Tc1 conditions can restore perforin expression in CD8⁺ T cells from sentinel nodes.

<p>CD8⁺ T cells sorted from sentinel node (SN) were cultured in Tc1 conditions <i>in vitro</i> for seven days in order to rescue perforin expression. These SN-derived CD8⁺ T cells were stimulated with anti-CD3 and anti-CD28 stimulating antibodies with the presence of IL-12 and IL-2 cytokines, as well as an anti-IL-4 neutralizing antibody. At the end of the culture, cells were analyzed by flow cytometry and RT-qPCR. (<b>A</b>) Dot plots showed the flow cytometry data from a representative patient for granzyme B vs. perforin expression, before and after the stimulation. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. (<b>B</b>) Flow cytometry result of T-bet expression percentage from CD8⁺ T cells pre- and post-stimulation was analyzed. The frequency of T-bet expression was calculated from CD8⁺ T cells. (<b>C</b>) <i>TBX21</i> and <i>GATA3</i> gene expression analysis was done by RT-qPCR from cells in different culture conditions. <i>RPII</i> gene was used as housekeeping gene and the fold change was calculated based on cells without IL-12 and anti-IL-4 as control using 2^-ΔΔCt method.