Panel A shows a series of flow cytometry dot plots. The first plot displays forward scatter (FSC-A) versus side scatter (SSC-A) with a population gated at 93.3 percent. The second plot shows SSC-A versus FSC-A with a gated population at 52.4 percent. The third plot presents CD3e versus viability with a gated population at 78.2 percent. The fourth plot shows CD44 versus CD8a with a gated population at 2.63 percent. The fifth plot displays SIINFEKL versus CD8 with a gated population at 82.7 percent. The sixth plot shows CD69 versus CD103 with gated populations at 2.42 percent and 93.5 percent. Panel B consists of six scatter plots comparing the number of IV positive and IV negative CD8 positive T cells, CD4 positive T cells, and B cells in the nasal cavity and lung of vehicle (PBS) and FTY720 treated mice. The axes are labeled with log10 counts. Panel C includes three scatter plots showing the number of IV negative CD4 positive T cells in the nasal cavity and lung, and total CD4 positive T cells in the blood of isotype plus PBS and anti CD4 plus FTY720 treated mice. The axes are labeled with log10 counts.
Flow cytometry gating strategy and confirmation of FTY720 and anti-CD4 depletion efficacy. (A) Gating strategy for identification of IV− CD8+ SenNP+ CD69+ CD103+ T cells in Figs. 2, 3, 4, and 5. (B) The number of IV+ (top row) and IV− (bottom row) total CD8 T cells (left column), CD4 T cells (middle column), and B cells (right column) in the nasal cavity and lung of vehicle (PBS) (n = 8) or FTY720-treated Ad-SenNP-immunized mice (n = 8). (C) The number of IV− CD4 T cells in the nasal cavity and lung, and total CD4 in the blood, of isotype + PBS (n = 4) and anti-CD4 + FTY720–treated mice (n = 5). Statistical significance was determined using a two-sided Mann–Whitney test with *P < 0.05, **P < 0.01, ***P < 0.001, and NS for not significant.