LAMP/<i>gag</i> immunization induces polyfunctional and memory CD4⁺ T cells.

<p>Balb/c mice were immunized twice with the indicated plasmids. Fifteen days after the second immunization, the splenocytes were cultured with CD4- or CD8-restricted Gag peptides and the phenotype and cytokine production were analyzed. <b>A</b>–<b>B</b>) The cells were stained with PercP-anti-CD4 and APC-anti-IFN-γ, or with PercP-anti-CD4, APC-anti-IL-2 and PE-anti-TNF-α, and analyzed by FACS. Dot blots indicate CD4 and IFN-γ staining (<b>A</b>) or TNF-α and IL-2 staining among CD4⁺ cells (<b>B). C</b>) Culture supernatants were collected and the amount of TNF-α was analyzed by ELISA. <b>D</b>–<b>E</b>) Cells were stained with PercP-anti-CD4, PE-anti-CCR7 and APC-anti- IFN-γ. CD4⁺ cells were gated and the percentage and/or mean fluorescence intensity (MFI) of CD4⁺CCR7⁺IFN-γ⁺ cells were analyzed by FACS. <b>D</b>) Histograms indicate CCR7 expression among CD4⁺ cells. pITR vectors are in black; <i>gag_N</i> are in dashed line and LAMP/<i>gag</i> are in line histogram. The numbers indicated MFI values. <b>E</b>) Dot blots indicate the percentage of CCR7 and IFN-γ expression among CD4⁺ cells. Numbers indicate the percentage of IFN-γ⁺ cells among CCR7^hi and CCR7^l°CD4⁺ gated cells. The data are representative of three independent experiments. * p<0,05.