Cd28 apc

Flow cytometric analysis was performed up to 3 times pre-transplant and serially post-transplant to characterize peripheral blood immune cell phenotypes. Total T cells and T cell subsets were quantified by complete blood cell count and flow cytometry. Fresh PBMCs were isolated by Ficoll density gradient centrifugation (BD Biosciences, Franklin Lakes, NJ). PBMCs were stained with the following mAbs: CD3 PacBlue, CD95 V450, CD3 Alexa 700, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD25 PE-Cy7, IFNy PE-Cy7, CD28 APC, TNF APC, VLA-4 APC, CD11a PE, CD45RA FITC, CD40 FITC, CCR7 APC, CD20 APC (all BD Biosciences). PBMCs (1.5×10⁶) were incubated with appropriately titered antibodies for 15min at 20°C and washed twice. Samples were acquired immediately on a BD LSR II multicolor flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). For the stimulation assay, 1.5×10⁶ PBMCs were cultured in RPMI 1640 (Corning cellgro, Manassas, VA) supplemented with 10% fetal bovine serum and stimulated with 10µM phorbol 12-myristate 13-acetate (PMA) and 200nM ionomycin (Sigma-Aldrich, St. Louis, MO), with 1ul/ml GolgiPlug protein transport inhibitor for 5 h, +/− IL-15 (10ng/mL). PBMCs were were processed with BD Cytofix/Cytoperm Plus kit (BD 555028) per the manufactures recommendation prior to data acquisition.

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)}

PBMC were thawed in warm media, washed twice and stained with three separate antihuman antibody cocktails containing: (1 ) anti-CD3 AmCyan, CD4 Pacific Blue, CD8 APCH7, CD28 APC; (2 ) CD3 AmCyan, CD4 Pacific Blue, CD8 APCH7, CD27 PE, CD45RA PE-Cy5; (3 (link)) CD3 AmCyan, CD19 Alexa Fluor700, CD56 PE, CD33 PE-Cy7, TCR APC, all reagents from BD Biosciences. Additional information for these antibodies can be found on ImmPort (https://immport.niaid.nih.gov/) under accession number SDY212. Incubation with antibodies was performed for 40 min at 4ºC. Cells were washed with FACS buffer (PBS supplemented with 2% FBS and 0.1% Na Azide), and resuspended in 200 µL FACS buffer. Data was collected using DIVA software in an LRSII instrument (BD Biosciences). Analysis was performed using FlowJo 8.8.6 by gating on live cells based on forward vs side scatter profiles, then using double gating for singlet discrimination, followed by cell subset-specific gating.