Tonsillar Tfh and Peripheral Tph Cells Co-Culture

Tonsillar follicular helper T cells (Tfh, CD19⁻CD4⁺CD45RA⁻CXCR5^high) from patients who underwent routine tonsillectomy or peripheral memory T helper cells from bronchoalveolar lavage (BAL) of sarcoidosis patients (mostly peripheral helper T cells, Tph, CD19⁻CD4⁺CD45RA⁻) were sorted on an ARIA II flow cytometry sorter (Becton Dickinson). Patient samples were obtained from the Unfallkrankenhaus Marzahn (tonsils) or the Charité Universitätsmedizin Berlin (BAL). Sorted T cells were co-cultured for 7 days with heterologous tonsillar memory B cells (CD19⁺CD4^−IgD⁻CD38⁻) at a 1:1 ratio in the presence of 4 ng/ml staphylococcal enterotoxin B (Toxin Technology) as described previously^{36 (link)}. To block T cell help, 20 μg/ml anti-CD40L antibody (clone TRAP1) and/or 10 μg/ml recombinant soluble IL-21 receptor (R&D Systems, Cat. 9249-R2) were added to the culture. Cells were acquired on an LSR II Fortessa flow cytometer (Becton Dickinson) and analysed using FlowJo version 10 software (Tree Star Inc.).
All antibodies utilised in the study are listed in Supplementary Table 3 for reference.