Cd8 pacific orange

Heparinized whole blood (100 μl) was cultured in 96 U bottom culture plate and incubated with PMA/Ionomycin (10 ng/ml and 100 µg/ml, Sigma-Aldrich, UK), P. falciparum 3D7 schizonts lysate (5 µg/ml) and mycobacterium purified protein derivative (10 µg/ml, Statens Serum Institut, Denmark) in parallel with a non-stimulated background control for 24 h. Harvested cells were stained with surface markersCD3-APC-H7, CD4-PerCP (all from BD Biosciences, San Jose, CA, USA), and CD8-Pacific Orange (Invitrogen™ Life technologies, Carlsbad, CA, USA) and lysed as above. The cells were the permeabilized with 500 μl of 1× BD Cytofix/Cytoperm (BD Cytofix/Cytoperm™, San Diego, California) and then stained with intracellular markers IFN-γ-FITC (BD FastImmune™, San Jose, CA, USA) and TNF-PE (BD Biosciences, San Jose, California) for 30 min. The cells were then acquired as above and minimum of 10,000 CD4⁺ T cell events were collected per sample. Single stained BD comp beads were processed and acquired in parallel to samples each day and used for compensation. Analysis was conducted using FlowJo software version 9.7.5 (Tree Star Inc., San Carlos, CA, USA).

T cell analyses were performed in purified peripheral blood mononuclear cells (PBMCs). A primary stain with unlabelled murine anti-human antibodies directed against PD-1 (clone EH12.2H7) or isotype control followed with secondary phycoerythrin (PE)-labelled goat-anti-mouse immunoglobulin antibody (Dako, Glostrup, Denmark) was followed by primary labelled surface stains as follows: CD4 (Pacific Blue); CD5 (allophycocyanin (APC)-AlexaFluor 700); CD25 (fluorescein isothiocyanate, FITC); CD45RA (energy-coupled dye, ECD or FITC); CD62L (PE or phycoerythrin-cyanin 5, PC5); CD127 (PE) (all from Beckman Coulter, CA, USA); CD8 (Pacific Orange) (Invitrogen, MD, USA); CD183 (APC) and CD194 (phycoerythrin-cyanin 7, PC7) (all from BD Biosciences, CA, USA). Analyses were done on a Gallios flow cytometer and T cell subsets were defined as follows: naïve (CD62L+CD45RA+), central memory (CM, CD62L−CD45RA+), effector memory (EM, CD62L−CD45RA−), TEMRA (CD62L+CD45RA−), TH1 (CD183+CD194−), TH2 (CD183−CD194+) and Treg (CD25+CD127−). Statistical analyses were performed using unpaired t tests for all subgroup results. A simple Bonferroni correction was applied to adjust for multiple testing, leading to a necessary p value of < 0.0025 for the definition of significance.

The following monoclonal antibodies and cell dyes were used: CD45.1 FITC, CD45.2 AlexaFluor700, CD45.2 FITC, TCR-beta APC, CD4 PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE-Cy7, L-selectin eFluor450, CD122 biotin (all eBioscience); CD8 Pacific Orange, streptavidin PE-TexasRed, LIVE/DEAD blue (all Invitrogen); and CD45.1 Brilliant Violet 650, CD4 Brilliant Violet 711, and TCR-beta PerCP-Cy5.5 (all BioLegend). Samples were acquired on LSR-II, LSRFortessa, or Fortessa X20 flow cytometers (BD), and analysis was performed with FlowJo software (Treestar).

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.

For cytokine analysis, 2 x 10⁶ splenic cells were plated into a 96-well plate. Cells were suspended in RMPI 1640 culture medium and incubated for four hours utilizing phorbol 12-myristate 13-acetate (30ng/mL) and ionomycin (400ng/mL) with 10μg/mL of Brefeldin A at 37°C. After stimulation, cells were then stained for the following intracellular and extracellular markers CD4—Pacific Blue (BD Biosciences, clone RM4-5), CD8—Pacific Orange (Invitrogen, clone MCD0830), CD25—FITC (BioLegend, clone PC61), CD3—Alexa 700 (BD Biosciences, 500A2), FOXP3—APC (eBioscience, FJK-16S), IL-2—FITC (BD Pharmingen, clone JES6-5H4), IL-4—PE (BioLegend, clone 11B11), CCR4—PE Cy7 (BioLegend, clone 2G12), CXCR3—APC (BioLegend, clone CXCR3-173), and IL-17A—PE (eBioscience, clone eBio17B7). Samples were run on an LSR II flow cytometer (BD Biosciences) and subsequent data was analyzed with FlowJo 10.0.8rl software (Tree Star, San Carlos, CA).