Anti human igm

For co-cultures of in vitro polarized T cells (Fig. 4f–h and Extended Data Fig. 5g–i). CD4 T cells were purified using MACS (Miltenyi Biotec) from healthy donor PBMCs and cultured in the presence of plate-bound anti-CD3 (OKT3, 10 μg ml⁻¹) and anti-CD28 (CD28.2, 5 μg ml⁻¹) antibodies in complete RPMI alone (unstimulated control) or with (1) IL-2 (50 ng ml⁻¹), IL-12 (5 ng ml⁻¹) and anti-IL-4 (1 μg ml⁻¹, R&D MAB204-SP) (T_H1 conditions); (2) IL-6 (50 ng ml⁻¹) with or without IL-2 (50 ng ml⁻¹); or (3) plasma from HC individuals or individuals with DS (1:2 ratio). After 4 days, T cells were washed and irradiated (5,000 rad) and co-cultured with freshly MACS-sorted naive B cells from the same donor. Cells were co-cultured for 3–6 days in complete RPMI supplemented with IL-21 (50 ng ml⁻¹), IL-2 (50 ng ml⁻¹) and anti-human IgM (5 μg ml⁻¹, Southern Biotech) and collected for flow cytometry. The supernatants were collected for IFNγ ELISA quantification (BioLegend) according to the manufacturer’s instructions.
For T cell–B cell co-cultures from HC donors and donors with DS (Fig. 4i,j), CD4 T cells were MACS-purified and co-incubated with sort-purified CD19⁺CD38⁻CD27⁻CD11c⁻ B cells on the same day. B and T cells were co-cultured for 3 days in complete RPMI supplemented with IL-21, IL-2 and anti-human IgM as above and collected for flow cytometry.