αcd38 pe cy7

For all experiments, frozen PBMCs were thawed and rested for 16-18h in complete RPMI 1640 media at 37°C in a 5% CO₂ incubator before starting the experiments. Phenotypic analyses of PBMCs were performed using surface antibody staining for 30 minutes at 4°C, using combinations of the following anti-human monoclonal antibodies: αCD3-PE/Dazzle594 (OKT3), αCD3-FITC (OKT3), αCD38-PE/Cy7 (HB-7), αCD19-BV650 (HIB19), αCD56-BV750 (5.1H11), αCD4-PerCP/Cy5.5 (OKT4), αCD8-AF700 (SK1), αCCR7-APC/Fire (G043H7), αCD45RO-BV711 (UCHL1), all purchased from BioLegend. After staining the cells with antibodies for the phenotypic markers cells were fixed in 1% paraformaldehyde and acquired in a flow cytometer. The functional capacity of T cells was determined by analyzing the intracellular cytokine production upon stimulation with overlapping HIV-Gag polyprotein peptides using αIFNγ-APC (4S.B3) and αTNFα-PE (MAb11) antibodies from BioLegend. After staining the cells for surface markers, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) followed by intra cellular cytokine staining and finally fixed in 1% paraformaldehyde. Live/dead cell discrimination was performed using Zombie Aqua fixable viability Kit (BioLegend). Data were acquired and compensated using Cytek Aurora (Cytek Biosciences) flow cytometer and further analyzed with FlowJo (version 10.7.1).

A mouse MerTK-specific ASO and control ASOs were produced by Ionis Pharmaceuticals, Inc. Anti-mouse CTLA-4 (Catalog# BP0164) and anti-mouse PD-1 (Catalog# BE0146) were purchased from BioXCell. Liberase (Catalog #05401127001) and DNAse (Catalog #4716728001) were purchased from Roche and Sigma-Aldrich, respectively. Flow cytometry antibodies, including αCD45-PerCP Cy5.5 (Catalog# 103131), αCD4-PE/Dazzle594 (Catalog# 100456), αCD8-FITC (Catalog# 100706), αGranzyme B (GrB)-Pacific Blue (Catalog# 515408), αGr1-BV510 (Catalog# 108437), αCD11b-APC Fire750 (Catalog# 101262), αF4/80-Alexa Fluor 700 (Catalog# 123130), αCD38-PE-Cy7 (Catalog# 102718), αCD206-PE (Catalog# 141706), and αMertK-APC (Catalog# 151507) were ordered from BioLegend.

αCD2-biotin (clone: RPA-2.10), αCD14-biotin (clone: HCD14), αCD16-biotin (clone: 3G8), αCD19-biotin (clone: 561), αCD235a-biotin (clone: HIR2), αCD3-biotin (clone: OKT3a), αCD34-APC (clone: 561), αCD38-PE/Cy7 (clone: HIT2), αCD4-APC/Cy7 (clone: RPA-T4), αCD8a-Pacific Blue (clone: RPA-T8), αCD90-PerCP/Cy5.5 (clone: 5E10), αCD272-FITC (αBTLA; clone: MIH26), αIgG2-FITC (clone: MOPC-173), αCD200R-PE (clone: OX-108), αIgG1-PE (clone: MOPC-21), αCD160-PerCP/Cy5.5 (clone: BY55), αIgM-PerCP/Cy5.5 (clone: MM-30), αCD96-APC (clone: NK92.39), αIgG1-APC (MOPC-21), αCD28-PE/Cy7 (clone: CD28.2), αIgG1-PE/Cy7 (MOPC-21) and αStreptavidin-FITC were from BioLegend, USA. αCD45-PE (clone: HI30) was from eBioscience, USA. αIgG1-PE (clone: IS11-12E4.23.20) was from Miltenyi Biotec, Germany. Samples were analyzed on BD LSRFortessa™ (BD Biosciences, USA). Data was analyzed using FlowJo software v.10.7 (FlowJo, LLC, USA).