Cd56 pe cy7

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.

Analysis of CD80 or CD86 expression on monocytes was done with CD14-APC (BD, 555399), CD80-PE (BD, 557227), or CD86-PE (BD, 555665). For detection of CD25, CD69, and TRAIL on NK cells, NK cells were identified as CD56⁺/CD3⁻. The following stainings were performed: CD25-PerCP-Cy5.5 (Biolegend, San Diego, CA, USA, 356112), CD56-APC (Miltenyi Biotec, 130-090-843), and CD3-FITC (BD, 555332). CD69-APC (BD, 555533), CD56-Pe-Cy7 (eBioscience, 25-0567-42), and CD3-FITC (BD, 555332). TRAIL-PE (BD, 550516), CD56-APC (Miltenyi Biotec, 130-090-843), and CD3-APC-Cy7 (BD, 557832). Samples were measured on a BD FACSCanto II with BD FACSDIVA 8 software.