**2. The Molecular Events That Mediate B-Cell Activation and the Role of Multivalency**

The activation of B cells and the subsequent downstream events that result in antibody production are consequences of the initial interaction between an antigen and the BCR. The signaling events initiated by this interaction, which are the subject of many excellent reviews [7–9], stimulate B cell proliferation and upregulate MHC Class II and the costimulatory molecules that permit subsequent interactions with T helper cells. B-cell activation is a quantitative phenomenon, in which the degree of activation is dependent on both the affinity of the BCR for its cognate antigen [10,11] and the valency of the antigen. The critical role of antigen valency in B-cell responses was first recognized by Renee and Howard Dintzis at the Johns Hopkins School of Medicine [12–14], who assessed the immunogenicity of a T-cell independent antigen consisting of a polymer (polyacrylamide) displaying a model hapten (dinitrophenol; DNP). The modular nature of this system allowed the dissection of antibody responses as a function of the valency and density of DNP display. They concluded that " ... the fundamental molecular event in the induction of the primary immune response is the linking together by a single antigen molecule of a critical number of separate hapten receptors into a molecularly connected entity", which they termed an immunon [12]. Numerous subsequent studies established this relationship between antigen valency and B-cell responsiveness, particularly in the context of T-cell-independent antibody responses [15,16].

The series of events that begins with the activation of naïve B cells by a T-cell-dependent antigen and ultimately result in differentiation to LLPCs are more complicated than what occurs with a T-cell independent antigen, yet antigen valency and density also play an important role in this process. Multivalent interactions promote BCR clustering and the formation of lipid rafts [17–20]. These, in turn, promote signaling to the B cell and receptor-mediated internalization of the antigen complex [21], steps critical for B cells to present antigen on MHC Class II and receive help from CD4 T cells. Accordingly, multivalency enhances BCR clustering [22], BCR/antigen internalization and antigen presentation [23], as well as the upregulation of costimulatory molecules that are important for subsequent interactions with T helper cells [24]. Thus, these multivalent interactions have a profound effect on early steps in B-cell activation and ultimately influence antibody production and other downstream events.

While the influence of multivalency on the early steps in B-cell activation have been extensively studied, less is known about how these events influence the establishment of germinal centers (GCs) and production of LLPCs. GCs are discrete anatomical sites within B-cell follicles in which B cells proliferate and undergo somatic hypermutation and affinity maturation. It is here that they differentiate

to memory cells and LLPCs. In GCs, B cells compete for binding with antigens displayed on follicular dendritic cells, and then present antigens to follicular T helper cells, which in turn provide survival signals to the B cell. Although this process does not require a multivalent antigen, enhanced B-cell crosslinking in GCs leads to increased GC B-cell proliferation and promotes differentiation to plasma cells, particularly in scenarios in which T help is limiting [25]. Thus, it is likely that multivalency can exert its stimulatory effects at multiple steps during the B-cell activation and differentiation process.
