Cd8 v450

Cells were stained for 30 min at 4 °C in PBS containing 0.01% NaN₃ and 0.5% BSA with directly labelled antibodies against: CXCR3 alexa fluor488, CCR5 PE, CCR4 PercP-Cy5.5, CCR7 PE-Cy7, CCR6 alexa647, CD4 APC-H7,CD8 V500, CD3 V500, CD69 PercP, CD45 V500, Foxp3 PercP-Cy5.5, Granzyme-A PE, CD8 V450 (all from BD Biosciences), CD3 FITC (Sanquin, Amsterdam, The Netherlands), CD4 PE-Cy7, CD28 APC, CD8 APC-efluor780, CD45RA efluor450, CD45RO PE (all from eBioscience Inc., San Diego, CA, USA), Granzyme-K Fitc (Immunotools, Friesoythe, Germany), Granzyme-B APC (Invitrogen, Thermo Fisher Scientific Inc.) and Perforin PercP-Cy5.5, IL-10 Pe-Cy7 (Biolegend, San Diego, CA, USA). For cytokine staining, we used the Th1/Th2/Th17 kit from BD Biosciences. Cells were analysed on an FACS Canto II (BD Biosciences) and data were analysed using the FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI to illustrate cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest was normalized to the gMFI of the negative population.

For baseline T cell frequencies 0.5(10)⁶ cells from baseline uninfected total lymphocyte samples were stained and analyzed as above with phenotype antibody panel as follows: CD95-APC (2μl, Biolegend 305611), CCR7-PE (2μl, BD 566742), CD28-PE Cy7 (2μl, Biolegend 302925), CD45RO-FITC (3μl, Biolegend 304204), CD45RA-PerCP Cy5.5 (2μl, Biolegend 304121), CD4-APC H7 (2μl, BD 560158), CD19-V510 (3μl, BD 562953), CD8-V450 (2.5μl, BD 561426).

PBMCs obtained at A5321 study entry were stained with the following monoclonal antibodies to evaluate surface PD-1 expression: CD3 APC-H7 (catalog 560176), CD4 PC5 (catalog 555348), CD8 V450 (catalog 560347), PD-1 (clone M1H4), A488 (catalog 557860) (all from BD Biosciences), and LIVE/DEAD Aqua (Invitrogen, Thermo Fisher Scientific). Cells were fixed in 1% paraformaldehyde and analyzed using a BD LSRFortessa (BD FACSDiva software) within 24 hours after staining. Lymphocytes were identified based on size and granularity. The lymphocyte population was filtered through side scatter area versus side scatter height histogram to eliminate doublets from the analysis. Single cells were analyzed using LIVE/DEAD Aqua dye exclusion, and then CD4⁺ and CD8⁺ populations were defined based on dual expression with CD3. These 2 populations were plotted against PD-1. Fluorescence minus one controls were used to define the PD-1⁺ T cell populations.

To evaluate levels of soluble biomarkers, frozen, longitudinal plasma samples were thawed and analyzed in batches that included all the samples for a participant. Plasma concentrations of interleukin (IL)-6, high-sensitivity CRP (hsCRP), soluble CD14 (sCD14), and soluble CD163 (sCD163) were quantified using enzyme-linked immunosorbent assay (ELISA) kits per manufacturer’s instructions (R&D, Minneapolis, MN). Duplicates of 20% of the samples were included in each ELISA plate. Results were analyzed using BioTek ELx800 ELISA reader and KCjunior software (version 1.10). Levels of T cell activation and cell-cycling in cryopreserved PBMC from each participant were determined in batch using multicolor flow cytometry. PBMC samples were thawed, washed, and stained with the following antibodies: Live/Dead Aqua (Invitrogen, Grand Island, NY), CD3 APC-H7, CD4 Alexa 488, CD8 V450, HLA-DR PE, and CD38 APC (all BD Biosciences, San Diego, CA). To evaluate cell cycling, samples were permeabilized (Permeabilizing Solution 2, BD Biosciences) and stained with Ki-67 PerCP-Cy5.5 (BD Biosciences). Cells were then fixed in 1% paraformaldehyde, and analyzed using BD LSR Fortessa (FACSDiva) within 24 hours of staining.