Figure 5. Monovalent antigen controls IgD BCR activation.

• A
  Schematic overview of reconstitution of BCR‐deficient Ramos (HL‐KO) cells with murine HEL‐specific BCR of IgM (HH10‐mu IgM) or IgD (HH10‐mu IgD) isotype, respectively.
• B
  Representative flow cytometric analysis of Ramos cells reconstituted with HH10‐mu IgM or HH10‐mu IgD, respectively. Cells were stained with α‐murine μHC, δHC, and κLC antibodies, respectively. EV‐transduced HL‐KO cells, expressing GFP only, were used as negative control (gray).
• C
  Representative flow cytometric analysis of HEL‐binding in reconstituted Ramos cells after staining with fluorescently labeled HEL. EV‐transduced HL‐KO cells, expressing GFP only, were used as negative control (gray).
• D
  Representative intracellular Ca²⁺ influx in HH10‐mu IgM‐ and HH10‐mu IgD‐expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL; both at a concentration of 1 μg/ml), or 10 μg/ml α‐mouse κLC antibody, respectively.
• E
  Representative intracellular Ca²⁺ influx of HH10‐mu IgM‐ (top) and HH10‐mu IgD‐ (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1:25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml.
• F
  Schematic overview of reconstitution of BCR‐deficient Ramos (HL‐KO) cells with HH10‐specific human IgM (HH10‐hu IgM) or IgD (HH10‐hu IgD) BCR isotype, respectively.
• G–J
  Identical to Fig 5B–E using HH10‐hu IgM‐ and HH10‐hu IgD‐expressing cells instead of HH10‐mu IgM‐ and HH10‐mu IgD‐expressing cells, respectively. Data shown in B–E and G–J are representative of at least three independent experiments.