Mojosort human cd4 t cell isolation kit

PBMC fraction was obtained by gradient centrifugation of blood samples using a standard Ficoll solution (PanEco, Russia), density 1.077. NK cells and CD4⁺ T cells were isolated from PBMC using NK cell Isolation Kit (Miltenyi Biotec, Germany) and MojoSort™ Human CD4 T Cell Isolation Kit (Biolegend, USA) by negative magnetic separation, according to the manufacturers’ protocols. NK cell and T cell purity reached 95-99%. Lymphocytes were cultivated in NK MACS medium (Miltenyi Biotec, Germany) supplemented with 10% FCS (HyClone, USA) and Antibiotic Antimycotic Solution (Sigma-Aldrich, USA).
Dendritic cells were obtained based on the standard protocol (23 (link)). Briefly, PBMC were plated at a concentration of 10⁶ cells/ml in RPMI-1640 medium (PanEco, Russia) supplemented with 10% FCS (HyClone, USA), 2 mmol/L of L-glutamine (PanEco, Russia) and Antibiotic Antimycotic Solution (Sigma-Aldrich, USA) for 24h, then gently washed from non-adherent cells and cultured for 10 days. IL-4 (50 ng/ml) and GM-CSF (60 ng/ml) (Sci-Store, Russia) were added at day 0, day 1 and then every 3 days, with the fresh medium. At day 10, more than 90% of cells had CD14^lowCD11c⁺HLA-DR^hiCD86^hi phenotype.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from anonymous, healthy donors (provided by UK National Health Service Blood and Transplant Service) using Ficoll Paque Plus (Sigma) according to manufacturer’s instructions. CD4+ T cells were isolated by negative selection using MojoSort Human CD4+ T Cell Isolation kit (Biolegend). Primary CD4+ T cells were maintained in RPMI medium 1640 (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Labtech), 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific), and 10 IU/mL IL-2 (Centre for AIDS Reagents [CFAR], National Institute of Biological Standards and Controls [NIBSC]). HEK293T and HeLa TZM-bl cell lines were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% FCS and 100 U/mL penicillin–streptomycin. Proviral constructs carrying functional nef alleles and nef-defective virus were previously described (16 (link)). Viral stocks were generated by transfecting HEK293T cells using Fugene 6 (Promega). After 48 h, virus was harvested, purified by sucrose gradient ultracentrifugation, and titrated by infectivity assay on HeLa TZM-bl reporter cells using Bright-Glo (Promega).

PBMC sample processing was performed at the same time for each triplet (PwMS at VIS1&VIS2 and HC), to minimize differences within processing. The MojoSort™ Human CD4 T Cell Isolation Kit (#480010, BioLegend, San Diego, CA, USA) was used for CD4⁺ T cell enrichment, in accordance with the manufacturer’s instructions.
To assess whether CD4⁺ T cell enrichment was achieved, flow cytometry was performed in a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) on a subset of samples (before and after magnetic enrichment). The following antibodies were used: CD14-eFluor450 (#48014941, clone 61D3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD19-Alexa Fluor 647 (#302222, clone HIB19, BioLegend, San Diego, CA, USA); CD3-PerCP-Cy5.5 (#45003741, clone OKT3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD4-BV711 (#317439, clone OKT4, BioLegend, San Diego, CA, USA); and CD56-PE-Cy7 (#25056741, clone CMSSB/NCAM, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA).
Regarding the gating strategy, CD4⁺ T cells were defined as CD3⁺CD4⁺CD19^-CD56^- single cells (see Supplementary Figs. S6, 7 online). A detailed protocol and additional details regarding the methodology used for CD4⁺ T cell enrichment and flow cytometry may be found in Sect. 2 of the Supplementary Information.