 U/ml human IL-2 and 10 mg/ml brefeldin A. The clinical data (tumor severity and phenotype) of all patients were then correlated with both the total frequency and the subset (CD4+; CD8+; CD4-CD8- [DN]; CD4+Cd8+[DP]) of circulating iNKT cells. Differences were considered statistically significant when p<0.05.
We found a significant increase in the frequency of iNKT cells (Vα24Vβ11 positive cells in the CD3+ gated region) in MPM patients in comparison to controls (0.0388% vs. 0.0139%,expressed as a percentage of total T lymphocytes, p= 0.0013). Skewed subsets of circulating iNKT cells in MPM patients, compared to controls, were also determined, with a low frequency of circulating DN iNKT cells (p= 0.045 ), and an high frequency of DP iNKT cells ( p= 0.027 ), respectively.On the contrary, no significant difference in the expression of intracellular IFN-γ and IL-4 in the different iNKT cell subsets from MPM patients or healthy controls were measured. Whenpatients were reclassified on the basis of histological type and clinical stage of tumor, no significant correlation was revealed between iNKT cells and MPM classes.
This study demonstrates the presence of differences in the frequency and subset of circulating iNKT cells in MPM patients. The role of these cells in this disease remains to be clarified.
This study was supported by Piedmont County Grants for Research (prot. N°36529, 2009, Italy).
References
Invariant natural killer (iNKT) cells are a subset of T cells that recognize glycolipid antigens presented by the CD1d molecules. Upon activation, iNKT cells rapidly produce both Th1- and Th2-specific cytokines, such as interferon (IFN)-γ and interleukin (IL)-4, and exhibit both antigen-specific and NK-like cytolytic activities. Their peculiar capacity to produce cytokines provides iNKT cells with the ability to link the innate and adaptive immune responses.iNKT cells promote antimicrobial and anti-tumor immunity, actively induce T cell tolerance and protect against autoimmune diseases. Various iNKT cell alterations (frequency, phenotype, activity) have been reported in cancer patients (Molling et al., 2008), but no data are available on the iNKT cells in MPM patients.
The aims of the present work were to analyze the frequency and/or the subset ratio of iNKT cells in MPM patients, compared to healthy volunteers, and to evaluate possible correlations between these cells and the state and/or severity of MPM. We analyzed the cell surface expressions of CD3, CD4, CD8 and Vα24Vβ11 (specific iNKT cell markers) on peripheral blood mononuclear cells (PBMC), isolated from patients with MPM and matched healthy volunteers. We then evaluated the intracellular expression of IFN-γ and IL-4 cytokines by flow cytometry (FACSVantage-SE) after 2.5 ng/ml PMA and 1mg/ml ionomycin stimulation for 4 h at 37°C in the presence of 5 U/ml human IL-2 and 10 mg/ml brefeldin A. The clinical data (tumor severity and phenotype) of all patients were then correlated with both the total frequency and the subset (CD4+; CD8+; CD4-CD8- [DN]; CD4+Cd8+[DP]) of circulating iNKT cells. Differences were considered statistically significant when p<0.05.
We found a significant increase in the frequency of iNKT cells (Vα24Vβ11 positive cells in the CD3+ gated region) in MPM patients in comparison to controls (0.0388% vs. 0.0139%,expressed as a percentage of total T lymphocytes, p= 0.0013). Skewed subsets of circulating iNKT cells in MPM patients, compared to controls, were also determined, with a low frequency of circulating DN iNKT cells (p= 0.045 ), and an high frequency of DP iNKT cells ( p= 0.027 ), respectively.On the contrary, no significant difference in the expression of intracellular IFN-γ and IL-4 in the different iNKT cell subsets from MPM patients or healthy controls were measured. Whenpatients were reclassified on the basis of histological type and clinical stage of tumor, no significant correlation was revealed between iNKT cells and MPM classes.
This study demonstrates the presence of differences in the frequency and subset of circulating iNKT cells in MPM patients. The role of these cells in this disease remains to be clarified.
This study was supported by Piedmont County Grants for Research (prot. N°36529, 2009, Italy).
References
Grégoire M.(2010)- Cell Adh Migr.4:153-61.
Molling JW et al.(2008)- Clin Immunol.129:182-94.