In conclusion, our data show that HIV infection is usually associated with a complex pattern of em M

In conclusion, our data show that HIV infection is usually associated with a complex pattern of em M.tb /em -specific T cell impairment affecting both CD4+ and CD8+ T cell populations, and suggest that depletion of antigen-specific Th1 and Th17 CD4+ T cells may directly lead to TB disease susceptibility compounded by loss of corresponding CD8+ T cell function. ? Summary HIV illness is associated with a complex pattern of em M. (Alpha crystallin 16kDa) recombinant protein (Rv2031c; 10g/mL; Lionex, Germany), cytomegalovirus lysate (CMV; 5g/mL, Virusys, USA). The recombinant proteins were indicated and purified from E.coli, had purity of 95% and endotoxin material below 25 IU/mg (Limulus Amebocyte Lysate assay). Brefeldin A (5 g/mL; Sigma-Aldrich, UK) was added after six hours and further incubated for five hours. Red blood cells were lysed and white cells fixed before long-term storage in liquid nitrogen. Circulation cytometry and intracellular cytokine staining (ICS) Cryopreserved white cells were stained as previously Vitamin CK3 explained [26]. The following conjugated monoclonal antibodies directed at cell-surface markers and intracellular cytokines were used: CD3-eFluor 450 (clone UCHT1), IFN-PE/Cy7 (clone 4S.B3) and TNF-PerCP/Cy5.5 (clone MAb11) (eBioscience, UK); CD4-APC (clone RPA-T4), CD14-APC/Cy7 (clone HCD14), CD19-APC-Cy7 (clone HIB19) and IL-17-Alexa Fluor 488 (clone BL168) (BioLegend, UK), CD8-VioGreen (clone BW135/80) and IL-2-PE (clone N7.48A) (Miltenyi Biotec, Germany). Stained cells were acquired on a MACSQuant circulation cytometer (Miltenyi Biotec, Germany) Vitamin CK3 and all events were captured. At least 50,000 events were captured for each sample included in the analysis. Data were analysed using FlowJo vX (Tree Celebrity, USA). The gating strategy is demonstrated in Supplementary Number 1. Boolean cytokine combination gates were created using FlowJo vX and analysed with PESTLE (v1.7; National Institutes of Health (NIH), USA) and SPICE software (v5.35; NIH, USA). A positive T cell cytokine response was defined as a rate of recurrence of total cytokine-positive CD4+ and/or CD8+ T cells at least 0.1% of total CD4+/CD8+ T cells after background subtraction and at least twice the frequency of total cytokine-positive CD4+/CD8+ T cells in the negative control. Vitamin CK3 As opposed to BCG lacks the RD1-region encoding the genes for the proteins ESAT-6 and CFP-10. In addition, Rv2031c and components of PPD can be found in mycobacteria other than Therefore, as per approved convention, we classified individuals with LTBI only if demonstrating a cytokine-positive T cell response to ESAT-6 and CFP-10 as used in combination in our assay. Statistical Analyses Standard statistical methods for parametric and non-parametric data were used as appropriate. Specific checks are detailed in the results and include Fisher-exact T checks, Kruskal-Wallis checks and Mann-Whitney checks (Prism 5 for Mac pc OS X v5.0a; GraphPad, USA). Partial permutation analyses were determined using SPICE (v5.35; NIH, USA). Multivariate logistic regression was performed using SPSS (v22; IBM, USA). Results Participant characteristics 44 HIV-uninfected and 147 HIV-infected individuals were analyzed (Table 1). Of the second option, CD4+ T cell counts ranged from 200 cells/L (n=51), 201-350 (n=48) and 350 (n=48). 14 HIV-infected individuals experienced a history of active TB; there were no earlier Rabbit Polyclonal to Cytochrome P450 26A1 TB instances in the HIV-uninfected group. All were believed to have received BCG vaccination in child years, although only 20 (45%) HIV-uninfected and 89 (61%) HIV-infected individuals experienced a BCG vaccination scar. HIV-infected subjects were stratified by CD4+ T cell count. Table 1 Demographics of Study Participants BMI = Body mass index; TB = tuberculosis; BCG = Bacillus Calmette-Guerin, N/A = not applicable. *Data indicated as median (IQR) Impairment of PPD and Rv2031c-specific CD4+ T cell reactions in HIV-infected individuals As expected, median total CD4+ T cell counts were significantly reduced HIV-infected than uninfected individuals, whilst CD8+ T cell counts were higher (P 0.001 for those, Mann-Whitney) (Table 1). The median CD4:CD8 ratio of 1 1.63 (IQR 1.02 C 2.23) in the HIV-uninfected group was as expected for healthy African individuals [27,28], but was significantly reduced.