B cells use B-cell receptors (BCRs) to sense antigens that are usually presented on substrates with different stiffness. However, it is not known how substrate stiffness affects B-cell proliferation, class switch, and in vivo antibody responses. We addressed these questions using polydimethylsiloxane (PDMS) substrates with different stiffness (20 or 1100 kPa). Live cell imaging experiments suggested that antigens on stiffer substrates more efficiently trigger the synaptic accumulation of BCR and phospho-Syk molecules compared with antigens on softer substrates. In vitro expansion of mouse primary B cells shows different preferences for substrate stiffness when stimulated by different expansion stimuli. LPS equally drives B-cell proliferation on stiffer or softer substrates. Anti-CD40 antibodies enhance B-cell proliferation on stiffer substrates, while antigens enhance B-cell proliferation on softer substrates through a mechanism involving the enhanced phosphorylation of PI3K, Akt, and FoxO1. In vitro class switch differentiation of B cells prefers softer substrates. Lastly, NP67-Ficoll on softer substrates accounted for an enhanced antibody response in vivo. Thus, substrate stiffness regulates B-cell activation, proliferation, class switch, and T cell independent antibody responses in vivo, suggesting its broad application in manipulating the fate of B cells in vitro and in vivo. Eur. J. Immunol. 2015Immunol. . 45: 1621Immunol. -1634 Ig (mIg) and a heterodimer of Igα and Igβ in a 1 mIg:1 Igα-Igβ heterodimer stoichiometry [1,2]. Upon BCR engagement, the B cell initiates its activation that is followed by proliferation and differentiation into plasma cells and memory B cells [3]. It is appreciated that the antigens that encounter with B cells in vivo show a various degree of diversity based on their presentation forms, including antigen density [4,5], antigen affinity [4,5], antigen valency [6][7][8], and antigen mobility [9]. The early studies of ours and others showed that the initiation of B-cell activation is sensitive to antigen presentation forms, suggesting the sophisticated capability of B cells to sense the biophysical features of the antigens. For a long time, it has been neglected that the antigens encountered by B cells in vivo are actually presented on substrates with various stiffness features [10]. Stiffness usually represents the rigidity of an object, showing the extent that such an object resists deformation changes in response to an applied force. Young's modulus is usually used to describe the stiffness of an elastic substrate. For instance, the Young's modulus value of different virus particles is about 45-1000 MPa [11], while most of the mammalian cells show medium stiffness features from 0.01 to 1000 kPa [12]. Secreted soluble pathogen antigens in our plasma only exhibit very low stiffness of no more than 100 Pa [13]. Thus, substrates presenting the antigens on rigid virus capsid vesicles exhibit high stiffness features; those on the membrane of the virus-infected host cells show medium le...

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