Bv510 labeled anti cd44

The PE-labeled MHC-II Ag85B tetramer I-A(b)/FQDAYNAAGGHNAVF and control tetramer I-A(b)/PVSKMRMATPLLMQA were provided by the NIH Tetramer core facility at Emory University (Atlanta, GA). Isolated PBMCs, splenocytes, and lung lymphocytes were incubated with fixable viability stain 780 (BD Biosciences), APC-labeled anti-CD3 (Biolegend), PerCP-Cy5.5-labeled anti-CD8 (eBioscience), FITC-labeled anti-CD4 (Biolegend), and BV510-labeled anti-CD44 (Biolegend), together with either control or Ag85B PE-labeled MHC-II tetramer. To detect tissue-resident CD4 T cells in the lung, lung lymphocytes were incubated with the same cocktails, together with APC-Cy7-labeled anti-CD69 (Biolegend). Cells were washed three times with PBS/2% PBS. Sample acquisition was performed on FACSCelesta and data analyzed using FlowJo.

The PE-labeled MHC-I tetramer H-2K(b)/EsxH epitope IMYNYPAM was provided by the NIH Tetramer core facility at Emory University (Atlanta, GA). Isolated PBMCs, splenocytes, and lung lymphocytes were incubated with PE-labeled EsxH/H-2K(b) MHC-I tetramer, fixable viability stain 780 (BD Biosciences), APC-labeled anti-CD3 (Biolegend), PerCP-Cy5.5-labeled anti-CD8 (eBioscience), FITC-labeled anti-CD4 (Biolegend), and BV510-labeled anti-CD44 (Biolegend) for one hour at room temperature. Cells were washed three times with PBS/2% PBS. Sample acquisition was performed on FACSCelesta and data analyzed using FlowJo.