Harrison (Harvard Medical School), H. elicited, as is the case for HIV-1 and influenza. The average affinity of specific antibodies increases dramatically over the course of an immune response (1, 2). This phenomenon is known as sequencing (Scale bar, 500 m). (G) Quantification of clonal dominance in multiple GCs. Data obtained as in (F), with clonal identity assigned based on sequence. Each symbol represents one GC, with 2 GCs sequenced per LN (full clonality charts in fig. S2). Given is the percentage of cells belonging to the most abundant clone. Data are from 5 mice, 3 independent experiments. Quantification of cell colors in GC dark zones at 15 days SKQ1 Bromide (Visomitin) after immunization (Fig. 1D) revealed a wide range of color dominances (from 20.2% to 89.7%; median, 44.0%), and a relative paucity of predominantly single-colored GCs (6 of 40 (15%) GCs with color dominance > 70%; Fig. 1E). To validate these estimates, we used in-situ photoactivation followed by FACS sorting (9, 18) to obtain single B cells from individual GCs, the clonal identity of which we then determined by mRNA sequencing (Fig. 1F). This approach again showed varying degrees of clonal dominance among individual GCs (3/12 (25%) GCs with dominance >70%; SKQ1 Bromide (Visomitin) range, 22.5% to 87.5%; median, 43.0%; Fig. 1G and fig. S2). It also revealed the frequent presence of clones that were shared between two individual GCs in the same LN, indicative of synchronous origin (colored slices in fig. S2). We conclude that GCs display variable degrees clonal Rabbit Polyclonal to MAEA dominance, even when induced synchronously by immunization. While predominantly monoclonal GCs do exist, these are relatively rare at the time point assayed. Early GCs are highly diverse The diversity of clonal dominance levels among mature GCs led us to question the generalizability of reports proposing that GCs are seeded pauciclonally (11, 12, 19). To address this, we generated mice expressing the photoactivatable-GFP transgene along with Cre recombinase driven by the endogenous locus (sequencing (Fig. 2A and fig. S3A). Early GCs were highly and uniformly polyclonal, with 23 to 46 unique VDJ rearrangements detected when sequencing 34 to 77 single cells per GC (Fig. 2B). Extrapolation of these numbers using the Chao1 and ACE estimators of species richness (21) gave rough estimates of GC clonality that ranged from ~50 to ~200 clones per GC (median Chao1, 102 clones) for the 4 pairs of GCs analyzed (Fig. 2C). Overall mutation and class switching levels were low at this time point (fig. S3B), confirming that these were indeed early GCs. We were again able to SKQ1 Bromide (Visomitin) identify B cell clones that were shared between two GCs in the same pLN (colored slices, Fig. 2B; mean 15.8% (SD 6.4) of clones found in one GC were also found in its neighbor). Similar experiments using other antigens showed that, although the extent of early GC diversity can vary depending on the immunizing antigen, high diversity is not SKQ1 Bromide (Visomitin) a unique feature of the response to CGG (Fig. 2D and fig. S3CCE). Diversity was also not the result of non-specific recruitment of B cells to early GCs (fig. S3C and fig S4). We conclude that early GCs are highly diverse, containing tens to hundreds of distinct clones, depending on the antigen used for immunization. The progression from uniform diversity SKQ1 Bromide (Visomitin) (Fig. 2) to variable degrees of clonal dominance (Fig. 1) in the CGG response implies that individual GCs display different rates of homogenizing selection acting subsequently to this coalescence. Open in a separate window Figure 2 Clonal diversity in early GCs(A) Photoactivation of single early GC clusters. photo activatable-GFP-transgenic. AicdaCre. Rosa26lox-stop-lox-tdTomato mice were immunized in the footpad with 10 g CGG in alum and imaged 6 days later. FDC networks were marked by injection of labeled antibody to CD35. Left panels show images of a single tdTomato+ cluster (arrowheads) within a pLN prior to and after photoactivation (Scale bar, 200 m). Right panels show dissection of a single pLN with two photoactivated GCs (arrowheads) into two fragments, each of which is separately processed for cell sorting (Scale bar, 500 m). (B) Pie charts showing clonal diversity in early GCs. Each slice represents one clone. Colored slices represent clones that were found in both GCs (upper and lower pie charts) from the same pLN. Numbers in the center of each chart are (number of clones observed/total number of cells sequenced). Clonal identity was assigned based on sequence. Pairs are from 4 different mice in 3 independent experiments. (C) Estimation of total clonal richness in.