Figure 1

Representative flow cytometry protocol to determine the frequencies of CD8⁺T-lymphocyte subsets and apoptosis. Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients were stained ex vivo and after antigenic-stimulated cultures with CD3-FITC, CD8-APC, CD45RA-ECD, CD27-PE-Cy7 and 7-AAD. The lymphocytes were gated on forward (FSC) vs side (SSC) scatter dot-plot (A), backgated from CD3⁺ histogram and doublets were excluded by a density plot of FSC Area vs FSC Width (B). CD27 vs CD45RA dot plot (D) gated on CD3⁺CD8⁺ region (C) was used to define the frequencies of effector and naïve CD8⁺ T lymphocytes. Frequency of apoptotic cells (7AAD^low) was determined by FSC vs 7AAD dot-plot gated on CD3⁺CD8⁺ cells (total CD8⁺ T lymphocytes) (E) and on CD45RA⁺CD27^neg cells (effector CD8⁺ T lymphocytes) (F).