Ki67 pe cy7

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)}

The following antibodies from BD Biosciences (Franklin Lakes, NJ) were used for staining: CD8-FITC (53.67), CD8-APC (KT15), Foxp3-PE (FJK-16s), CD3-AF700 (17A2), CD44-PerCP-Cy5.5 (IM7), CD4-FITC (RM4– 5), CD62L-BV421 (MEL-14), CD25-APC (PC61), Ki67-PE-Cy7 (SolA15), CTLA-4-PE (UC10–4F10–11), PD-1-BV421 (RMP1–30), granzyme B-PE (NGZB), OX40-BV711 (OX86), GITR-BV510 (DTA-1). MHC class I-restricted (H2-Db) PE-labeled CEA-tetramer (sequence: EAQNTTYL) was purchased from MBL International Corporation (Woburn, MA). Live/Dead fixable aqua stain and transcription factor staining buffer set were purchased from Thermo Fisher (Waltham, MA). Flow cytometry was performed on BD LSRFortessa or BD FACSVerse (BD Biosciences) and analyzed using FlowJo v.9.7.6 or v.10.5.3 (TreeStar). Cell viability was examined using trypan blue staining prior to data acquisition. Live cells were gated via forward and side scatter. Isotype control staining was <5% for all samples analyzed.