Supplementary Figure 6: Themis2 supports the positive selection of germinal center B cells and determines the threshold for activation by membrane bound antigen.
From: Themis2 lowers the threshold for B cell activation during positive selection
(a) CD45.1 wild-type and CD45.2 wild-type or Themis2-/- IgHEL B cells were transferred into B6 mice 1 day before IP immunization with SRBCs conjugated to HEL. Percentage of wild-type and Themis2-/- CD45.2 IgHEL B cells at input and 8 days after immunization with SRBC-HEL, where columns show means and s.e.m. and comparison by unpaired t test, **P<0.01. Symbols represent individual mice at 8 days or the separate input samples. Data were combined from 3 experiments. (b) Scheme illustrating the approach to test the activation of WT and Themis2-/- IgHEL B cells by membrane bound antigens of varying affinity and abundance. pBMN retrovirus expressing mHEL variants linked to 3 tandem intracellular GFP molecules was stably transfected into NIH3T3 cells with or without an ICAM-1 construct and incubated overnight with CD45.2 and CD45.1 allotype-marked WT:WT or WT:Themis2-/- IgHEL B cells. (c) Percentage of IgHEL B cells activated by overnight incubation with NIH3T3 cell clones expressing a constant level of ICAM-1 and different levels of mHEL (2 clones), and variants mHEL2X and mHEL3X (2 clones each). Data are representative of three separate experiments with different clones. Symbols represent individual samples and bars means.
