Human t cell enrichment cocktail

Peripheral blood and leukapheresis samples from 22 blood donors younger than 35 years and 24 donors older than 65 years, de-identified except for age range, were purchased from the Stanford Blood Center. In addition, peripheral blood samples were obtained from 15 volunteers, who did not have evidence for an acute or uncontrolled chronic disease and who did not have a history of an autoimmune or a malignant disease. T cells were isolated directly from blood with Human T Cell Enrichment Cocktail (STEMCELL Technologies, Canada). CD4 naïve (CD3+CD4+CD62L+CD45RA+CD28+), central memory (CD3+CD4+CD62L+CD45RA–CD28+), and effector memory (CD3+CD4+CD62L-CD45RA–CD28+) T cells were further purified by fluorescence activated cell sorting (FACS) using a BD Aria 3 cell sorter.

Healthy volunteer T cells were prepared from healthy blood donors by immunodensity negative selection (Human T cell Enrichment Cocktail, Stem Cell Technologies, catalog 15021) and labeled with 1 μM CSFE (Invitrogen). T cells were cocultured with sorted macrophages or DCs at a 25:1 ratio for 7 days, incubated with antibodies to CD3, CD4, CD8, HLA-DR, and CD69 (Supplemental Table 3), and analyzed by flow cytometry.

CD4 T cells were isolated using Human T cell Enrichment Cocktail (STEMCELL Technologies, Canada), followed by purification of naive (CD3⁺CD4⁺CD62L⁺CD45RA⁺CD28⁺), CM (CD3⁺CD4⁺CD62L⁺CD45RA⁻CD28⁺), and effector memory (CD3⁺CD4⁺CD62L⁻CD45RA⁻CD28⁺) subsets by fluorescence-activated cell sorting. ATAC-seq libraries were generated using 50,000 cells for each subset. Cells were first washed with cold PBS and RSB buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂), followed by washing with RSB buffer containing 0.1% NP-40 and 0.1% Tween-20. Subsequently, cell pellets were resuspended in a transposase reaction mix (25 μl 2× TD buffer, 2.5 μl transposase (Illumina), and 22.5 μl nuclease-free water) and incubated at 37 °C for 30 min. DNA fragments were purified using Qiagen MiniElute Kit and the library was amplified with Nextera PCR primers. Library quality was checked on bioanalyzer and sequenced on Illumina NextSeq 500. The sequencing reads were processed by trimming adapters and low-quality reads using in-house scripts and aligned to human reference genome hg19. Peaks were identified for each sample using macs2 and a consensus peak set was determined consisting of peaks present in at least three samples. Reads were converted into bigwig format for visualization of the genomic tracks, which were normalized to total reads mapped within the consensus peak set⁶⁴ .