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. 2020 Nov 4;39(24):e105594. doi: 10.15252/embj.2020105594

Figure EV1. Mouse model to study immunomodulatory properties of NEF on antigen‐specific interaction between B cells and T cells.

Figure EV1

  1. TCR‐MHC class II crosslinking by superantigen (SEB) initiates signaling exclusively on the T‐cell side, precluding investigation of B‐cell responses upon IS formation.
  2. Presentation of endogenously processed cognate peptide (OVA323–339) on B‐cell MHC class II to CD4OT‐II cells allows bidirectional signaling in B and T cells enabling analysis of B‐cell downstream functions.
  3. Retrovirally transduced CD4OT‐II cells were co‐cultured with BHEL cells pulsed with HEL‐OVA in the presence of LPS or adoptively transferred into recipient mice. HEL‐OVA is internalized via BCR‐mediated phagocytosis, processed and presented on MHC class II of B cells. This results in the establishment of an Ag‐specific IS between B cells and transduced/NEF‐expressing CD4OT‐II cells. Upon IS formation, B‐cell signaling is initiated resulting in B‐cell activation, proliferation, and differentiation. This model allows investigation of initial events modulated by NEF during B‐cell activation. For analyzing the effect of NEF on antibody production, SMARTA mice (T cells with TCR specific to irrelevant Ag) were used as recipients for HEL‐OVA/CFA immunization. HEL‐specific IgM and IgG ELISA was performed on serum samples extracted from blood collected on indicated days.