Cfp 10

For T cell assays, overlapping peptide pools targeting ESAT-6 (BEI Resources Cat#NR-50711), CFP-10 (BEI Resources Cat#NR-50712), Ag85A (BEI Resources Cat#NR-34827), Ag85B (BEI Resources Cat#NR-34828), and TB10.4 (BEI Resources Cat#NR-34826) as well as H37Rv M.tb whole cell lysate (BEI Resources Cat#NR-14822) were used. Staphylococcal Enterotoxin Type B (SEB) (List Biological Laboratories Cat#122) was a positive assay control, and dimethyl sulfoxide (DMSO) (Sigma-Aldrich Cat#D8418) was a negative assay control.
For antibody assays, M.tb antigens tested were: purified protein derivative (PPD) (Statens Serum Institute), Ag85A and B in a 1:1 ratio (BEI Resources Cat#NR-49427 and #NR-53526), recombinant ESAT-6 (BEI Resources Cat#NR-49424) and CFP-10 (BEI Resources Cat#NR-49425) in a 1:1 ratio, HspX (BEI Resources Cat#NR-49428), 1-tuberculosyladenosine (1-TbAd) (provided by Dr. Branch Moody), and lipoarabinomannan (LAM) (BEI Resources Cat#NR-14848). An equal mixture of influenza antigens from HA1(B/Brisbane/60/2008) and HA1(H1N1) (A/New Caledonia/20/99) (Immune Technology Corp ITIT-003-001p and IT-003-B3p) was used as a positive assay control.

For flow cytometry, we used anti-CD3 FITC (BioLegend, USA, Cat#300440) and PerCP (BD Biosciences, USA, Cat#347344), anti-CD56 FITC (BioLegend, USA, Cat#318304) and APC (BD Biosciences, USA, Cat#555518), anti-CD16 PE (BioLegend, USA, Cat#302008); for staining memory cells anti-KLRG1 FITC (BioLegend, USA, Cat#138410), anti-CD27 PE (BD Biosciences, USA, Cat#555441); for intracellular staining anti-IL-32α (R&D Systems, USA, Cat#IC30402A) and anti-IFN-γ APC (BioLegend, USA, Cat#502512) antibodies were used. Magnetic beads conjugated to anti -CD56 (Miltenyi Biotec, Germany, Cat# 120-000-307) and -CD14 antibodies (Miltenyi Biotec, Germany, Cat# 120-000-305) were used for positive selection of NK cells and monocytes respectively.
For in vitro stimulation assays, we used ESAT-6 (BEI Resources, USA, Cat#NR-50711) and CFP-10 (BEI Resources, USA, Cat#NR-50712) peptide pools consisting of 21 and 22 individual peptides belonging to 6-kDa ESAT-6 and 10-kDa CFP-10 respectively. γ-irradiated Mtb H37Rv whole cells were used for some experiments (BEI resources, USA, Cat#49098).

Purified protein derivative (PPD; Statens Serum Institute, 10 μg/mL), recombinant early-secreted antigen 6 (ESAT-6, 10 μg/mL) and culture filtrate protein 10 (CFP-10, 10 μg/mL) peptide pools [BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)] were the antigens used in the study. The human immunodeficiency virus Gag peptide pool (HIVPP; AIDS Reagent Program, Division of AIDS, NIAID, NIH, 10 μg/mL) was used as non-TB specific antigen stimuli i.e., negative control. Finally, the combination of Phorbol 12-myristate 13-acetate (P)–ionomycin (P/I; Calbiochem, 12.5 ng/mL and 125 ng/mL) was used as a positive control.