Fig. 1: representative flow cytometry protocol to determine Vβ repertoire of total CD8+ T-lymphocyte and subpopulations. Peripheral blood mononuclear cells from cutaneous leishmaniasis patients were stained ex vivo with CD3-allophycocyanine-cyanine (APC) 7, CD8-APC, CD45RA-PE-Texas Red, CD27-PE-cyanine 7 and 24 different anti-Vβ monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE) and FITC-PE. The lymphocytes were gated on forward (FSC) vs. side (SSC) scatter dot-plot (A) and CD8+ T-lymphocytes were defined by double positive staining to CD3 and CD8 (CD3+CD8+) (B). Based on lymphocyte gate we analysed the expression of three different Vβ families (in each of the 8 tubes) (C) and CD8+ T lymphocyte subsets were defined by CD45RA and CD27 staining as late-differentiated effector (LDE) (CD45RA+CD27-), early-differentiated (naïve) (CD27+CD45RA+), late-differentiated effector memory (EM) (CD45RA-CD27-) and central memory (CM) (CD45RA-CD27+) (D). We also performed the Vβ analysis of naïve (E), LDE (F), EM (G) and CM (H) CD8+ T-lymphocytes.