Cd4 apc h7

One day post isolation FACS analysis was used to determine the percentages of various CD4+ T cell subsets, like naïve (TN), effector memory (TEM), central memory (TCM), T helper 17 cells (Th17), follicular helper T cells (Tfh) and regulatory T cells (Treg). For this the following antibodies were used: CD4-APC H7 (BD Biosciences, 560158), CD45RA-PE Cy7 (BD Biosciences, 560675), CD45RO-PerCP Cy5.5 (BD Biosciences, 560607), CCR7-APC (BioLegend, 353214), CCR6-PE (BD Biosciences, 559562), CXCR5-PerCP Cy5.5 (BioLegend, 335001), CD127-PE Cy7 (BD Biosciences, 560822), CD25-Horizon V450 (BD Biosciences, 560355). Further, the expression of different receptors on unstimulated as well as stimulated CD4+ T cells was determined using the following antibodies: CD4-APC H7, TCR-CD3-APC H7 (BD Biosciences, 560275), CD28-PerCP Cy5.5 (BD Biosciences, 560685), MHC-I-Horizon V450 (BD Biosciences, 561346), FAS-PE (BD Biosciences, 556641), FAS-L-PE (BioLegend, 306407), PD1-APC (BD Biosciences, 558694), PD1-L-PE Cy7 (BD Biosciences, 558017), TRAIL-PE (BD Biosciences, 550516), CTLA-4-PE (BD Biosciences, 555853), CD69-Horizon V450 (BD Biosciences, 560740), CD25-PE Cy7 (BD Biosciences, 557741), CXCR4-PerCP Cy5.5 (BD Biosciences, 560670) and CCR5-PE Cy7 (BD Biosciences, 557752). Cells were analyzed using the BD FACS Canto II with FACSDiva software.

Purified PBMCs were thawed and stained with the following conjugated monoclonal antibodies: CD19- and CXCR5-Alexa 488, CD27-PerCP-Cy5.5, CD21- and CD45RO-APC, IgD, and CD4-APC-H7 (all from BD Biosciences, Buccinasco, Italy) and CD3- and CD10-Pe/Cy7 (BD Biosciences and Biolegend, respectively). Multicolor flow cytometry was performed on a FACSCanto, interfaced to a FacsDiva software (BD Biosciences) and analyzed through Flow-Jo software version 8.8.3 (Three Star Inc., Ashland, Oregon, USA). The different CD19+ B-cell subpopulations were defined as follows: resting memory (CD27+CD21+), switched memory (CD27+IgD−), unswitched-memory (CD27+IgD+), naïve (CD27−IgD+), double negative (DN) (CD27−IgD−), and mature activated (MA) (CD10−CD21−).

The cells were thawed and washed with PBS first and PBS/1% BSA later and then stained with surface antibodies for 30–60 minutes. Surface antibodies used were CD3 - Amcyan, CD4 - APC-H7 and CD8 - PE-Cy7 (all from BD Biosciences). The cells were washed and permeabilized with BD Perm/Wash buffer (BD Biosciences) and stained with intracellular cytokines for an additional 30 min before washing and acquisition. Cytokine antibodies used were IL-10 (BD Pharmingen), IL-19, IL-24 and IL-26 (R&D Systems). Flow cytometry was performed on a FACS Canto II flow cytometer with FACSDiva software v.6 (Becton Dickinson). The lymphocyte gating was set by forward and side scatter and 100,000 lymphocyte events were acquired. FMO gating was used for intracellular cytokine detection. Data were collected and analyzed using Flow Jo software. All data are depicted as frequency of CD4⁺ or CD8⁺ T cells expressing cytokine(s). Values following media stimulation are depicted as baseline frequency while frequencies following stimulation with antigens or anti-CD3 are depicted as net frequencies (with baseline values subtracted).