Background cytokine production in the negative control of the ICS assay was subtracted from each stimulated http://www.selleckchem.com/products/epacadostat-incb024360.html condition. We defined the production of any cytokine (IFNγ and/or TNFα and/or IL2) as a positive CD4+ and CD8+ T-cell response, with 0.02% as the detection limit corresponding to at least 20 analyzed events. All donors were responsive to SEB positive control in the ICS assay. Functional characterization of the cytokine-producing

subset proportion was only performed in subjects with a positive cytokine response to RD1 antigens. Phenotypical analysis of different antigen-memory response of CD4+ and CD8+ T-cells was evaluated by flow cytometry according to the expression of the memory/effector surface markers HSP inhibitor CD45RA and CCR7. For a restricted number of samples (three HIV–LTBI and two HIV–TB) the phenotype was evaluated using the mAb CD45RA and CD2729; the results are not shown because it was not possible to combine them with CD45RA and CCR7 data. The memory status of antigen-specific CD4+ and CD8+ T-cells was evaluated on differently gated CD4+ and CD8+ T-cells. In particular, the phenotypical analysis was performed within the gates defined as total CD4+ T-cell response and total CD8+ T-cell response, identifying CD4+ and CD8+ T-cells as producing any cytokines. The analysis of the total enrolled subjects was performed by TC without knowing the LTBI status of the patients (active TB status was known

based on the initial positive AFB smear staining). Independent blind analyses were then repeated by EP using the same gating strategy. Concordance of the analyses was 90% and agreement was achieved by discussion. As described above, FACS analysis was performed on all enrolled subjects, therefore, results from subjects without LTBI were available. In these subjects, the amount of the specific response to Mtb antigens and the percentage not of the cytokine-positive cells was evaluated and the results were comparable to those found in the unstimulated

samples (data not shown). Data were analyzed using SPSS software (Version 19 for Windows, Italy SRL, Bologna, Italy). For continuous measures, medians and interquartile range (IQR) were calculated and the Mann–Whitney U test was used. For non-continuous measures the Chi-square test was used. P values as ≤0.05 were considered significant. Eighty-six HIV-infected patients naïve to ART were studied between September 2012 and February 2014. Twelve of these patients were enrolled as HIV–TB. Among the 74 subjects without active TB, 15 resulted HIV–LTBI and were characterized by not reporting any clinical symptoms related to active TB, no radiological evidence of TB lung lesions and no Mtb isolation from sputa (Table 1). Eighty-five percent of the enrolled subjects were BCG-vaccinated, almost 41% from South America and 29.5% from Eastern Europe, and no significant difference of gender or age was observed.