Cd8 pe txred

Flow cytometry was performed on whole blood and BAL samples obtained from all 21 animals as previously described.^{25 , 33 (link)} For T cell phenotyping the following antibodies were used: CD3 V500 (1:50, clone SP34-2), CD4 PerCP-Cy5.5 (1:10, clone L200), CD8 PE-TxRed (1:30, clone RPA-T8), CD28 APC (1:5, clone CD28.2), CD69 APC-Cy7 (1:20, clone FN50), CD95 PE-Cy5 (1:5, clone DX2), CD183 AL488 (1:10, clone 1C6/CXCR3), CD184 PE-Cy5 (1:5, clone 12G5), CD195 APC (1:5, clone 3A9), CD197 PE-Cy7 (1:20, clone 3D12), HLA-DR APC-Cy7 (1:75, clone L243), and Ki67 PE-Cy7 (1:50, clone B56) all purchased from BD Biosciences (San Jose, CA, USA). Flow cytometry analyses were conducted by gating first on lymphocytes followed by the elimination of B cells by gating for CD20. The remaining cells were gated for the selection of T cells using CD3, followed by gating into CD3⁺CD4⁺ and CD3⁺CD8⁺ subpopulations. The frequencies of CD4⁺ and CD8⁺ T cells expressing activation and homing markers were compared using Ki67, CXCR3, CCR5, and CCR7.^{70 (link)} The levels of Foxp3⁺ were determined as a measure of the T_reg response. Finally, the extent of CD4⁺ and CD8⁺ cells belonging to either central memory (T_CM) or effector memory (T_EM) relative to the naïve T cell population were measured using a combination of CD28 and CD95 markers.^{37 (link)}