Cd4 pacific blue

We used the following conjugated antibodies: CD38-fluorescein isothiocyanate, CD34-phycoerythrin (PE)-cyanin5 (Cy5), CD4-Pacific Blue, CD19-allophycocyanin (APC), CD3-Alexa Fluor 700, CD8-APC-H7, CCR7-Alexa Fluor 647, CD79a-APC from BD Biosciences (San Jose, CA); CD56-PE, CD45RA-PE-TexasRed, CD7-PE-Cy7, CD1a-PE, CD5-PE-Cy5 from Beckman Coulter (Brea, CA); CD31-PE from Miltenyi Biotech (Bergisch Gladbach, Germany) and CD27-PE-Cy7 from eBioscience (San Diego, CA). We obtained LIVE/DEAD aqua fluorescent dye from Molecular Probes (Invitrogen, Carlsbad, CA). The Fam-FLICA-FITC probe against active caspase-1 was used according to the manufacturers’ instructions (ImmunoChemistry, Bloomington, MN). Standard protocols were used for all types of staining. Data were acquired with an LSRII Flow Cytometer (BB Biosciences), and analyzed with FlowJo v7.6.5 (Treestar, Ashland, OR).

Freshly isolated decidual CD4+ T and Peripheral blood-CD4+ T cells were stained simultaneously with CD3-PE-Cy7, CD4-pacific blue, CD161-APC (BD Biosciences, Franklin Lakes, New Jersey) and either CCR3-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), IL-23R-PerCP, CCR4-mouse PE, CCR8-rat PE, CCR6-PE, CCR8-rat-PE, CXCR3 mouse-PE (R&D systems, Minneapolis, MN), or CRTH2 rat-PE (Myltenyi Biotech, Bergisch Gladbach, Germany) mAbs or their respective isotype controls: IgG1 mouse PE-Cy7, IgG1 mouse-pacific blue, IgG1 mouse APC, IgG2a rat-FITC, IgG2b mouse-PerCP, IgG1 mouse-PE, IgG2a rat-PE (BD Biosciences, Franklin Lakes, New Jersey), IgG2b-mouse PE, IgG2b-rat PE (R&D systems, Minneapolis, MN). Stained cells were acquired on a BD Biosciences LSR II flow cytometer (BD Biosciences, Franklin Lakes, New Jersey) (Data were analyzed with BD Biosciences FACSDiva software version 6.2.

Following co-culture, cells were immediately fixed with BD Lyse/Fix buffer for 10 minutes (BD Biosciences), washed and then pelleted. Cells were then surfaced stained with CD4 Pacific Blue and CD8-APC H7 (BD Biosciences) for 20 minutes. For phosphorylated STAT-3 and -5 detection, samples were permeabilized with ice cold BD Perm Buffer III for 30 minutes, washed with PBS, and then incubated with phospho-STAT3 Alexa Fluor 488 or PE and phospho-STAT-5 Alexa Fluor 647 for 30 minutes. Cells were acquired on an LSR II flow cytometer (BD Biosciences); 50,000 events per condition were collected.

Cryopreserved PBMC was thawed and batch analysed to ensure consistency, using an LSRII flow cytometer (BD Biosciences) at Uganda Virus Research Institute/Medical Research Council basic science laboratory. Cell surface staining was used to phenotype the markers for T-cell activation and exhaustion using antibodies for CD3- Amcyan, CD4- Pacific blue, HLADR-PerCP cy5.5, CD38-APC, PD-1-PE and Live/dead marker-FITC (BD Biosciences). Overall, at least 500 000 events in the CD3-positive gate were collected. The gating was standardised and set using fluorescence minus one controls for HLADR, CD38 and PD1 as shown in Figure 2. Percentages of activated T cells were determined by the proportion of CD3 + CD4 + (CD8) CD38 + HLADR+ T cells, and immune exhaustion was determined by the percentage of T cells expressing programmed cell death marker-1 (CD3 + CD4 + (CD8) PD-1) as shown in Figure 2.

Freshly drawn blood samples (30 μl) were incubated with fluorochrome-labelled antibodies in a total volume of 100 μl FACS buffer (PBS/1% fetal calf serum (FCS)) for 45 min in the dark at 4 °C. Red blood cells were lysed by incubating the sample with 2 ml of 1X red blood cell lysis solution (BD Biosciences, Oxford, UK) for 10 min at room temperature (RT). After thorough washes samples were fixed in 1% paraformaldehyde (PFA) for 30 min in the dark at 4 °C. Samples were then washed and resuspended in PBS. All samples were examined by flow cytometry (FACS CANTO II; BD Biosciences), and data analysed blindly using the FlowJo software (version 10.07 for Windows, Tree Star, Ashland, OR, USA). The following anti-human antibodies were used for identification of leukocyte subsets: CD14-APC-Cy7, CD19-FITC, CD16-Pacific Blue, CD56-PE, CD8-PE-Cy7, CD4-Pacific Blue, CD11b-APC (all from BD Biosciences, UK) and HLA-DR-PE (eBioscience, UK). Live cells were gated for further analysis of leukocyte subsets. Different subsets of leukocytes were identified by relative cell size and granularity (FSC/SSC) or cell surface markers (Fig. 1). The neutrophil/lymphocyte ratio (NLR) was calculated by dividing the percentage of neutrophils by the percentage of lymphocytes.

For phenotypic analysis, animals were sacrificed at 24hrs utilizing CO₂ euthanasia and their spleens were harvested. The spleens were processed into single-cell suspensions and the number of cells per mL of suspension was obtained via a Nexcelom Auto Cellometer. 2x10⁶ cells were plated into a 96-well plate and stained for extracellular markers CD4—Pacific Blue (BD Biosciences, clone RM4-5), CD8—Pacific Orange (Invitrogen, clone MCD0830), CD3—APCCy7 (BioLegend, clone 17A2), CD44—PerCP (BioLegend, IM7), CD62L—APC (eBioscience, clone MEL-14), CD43—FITC (BioLegend, clone 1B11), and CD43—PE (BD Pharmingen, S7). TruCount Beads from BD Pharmingen were prepared according to the manufacturer’s instructions and used to determine absolute cell counts.