Ova peptide 323 339

Primary cells were isolated by mechanical disruption of cell suspensions of spleen (and also lymph nodes in the case of CD4⁺ T‐cell isolation). B cells from DICER‐KO and C57/BL6 wild‐type mice were isolated by immunomagnetic depletion with anti‐CD43 beads (Miltenyi). During the 24 h before co‐culture, isolated B cells were cultured in EV‐depleted complete RPMI medium [EV‐free RPMI supplemented with 10% EV‐depleted fetal bovine serum (discarding the pellet after ultracentrifugation for 16 h at 100,000 g), 50 μM β‐mercaptoethanol (Invitrogen), 10 mM Hepes (Invitrogen), and antibiotics]. The same batch of ultracentrifuged EV‐depleted serum was used for all experiments to minimize variability and checked by NanoSight for EV depletion. For B‐cell pre‐activation, this medium was supplemented either with 25 μg/ml LPS (Sigma) and 10 ng/ml IL‐4 (Peprotech) or with 10 μg/ml CD40 and 10 μg/ml F(ab)’2 fragments of goat anti‐mouse IgM (Jackson Immunoresearch). Regardless of the activation method, the B cells were incubated in the presence or absence of 5 μg/ml OVA peptide 323–339 (GenScript). For OT‐II‐derived CD4⁺ T‐cell isolation, splenocytes and lymph node cells were incubated for 16 h in EXO‐free RPMI medium supplemented with 5 μg/ml of OVA, and T cells were then purified using the CD4⁺ T‐cell isolation kit (Miltenyi Biotec). Antibodies and primers are listed in Appendix Table S2.

CD4⁺ T cells were isolated from the spleens of C57BL/6J as described and stimulated with the indicated concentrations of anti-CD3ε and 1 μg/ml of anti-CD28 (BD Pharmingen) for 48 h. Cells were analyzed by flow cytometry and IL-2 was detected in the supernatants by an ELISA. For co-culture experiments, empty vector Chinese Hamster Ovary – Antigen-presenting cells (CHO-APCs) were fixed by treating with Mitomycin C (Sigma) 37°C for 45 min 6 × 10⁵ cells/ml of CHO-APCs were pulsed with the indicated concentrations of ova-peptide 323–339 (Genscript). CD4⁺ T cells were isolated from spleens of BALB/c-Tg(DO11.10)10Loh/J and mixed with peptide pulsed APCs at 2.5 × 10⁶ cells/ml in each well of a 12-well plate. Cells were harvested after 48 h and analyzed by flow cytometry after gating on CD4⁺ T cells expressing the DO.11.10-TCR. Supernatants were used for detection of IL-2 by an ELISA.