Adoptive Transfer of B and CD4+ T Cells

B cells and CD4⁺ T cells were isolated from nonexposed or KEL^lo RBC–exposed C57BL/6 recipients. To isolate each population, splenocytes from each group were harvested by sterile mechanical disruption of the spleen and lysis of RBCs. B cells or CD4⁺ T cells were then negatively enriched using a mouse Pan B Cell Isolation Kit II or a mouse CD4⁺ T Cell Isolation Kit (both from Miltenyi Biotec). CD4⁺ T cells or B cells were adoptively transferred into C57BL/6 mice, as done previously (10⁷ CD4⁺ T cells or B cells into C57BL/6 recipients or 5 × 10⁷ B cells into B cell–deficient μMT mice) (25 (link)–30 (link)), followed by KEL RBC transfusion. Following transfer, all recipients were challenged with KEL RBCs. For cellular transfer into μMT mice, recipients were conditioned with gamma radiation treatment (300 rad) 24 h prior to B cell transfer, as done previously (31 (link)).