Cd19 alexa fluor 647

Single-cell suspensions from spleens, bone marrow, or peripheral lymph nodes were blocked for 30 min using FBS. Cell-surface staining was achieved by incubating cells at 4°C for 30 min with fluorescence-conjugated anti–mouse antibodies (clone; source): XBP1s–Alexa Fluor 647 (Q3-695; BD), XBP1s-phycoerythrin (PE; Q3-695; BD), B220–Alexa Fluor 488 (RA3-6B2; BioLegend), B220-BV605 (RA3-6B2; BioLegend), CD43-PE (eBioR2/60; eBioscience), CD19–Alexa Fluor 647 (6D5; BioLegend), IgM-PE-Cy7 (RMM-1; BioLegend), IgD-FITC (11-26c.2a; BioLegend), GL7-PE (GL7; BioLegend), AA4.1-PE-Cy7 (AA4.1; BioLegend), CD1d-PerCP-Cy5.5 (1B1; BioLegend), CD23-FITC (B3B4; BioLegend), CD3-APC-Cy7 (145-2C11; BioLegend), CD4-BV605 (RM4-5; BioLegend), CD8α-PE-Cy7 (53–6.7; BioLegend), and CD138-PE (281–2; BioLegend). Viability staining was accomplished using DAPI exclusion during acquisition. Acquisition of B, T, and dendritic cell populations was performed on an LSRII cytometer (BD) harboring a custom configuration for the Wistar Institute. Cytometry data were analyzed using FlowJo software (7.6.1; Tree Star Inc.).

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.

Peritoneal cells were subjected to flow cytometry analysis using the antibodies CD19-Alexa Fluor 647 (rat anti-mouse IgG2a, clone: 6D5; BioLegend, San Diego, California, USA), CD5-PerCP (rat anti-mouse IgG2a, clone: 53–7.3; BD Pharmingen, San Jose, California, USA), and CD11b-PE (rat anti-mouse IgG2b, clone: M1/70; BD Pharmingen, San Jose, California, USA). Measurement was made using a CyAn ADP flow cytometer (Dako, Glostrup, Denmark). B-1a cells were characterized as CD19⁺CD5⁺CD11b⁺, B-1b cells as CD19⁺CD5^-CD11b⁺, and B-2 cells as CD19⁺CD5^-CD11b^-, using the CyAn ADP flow cytometer and Summit software version 4.3 (Dako, Glostrup, Denmark). Rat IgG2a FITC-conjugated isotype control (Pierce, Thermo Scientific, USA) and rat IgG2b FITC-conjugated isotype control (Pierce, Thermo Scientific, USA) were used as the isotype controls.