2). The early responding C12Id+ HA-specific B cells were also not significantly different with regard to expression of CD24, one of the markers identified by Linton et al. 41 to correspond with extrafollicular foci development, although we measured CD24 expression with an anti-CD24 mAb different from that used by that earlier study (data not shown). Thus, LN seem to lack a specific resident B-cell subpopulation comparable to the MZ B-cell population in the spleen that can facilitate rapid responses to early blood-borne infection. Instead,

follicular (C12Id+ HA-specific) B cells provide this rapid, Ibrutinib price strong (Fig. 1) and protective 2 extrafollicular B-cell response (Fig. 3). Various models have been proposed to explain the regulation of extra- versus intra-follicular B-cell responses 22, 26, 39–41, 46. The influenza virus model system described here and available tools to study the C12Id-specific responses provide an excellent opportunity to further analyze this important differentiation process in vivo in

the context of an infection. Our study identifies C12Id-expressing HA-specific B cells as predominant contributors to a strong extrafollicular B-cell response in regional MedLN, the site of much of the early Ab response to this virus. Predominant R428 in vitro participation of the C12Id-expressing HA-specific cells in extrafollicular responses is consistent with earlier findings that failed to find any mutated C12Id-sequences among HA-specific B cells 27, i.e. these cells showed no signs of affinity maturation. However, we show here that while C12Id+ cells vigorously participate in extrafollicular foci responses (Fig. 3), they can also initiate

germinal centers (Fig. 4). While we cannot completely rule out that C12Id+ B cells that mutated their BCR might have lost the idiotope that allowed us to stain these cells for FACS analysis, Cell press the earlier extensive sequence studies on B cells from mice at various times after immunization (26), make this trivial explanation somewhat unlikely. Given the recent studies by Paus et al. 22 that implicated BCR-affinity for antigen in the selection process, our studies might suggest that high-affinity interactions with antigen are a necessary, but likely not sufficient, signal for extrafollicular foci development as C12Id+ HA-specific cells are able to also initiate germinal centers (Figs. 3 and 4). Failure to expand this germinal center response during early infection, rather than an inability to initiate it, might result in the irregular kinetics and small frequencies of C12Id+ germinal center B cells observed here (Fig. 4). This interpretation is not only consistent with the presented data, but also with earlier studies using the (4-hydroxy-3-nitrophenyl) acetyl system, which demonstrated that extrafollicular foci and germinal center B cells can have a common precursor 25, 26, 39. The vigorous infection-induced extrafollicular foci response is likely supported and modulated by external signals.