It has been www.selleckchem.com/products/GDC-0449.html suggested that multifunctional CD4+T cells, able to produce simultaneously IFN-γ, IL-2 and TNF-α, are associated with protective immunity or a beneficial outcome in chronic infectious diseases, such as HIV [25–28] and HCV . We therefore evaluated the quality of Th1 responses
induced by LbAg and LaAg in healed CL patients, based on their ability to secrete these three major Th1-related cytokines at the single-cell level. Using multiparametric flow cytometry, seven distinct populations of cytokine-producing cells can be delineated based on any of the possible combinations of IFN-γ+, IL-2+ and TNF-α+ producers, and the relative frequency of these distinct populations defines the quality of the Th1 response. The percentages of cytokine-producing cells were shown to be higher in the healed CL patient group than in healthy controls, and we were able to observe statistically significant differences between those groups for triple-positive (3+) multifunctional LY294002 nmr T cells (with both LbAg and LaAg), IFN-γ single-positive cells after LaAg stimulation and for IFN-γ+IL-2+ cells stimulated with LbAg (Fig. 2a). When comparing the quality of the Th1 response elicited by each Leishmania antigen evaluated we could observe that LbAg induces significantly higher percentages of multifunctional CD4+T cells and
IFN-γ+IL-2+ cells than LaAg stimulation in the healed CL patient group (Fig. 2a). The quality of the Th1 response was also evaluated by analysing the contribution of each phenotype in the total Th1 response, and is represented pictorially by pie charts (Fig. 2b). This kind of representation demonstrates clearly that LbAg induced a major proportion of multifunctional CD4+T cells (in red – 28% of the total Th1 response evaluated) and double-positive CD4+T cells Glutathione peroxidase (in blue – comprising 44% of the total Th1 response), while LaAg induced
predominantly single-positive cells (68%). More than half of the single-positive cells induced by LaAg were IFN-γ single-positive. In the control group, the majority of responsive cells were single-positives (>60%), and no major differences were observed concerning LbAg and LaAg stimulation. Having shown that LbAg induced higher cytokine production by CD4+T cells than LaAg in healed CL patients (Fig. 1b), we also investigated the relative cytokine concentrations produced by all distinct Th1 phenotypes induced by LbAg and LaAg, measured as the geometric MFIs. The highest MFI values for all three cytokines were found among triple-positive multifunctional CD4+T cells (both after LbAg and LaAg stimulation) (Fig. 2c) and a progressive decrease in the MFIs for all cytokines was observed as the degree of functionality decreased (3+ to single-positives). MFIs for IFN-γ and IL-2 from multifunctional T cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulus (Fig. 2c).