Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Abstract

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.he immune system uses immune receptors to sense and acquire antigens. Antigen binding induces a series of dynamic changes in the biophysical behaviors and biochemical features of the immune receptors, and these changes determine the fate of a cell (1-3). However, it has been difficult to accurately capture and thus comprehensively investigate these changes because they usually occur very quickly after immune recognition (1, 4). For example, recent live cell imaging studies showed that the B lymphocytes swiftly accumulate the surface-expressed B-cell receptors (BCRs) into the contact interface between the B cells and the antigen-presenting substrates to form a specialized membrane structure, the B-cell immunological synapse (IS) (1, 4). Moreover, both our studies and those of others showed that these accumulation events are sensitive to the biochemical and biophysical features of the antigens that B cells likely encounter in vivo (4, 5). These features include but are not limited to antigen density (6, 7), antigen affinity (6, 7), antigen valency (8-13), the mobility of the antigen (14-17), the stiffness of the substrates presenting the antigen (18, 19) and the mechanical forces delivered to the BCRs by the antigens (20, 21). These facts highlight a long-standing question in immunology: how can the initiation of B-cell activation process the information of antigen s...

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“…These observations were also validated by using primary splenic B cells from IgH B1-8/B1-8 /Ig −/− transgenic mice expressing the 4-hydroxy-3nitrophenyl acetyl (NP)-specific -B1-8 BCR (B1-8 primary B cells) coupled with a photoactivatable caged-NP antigen system ( Fig. 1, J to M, and movie S2) (17).…”

Impairment on the lateral mobility induced by structural changes underlies the functional deficiency of the lupus-associated polymorphism FcγRIIB-T232

“…To directly confirm that high lateral mobility is required for the inhibitory functioning of FcγRIIB, we used a photoactivatable antigen system, caged 4-hydroxy-3-nitrophenylacetyl (NP), which was recently developed for the purpose of setting up a controllable trigger point for the recognition of BCRs and antigen (Wang et al, 2016). In such a system, caged NP can only be converted to antigenic form and thus can be recognized by B1-8-BCR-expressing B cells only after photoactivation by 405-nm photons ( Fig.…”

N-WASP regulates the mobility of the B cell receptor and co-receptors during signaling activation

“…Stone et al showed that the spatial positions of the BCR and Lyn, a signaling kinase, become correlated after antigen stimulation, and this correlation is accompanied by a reduction in diffusivity of both molecules 39 . Using a photoactivatable antigen, Wang et al 40 Inhibition of the Arp2/3 complex, which nucleates branched actin networks downstream of N-WASP, also resulted in an overall reduction of BCR diffusivity. This is consistent with previous work, which has shown that inhibition, or knockout of N-WASP induces higher and more prolonged signaling.…”