B220 bv786

Donor WT SW_HEL or FAS-deficient SW_HEL spleen cells were enriched with MACS B cell isolation kit (Miltenyi Biotec) and separated using an AutoMACS Pro separator. Recovered cells were identified as HEL-binding B cells after being stained with saturating levels of HEL (Sigma) at 200ng/ml followed by HyHEL9-Alexa Fluor 647 and B220 BV786 (BD Biosciences). 2 ×10⁵ donor OT-II Pik3cd GOF or OT-II WT spleen cells were injected i.v. together with 2 ×10⁵ WT SW_HEL or FAS-deficient SW_HEL B cells into CD45.1⁺ SAP KO recipient mice. On the following day of the transfer, recipient mice were immunised sub-cutaneously with 20 g HEL-OVA in Sigma Adjuvant System (SAS; Sigma) in the lower flank and base of the tail. Inguinal lymph nodes were harvested at day 7.

Spleens were mechanically disrupted, single-cell suspensions were generated, and red blood cells were lysed with ammonium chloride TRIS. Cells were resuspended in PBS containing 1% FBS and incubated with indicated antibodies. For analysis of cell surface markers, antibodies against the following molecules were used: B220-PE (RA3–6B2; BioLegend), B220-BV786 (RA3–6B2; BD Biosciences), B220-BV510 (RA3–6B2; BioLegend), CD4-BV711 (GK1.5; BioLegend), CD8 BV421 (53–6.7; BioLegend), CD138-PECy7 (281–2; BioLegend), CD69-BV786 (H1.2F3; BD Biosciences), CD86-PerCPCy5.5 (GL-1; BioLegend). After cell surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD) (as per manufacturer’s instructions) and stained with Dylight650-E4 anti-Ars/A1 idiotype (produced and conjugated in our laboratory) OR HEL-650 (produced and conjugated in our laboratory) and Alexa Fluor 488-anti-GFP (rabbit polyclonal; Life Technologies). Events were collected on a CyAn ADP (Dako) and subsequent analysis using FlowJo software (Tree Star).

Bone marrow, peripheral blood, and splenic B cells from cCD79 and wild type mice were analyzed using the following antibodies: hCD79A-PE (R&D Systems, FAB69201P); hCD79B-PE (abcam, ab33295); mCD79B-AF488 ([HM79], made in house); hCD79A-AF647 ([Curly-14], made in house); B220-BV786 (BD, 563894); B220-APC (BD, 103212); B220-PE (BD, 561878); CD43 ACP-Cy7 (BD, 562866); BP1-PE (BD, 553735); CD24-APC (BioLegend, 101814); IgD-FITC (BioLegend, 405704); IgM-BUV396 (BD, 564025); CD19-FITC (BioLegend, 152404); CD93-APC (BioLegend, 136510); CD23-FITC (BD, 561772); CD1d-PE (BD, 553846); CD95-PE (BD, 554258); GL7-FITC (BD, 553666); fab anti-mIgG (H+L)-AF647 (Jackson ImmunoResearch, 115–606-072); phospho-Syk (Y352)-PE (BD, 557881); PTEN-PE (BD, 560002); mouse IgG1 isotype control-PE (BD, 559320).