Cd28 ecd clone cd28 2

After isolation, cells were resuspended in phosphate-buffered saline (PBS) containing 2 mM EDTA and spun for the removal of contaminating platelets. Prior to sorting, peripheral CD4⁺ T cells were enriched with the use of magnetic beads and column purification (Miltenyi Biotec). Enriched peripheral CD4⁺ T cells were then stained with previously determined volumes of the following fluorescently conjugated MAbs: CD3-APC-Cy7 or CD3-AF700 (clone SP34-2), CCR7-PE-Cy7 (clone 3D12), CD8-APC-Cy7 (clone SK1), CD45RA-APC (clone 5H9), CD95-PE-Cy5 (clone DX2), and CD62L-PE (clone SK11) from BD Bioscience; CD28-ECD (clone CD28.2) from Beckman Coulter, and CD4-BrilliantViolet650 (clone OKT4) and CD8-BV421 (clone RPA-T8) from BioLegend. Circulating populations for sorting were defined as follows: naive, CD45RA⁺ CCR7⁺ CD95⁻ SCM, CD45RA⁺ CCR7⁺ CD95⁺ CD28⁺ CD62L⁺; CM, CD45RA⁻ CD95⁺ CCR7⁺ CD62L⁺; and EM, CD95⁺ CCR7⁻. Sorting was performed on a FACSAria LSR II (BD Biosciences) equipped with FACSDiva software.

Peripheral blood mononuclear cells were isolated from whole EDTA blood by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. Mononuclear cell preparations of intestinal biopsy and necropsy tissues were obtained by mechanical disruption and enzymatic dissociation followed by lymphocyte separation by centrifugation through Ficoll-Paque Plus (GE Healthcare), as described previously54 (link). Freshly isolated cells were immunophenotyped using the following antibody panel: CD4 Pacific Blue (clone OKT4; BioLegend; 1:50); CCR5 PE (clone 3A9; BD Biosciences; 1:20); CD28 ECD (clone CD28.2; Beckman Coulter, 1:20); CD95 PE-Cy5 (clone DX2; BD Biosciences; 1:10); CD8 PE-Cy7 (clone SK1: BD Biosciences; 1:100); CD38 APC (clone OK10; NIH Nonhuman Primate Reagent Resource; 1:25); CD3 APC-Cy7 (clone SP34-2; BD Biosciences; 1:100); and Ki67 FITC (clone B56; BD Biosciences; 1:20). Approximately 100,000 CD3+ T cells were acquired for each sample and data analysis was performed using FCS Express software. All dilutions are from the manufacturer's stock and based on 100 μl total staining volume.

Multicolor flow cytometric analysis was performed on whole blood (WB) or cell suspensions using predetermined optimal concentrations of the following fluorescently conjugated monoclonal antibodies (MAbs). For WB T cell analysis the following MAbs were used: CD3-allophycocyanin (APC)-Cy7 (clone SP34-2), CD95-phycoerythrin (PE)-Cy5 (clone DX2), Ki67-AF700 (clone B56), HLA-DR-peridinin chlorophyll protein (PerCP)-Cy5.5 (clone G46-6), CCR7-fluorescein isothiocyanate (FITC) (clone 150503), CCR5-APC (clone 3A9), CD62L (clone SK11), and CD45-RA-PE-Cy7 (clone L45) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-BV650 (clone OKT4), and PD-1-BV421 (clone EH12.2H7) from BioLegend; and CD28-ECD (clone CD28-2) from Beckman-Coulter. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on an AURORA flow cytometer driven by the SpectroFlo software package (Cytek). Analyses of the acquired data were performed using FlowJo version 10.0.4 software (TreeStar).