Cd4 t cell negative selection kit

PBMCs were isolated from whole blood of HS and PLHIV using Ficoll-Paque solution (GE Healthcare; Piscataway, NJ) and density gradient centrifugation separation. CD4 T cells were purified from PBMCs using CD4 T cell negative selection kit (Miltenyi Biotec; Auburn, CA). The cells were cultured for 24 h in RPMI-1640 media (Fisher Scientific; Pittsburgh, PA) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals; Flowery Branch, GA), 100 IU/ml penicillin, and 2 mM L-glutamine (Thermo Scientific; Logan, Utah) at 37°C and 5% CO₂ atmosphere. For TCR stimulation, CD4 T cells were cultured in media containing Dynabeads coated with human T-activator CD3/CD28 (Fisher Scientific). For knockdown and overexpression experiments, the cells were stimulated with soluble CD3/CD28 (BD biosciences; San Jose, CA) and recombinant human IL-2 (PeproTech; Rocky Hill, NJ) for 2 days prior to lentivirus infections and for another 3 days post transfection. For anti-PD-1 treatment and IL-2 administration experiment, CD4 T cells isolated from HS were cultured for 24 h in the presence of Dynabeads coated with human T cell activator anti-CD3/CD28, anti-PD-1 (nivolumab, 20 μg/ml; Selleckchem, TX), human IgG4 isotype control antibody antibodies (Biolegend), or IL-2 (100 units/ml; Biolegend).

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll density centrifugation (GE Healthcare, Piscataway, NJ). CD4⁺ T cells were isolated from PBMCs using the CD4⁺ T Cell Negative Selection Kit (Miltenyi Biotec Inc., Auburn, CA). T cells were cultured in RPMI 1640 medium containing 10% FBS (Atlanta Biologicals, Flowery Branch, GA), 100 IU/ml penicillin, and 2 mM L-glutamine (Thermo Scientific, Logan, Utah) at 37°C and 5% CO₂ atmosphere. Cells were harvested after day 1 or day 4 of culture for the detection of apoptosis and DNA damage. To test the role of ATM activation in repairing DNA damage and apoptosis, 10 μM ATM inhibitor (KU60019, Abcam, Cambridge, MA) or DMSO were added to the cultures at day 1, and the cells were collected after 48 h for measuring apoptosis and DNA damage. To determine the role of the PI3K pathway in ATM activation, purified CD4 T cells were cultured for 48 h in the presence or absence of 20 μM PI3K inhibitor (LY294002, Sigma), followed by flow cytometry or western blot analysis.

PBMCs from healthy donors samples were isolated using Ficoll-Hypaque gradient centrifugation. T cells were obtained using SepMate™ PBMC Isolation Tube (STEMCELL, Vancouver, Canada). Isolated T lymphocytes were maintained in RPMI 1640 or ImmunoCult™-XF T cell Expansion Media (STEMCELL, Vancouver, Canada) with 50 U/ml interleukin-2 (PeproTech, Cranbury, NJ, USA). CD3⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells were isolated using Pan T negative selection kit (Miltenyi Biotec, North Rhine-Westphalia, Germany), CD4⁺ T cell negative selection kit (Miltenyi Biotec, North Rhine-Westphalia, Germany) and CD8⁺ T-cell negative selection kit (Miltenyi Biotec, North Rhine-Westphalia, Germany), respectively, following the manufacturer’s instructions. Isolated T cells were activated by adding a CD3/28 T-cell activator (STEMCELL, Vancouver, Canada) and 50 U/ml interleukin-2. T cells were resuspended in CryoStor CS10(STEMCELL, Vancouver, Canada) at -80°C and thawed quickly in a 37°C water bath. All cells were grown in a humidified incubator at 37°C supplied with 5% CO₂ and tested regularly for Mycoplasma contamination.

Naive CD4⁺vβ10⁺CD25^–CD44^loCD62L^hi C7 T cells were sort purified after enrichment with a CD4⁺ T cell negative selection kit (Miltenyi Biotec). Dendritic and Thetis cell subsets were sort purified from Rorc^{Venus-creERT2} mice using gating strategy above with inclusion of CCR6 to distinguish TC I and TC II from TC III and TC IV. cDC2s were distinguished from their CCR7⁺ counterparts by CD11c^hiMHCII^int expression. T cells were co-cultured in triplicate at a ratio of 300 dendritic or Thetis cells to 1 × 10³ T cells in the presence of ESAT peptide (1 µg ml⁻¹; Invivogen), with the addition of 0.5 ng ml⁻¹ TGFβ1 (Peprotech), 100 IU ml⁻¹ of IL-2 (NCI). FOXP3 expression was assessed after 4 days of culture.

PBMCs were isolated from whole blood using Ficoll density centrifugation (GE Healthcare; Piscataway, NJ, USA). CD4 T cells were isolated from PBMCs using a CD4 T Cell Negative Selection Kit (Miltenyi; Cambridge, MA, USA). CD4 T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), 100 IU/mL penicillin, and 2 mM L-glutamine (Thermo Scientific, Logan, UT, USA), and maintained at 37 °C in a 5% CO₂ incubator.