We stimulated the peripheral blood mononuclear cells (PBMC) for 16 hours with Mtb-specific antigens: a) RD1 proteins that consist in a mix of recombinant proteins ESAT-6 and CFP-10 (Lionex, Braunschweig, Germany) used at 4 μg/ml (LPS contamination was for ESAT-6 less than 0.05 IU/mg and for CFP-10 was equal to 66.7 IU/mg, as reported by the manufacturer); b) a recombinant methylated HBHA which was purified from M. smegmatis pMV3-38 as previously described [22 (link)] (LPS contamination was 0.60 IU/mg of protein) and used at a final concentration of 5 μg/ml [19 ]. The unrelated antigen CMV lysate (strain AD169) (Experteam, Venice, Italy) was used at 5 μg/ml while as positive control the SEB (Sigma, St Louis, MO, USA) was used at 200 ng/ml.
A costimulation with anti-CD28 and anti-CD49d monoclonal antibodies (mAb) at 2 μg/ml each (BD Bioscence, San Jose, USA) was performed. The fluorescently conjugated mAb used were those listed below: anti-CD3 allophycocyanin (APC)-Vio770, anti-CD8 VioBlue, anti-CD4 peridinin chlorophyllprotein (PerCP)-Vio700, anti-CD45RA phycoerythrin (PE)-Vio770, anti-CCR7 VioGreen, anti-IFN-γ APC, anti-TNF-α fluorescein isothiocyanate (FITC) and anti-IL-2 PE (all mAb from Miltenyi Biotec).
A costimulation with anti-CD28 and anti-CD49d monoclonal antibodies (mAb) at 2 μg/ml each (BD Bioscence, San Jose, USA) was performed. The fluorescently conjugated mAb used were those listed below: anti-CD3 allophycocyanin (APC)-Vio770, anti-CD8 VioBlue, anti-CD4 peridinin chlorophyllprotein (PerCP)-Vio700, anti-CD45RA phycoerythrin (PE)-Vio770, anti-CCR7 VioGreen, anti-IFN-γ APC, anti-TNF-α fluorescein isothiocyanate (FITC) and anti-IL-2 PE (all mAb from Miltenyi Biotec).