These cells were permeabilized with 0·5% saponin solution in PBS/BSA (SAP buffer). After 1 h permeabilization at 4°C cells were incubated, for additional 30 min, with the

cytokine NSC 683864 concentration antibodies PE-Cy7-labelled anti-IFN-γ, fluorescein isothiocyanate (FITC)-labelled anti-TNF-α, APC-labelled anti-IL-2 and PE-labelled anti-IL-10, washed with SAP buffer and resuspended in PBS/BSA. All antibodies were purchased from e-Bioscience except when noted. A minimum of 50 000 events per sample were acquired inside the lymphocytes gate, based on size and granularity properties, in a CyAn ADP flow cytometer device (Beckman-Coulter/Dako, Brea, CA, USA) and analysed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Statistical comparisons were performed by a two-tailed Wilcoxon matched-pairs signed-ranks, Mann–Whitney U-test (in the comparison between patients and control groups) and Spearman’s correlation tests, using GraphPad Prism version 5·0 software (GraphPad Software, La Jolla, CA, USA). All cytokine frequencies, mean fluorescence intensity (MFI) and iMFI values reported are after background subtraction of the frequency, MFI or integrated MFI (iMFI) of the identically gated population of cells from the same sample Ruxolitinib supplier cultured without antigen. Statistical significance was

assigned to P ≤ 0·05. Single-parameter evaluation of cytokine producing CD4+ T cells: analysis via iMFI of cytokine-expressing Rho cells can make a difference The majority of studies that evaluate immune responses in human leishmaniasis usually estimate the frequency of antigen-specific IFN-γ and other Th1-related cytokine-producing cells, as a key immune correlate of a protective

response. In a former report, Darrah et al. [31] developed a metric approach in order to evaluate the total response of a given population of cytokine-producing cells that combine the magnitude and quality of T cell responses multiplying the frequency of cytokine-expressing cells by the cytokine MFI, termed iMFI. After applying this novel metric approach to our data we were able to detect more pronounced differences between healed CL patients and control groups for both Leishmania crude antigen preparations than when using only the frequencies of cytokine-positive cells (Fig. 1a and b). More significantly, we found that LbAg-stimulated CD4+T cells have considerably higher iMFIs for IFN-γ, TNF-α and IL-2 in comparison to LaAg (Fig. 1b) in the healed CL group, while only the frequencies of IL2+CD4+ T cells differ between both antigens in the same group (Fig. 1a). These findings indicate that LbAg induces higher cytokine production by CD4+T cells than LaAg, rather than a higher percentage of cytokine-producing cells.