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. 2016 Nov 14;213(12):2707–2727. doi: 10.1084/jem.20160528

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© 2016 Xu et al.

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Figure 5. — FcγRIIB-T232 restores the inhibitory function if sufficient responding time is given for FcγRIIB-T232 to interact with the ICs in a photoactivatable antigen system. (A) A schematic diagram depicting the PLBs presenting the surrogate ICs composed of biotin-conjugated caged NP and biotin-conjugated anti-FcγRIIB antibody (AT10) and the action of using 405-nm photos to trigger the interaction between uncaged NP and B1-8-BCRs. (B) A schematic diagram depicting the experimental procedure of using a caged NP–based photoactivatable antigen system to provide sufficient responding time for FcγRIIB-T232 to interact with the ICs. Cells were preincubated on PLBs presenting surrogate ICs for 180 s before the 3-s photoactivation by a 405-nm laser. The accumulation of B1-8-IgM-BCRs into the B cell immunological synapse was examined for 300 s by TIRF microscopy imaging after photoactivation. (C) Statistical quantification of the normalized mean fluorescence intensity to show the synaptic accumulation of B1-8-IgM-BCRs after photoactivation. Error bars represent mean ± SEM of 22 cells from one representative of three independent experiments. (D) Similarly as in B, a 60-s responding time was given. (E) Similarly as in C, statistical quantification is given for the experiments in D. Error bars represent mean ± SEM of 20 cells from one representative of three independent experiments.