Then, lymphocytes were sub-gated on Alexa-488-positive CD3+ T cells (gate R2; see Fig. 1b) to enumerate in the next step APC-positive CD8+ cells located in the lower right (LR) quadrant and PerCP-positive CD4+ cells located in the upper left (UL) quadrant (see Fig. 1b). Quadrant markers were set up on background staining with appropriate isotype controls.