PD-1 Blockade Promotes In Vivo Expansion of HIV-infected CD4 T Cell Clones under Antiretroviral Therapy
Description
Whole-transcriptome gene expression values and differential gene expression analyses from sorted T cell subsets in six people with HIV who received a single dose of anti-PD-1 antibody during antiretroviral therapy.
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Steps to reproduce
Fluorescence-Activated Cell Sorting For sorting of CD4 T cell subsets, freshly isolated PBMC were stained with viability dye and the following mAbs: CD3-APC-H7 (BD Biosciences Cat# 641406, RRID: AB_1645730), CD4-BV785 (BioLegend Cat# 317442, RRID: AB_2563242), CD8a-BV650 (BioLegend Cat# 301042, RRID: AB_2563505), CD14-APC (BioLegend Cat# 301808, RRID: AB_314190), CD19-APC (BioLegend Cat# 363006, RRID: AB_2564128), CD27-BV605 (BioLegend Cat# 302830, RRID: AB_2561450), CD45RO-BV570 (BioLegend Cat# 304226, RRID: AB_2563818), LAG-3-FITC (BioLegend Cat# 369308, RRID: AB_2629751), 2B4-BV421 (BioLegend Cat# 393504, RRID: AB_2734462), PD-L1-PE (BioLegend Cat# 329706, RRID: AB_940368), PD-1-BV711 (BioLegend Cat# 329928, RRID: AB_2562911), and TIGIT-BB700 (BD Biosciences Cat# 747846, RRID: AB_2872309). For sorting of total CD4 T cells, cryopreserved PBMC were stained with viability dyes and the following mAbs: CD3-APC-H7, CD4-BV785, CD8-PacBlue (Thermo Fisher Scientific Cat# MHCD0828, RRID: AB_10372958), CD14-APC, CD19-APC, CD27-BV605, CD45RO-BV570, and PD-1-BV711. Sorting was performed on a BD FACSAria or a BD FACSymphony S6. Cells were sorted by FACS into heat-inactivated fetal calf serum and kept on ice until further processing. Nucleic Acid Extraction Cells were sedimented by centrifugation at 400 x g for 7 minutes at 4°C, lysed in RNAzol RT (Molecular Research Center Cat# RN190) at <5 x 106 cells/mL, homogenized by pipetting, and stored at -80°C until extraction. For RNA and DNA extraction, 0.4 volume of sterile H2O was added to each lysate to allow aqueous and organic phase separation. Total RNA was extracted from the aqueous phase according to the manufacturer’s instructions. The organic phase of each lysate was solubilized in DNAzol (Molecular Research Center Cat# DN127) for DNA extraction according to the manufacturer’s instructions. Transcriptome Sequence Analysis Messenger RNA libraries were constructed using NEB Next Ultra II RNA Library Preparation Kit (New England Biolabs Cat# E7770) and sequenced in 2×75-base, paired-end runs on an Illumina HiSeq 4000. Raw sequence data were converted to fastq format using bcl2fastq tool from Illumina. Preprocessing, QC, and RNA-seq analyses were performed with default settings in CLC Genomics Workbench v.21 (GWB) using the human reference (GRCh38, ENSEMBL v. 100). Differential gene expression analysis was performed pairwise between time points for each cell subset using samples from different participants as biological replicates.
Institutions
- National Institute of Allergy and Infectious DiseasesMaryland, Bethesda