Figure 2: GA is internalized and presented on MHC Class II molecules to CD4+ T lymphocytes. Purified splenic B lymphocytes were incubated with anti- IgM-biotin or GA-biotin for the indicated time points, fixed and stained with streptavidin-488 for detection of extracellular IgM or GA followed by fixation and extensive washing. The cells were permeabilized and intracellular IgM or GA was detected using streptavidin-APC. (A) Top panel: Anti-IgM induces the internalization of surface IgM. Shown is the fold increase in extracellular and intracellular geometric mean fluorescent intensity (gMFI) of IgM on wildtype C57Bl/6 splenic B lymphocytes compared to streptavidin conjugates alone. Extracellular and intracellular IgM was used as a positive control for the assay. Bottom panel: GA is internalized and recycled back to the surface of B lymphocytes. Shown is the fold increase in extracellular and intracellular gMFI of GAhi B lymphocytes from wild-type C57Bl/6 mice compared to streptavidin conjugates alone. (B) Top panel: GA is not recycled back to the surface of B lymphocytes in MHC Class II deficient B lymphocytes. Shown is the fold increase in extracellular gMFI of GAhi B lymphocytes from wild-type C57Bl/6, BCR-HEL tg, or MHC Class II KO animals at the indicated time-points. Bottom panel: Intracellular GA accumulates in MHC Class II deficient B lymphocytes. Shown is the fold increase in intracellular gMFI of GAhi B lymphocytes from wildtype, BCR-HEL tg, or MHC Class II KO animals at the indicated time-points. (C) GA is processed and presented by MHC Class II to CD4+ T lymphocytes. Purified B lymphocytes from the indicated mouse strains were incubated with wild-type T cells in the presence or absence of GA, or ovalbumin as a positive control. Culture supernatants were harvested at multiple time-points post incubation and IL-2 was measured using the Mesoscale discovery platform. Shown is IL-2 production 72 hours post addition of the stimulus. Statistical analysis was performed using a one-way ANOVA with Bonferroni post-test.