Individuals with values above these

were identified as positive responders. Hence, 50% of healthy controls demonstrated positive IFN-γ responses compared to only 11% of individuals with latent infection and 0% for individuals with active TB infection (P = 0·02). Similar results were observed for IL-17- and IL-22-producing CD4+ T cells with P-values of 0·03 for both groups. One Epigenetics inhibitor individual with active TB had a very high proportion of IL-17-producing CD4+ T cells (83·2%), which was excluded from analysis due to suspected systematic error. Four out of 10 latent TB individuals co-expressed elevated proportions of IL-17+ CD4 T cells and IL-22+ CD4 T cells. Because Th17 cells produce IL-17 and IL-22 and recruit neutrophils to the site of inflammation [18,31], we determined if circulating neutrophils also produce IL-17 and IL-22. As neutrophils comprise approximately 90% of granulocytes, we measured the expression of IL-17 and IL-22 in total granulocytes. The granulocytes were gated according to size and granularity using forward-scatter and side-scatter by flow cytometry (Fig. 2a, left panel). CD4-CD8- cells were then gated from

the granulocyte-enriched cell population (Fig. 2a, middle panel) and analysed for IL-17 and IL-22 expression (Fig. 2a, right panel). The intracellular IL-22 Birinapant purchase was detected in a significant proportion of granulocytes from healthy individuals (20–90%). However, intracellular IL-17 was not detected in granulocytes from normal controls and individuals nearly with latent and active TB

infection (data not shown). The proportion of IL-22-expressing granulocytes was significantly lower in individuals with latent and active TB infection compared to healthy controls (P = 0·02; Fig. 2b). IL-22 expression in pure granulocytes isolated from blood was confirmed by counterstaining with another granulocyte marker CD15 (data not shown). To confirm whether IL-22 is transcribed in granulocytes, IL-22 mRNA expression was evaluated at the mRNA level by quantitative real-time PCR (qPCR) in granulocytes isolated from three healthy individuals. Granulocytes were either unstimulated or were stimulated with PMA for 4, 24 and 48 h. Surprisingly, IL-22 mRNA was not detected in unstimulated granulocytes after isolation. However, IL-22 was induced in granulocytes stimulated with PMA and ionomycin (Fig. 2c) with the peak expression at 24 h post-stimulation. To determine whether antigen-specific CD4+ T cells in latent and active TB subjects produce IL-17, IL-22 and IFN-γ in response to mycobacterial antigens, PBMC were stimulated with mycobacterial culture filtrate for 7 days prior to analysis of intracellular cytokines. The induction of cytokine expressing cells was calculated as a percentage increase following stimulation with mycobacterial antigens compared to the unstimulated cells.