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. Author manuscript; available in PMC: 2014 Aug 1.
Published in final edited form as: J Immunol. 2013 Jul 3;191(3):1260–1275. doi: 10.4049/jimmunol.1300770

Figure 2. Analysis of B-cell development and serum Ig subclass levels in C57BL/6 (WT), 4E10 VH+/+ and 48d VH+/+ KI strains.

Figure 2

(A-C). Representative FACS contour histograms (representative of three experiments) comparing BM, splenic, and peritoneal B-cell development in 4E10 VH+/+ and 48d VH+/+ KI mice (expressing the somatically mutated H chains of the polyreactive, MPER-specific bnAb 4E10 and the non-neutralizing HIV-1 mAb 48d, respectively). Also shown as controls are WT (C57BL/6) littermates; all mice were 8-12 wk females. A. Analysis of BM B-cell subsets, either less differentiated subpopulations (upper panels), based on Hardy subfractionation, more differentiated subpopulations (middle panels), as revealed by surface IgM and IgD staining as previously described (32), or based on surface L chain κ and λ1-3 expression (lower panels). Indicated B-cell subsets were pre-gated as singlet, live lymphocytes (upper panels), or as singlet, live, total (CD19+B220+) B-cells (middle and lower panels), respectively. Numbers indicate percentages of B-cells in each subset, with subsets defined as follows: pro/pre=pro-B/large pre-B (fractions A-C’; B220loCD43+), small pre=small pre-B (fraction D; B220loCD43−), imm/mat=mature+immature (fractions E-F; B220hiCD43−); imm=immature (IgMint/hiIgD−), T1/T2=transitional 1+2 (IgMhiIgDlo/int), and mat=mature (IgMintIgDhi). B. Analysis of splenic B-cell subpopulations, as revealed either by surface CD93 and CD23 expression of live lymphocytes (top row), and by surface CD21 and CD23 or IgM and IgD expression of live, total (CD19+B220+) B-cells (middle and lower rows, respectively). Numbers indicate percentages of B-cells in each gate, and B-cell subsets indicated in upper panels are defined as follows: T=transitional (B220+CD93+), Mat+MZ (B220+CD93−), whereas those indicated in the middle row are defined as: NF=newly formed i.e. transitional (CD21−CD23−), MZ=marginal zone (CD23intCD21hi), and Mat=mature follicular B2 (CD23hiCD21int). C. Analysis of peritoneal B-cell subsets as revealed by surface staining of live lymphocytes with B220 and CD5 with numbers in each gate indicated, and B-cell subsets are defined as follows: B1a (B220intCD5int), B1b (B220intCD5−), B2 (B220+CD5−). D. Analysis of total Ig subclass levels in 4E10 VH+/+ and 48d VH+/+ KI mice, or WT (C57BL/6) littermates. Serum samples were collected from 6–12 wk female naïve mice, and serum concentrations for all Ig subclasses were determined by ELISA analysis. Each symbol represents an individual mouse and horizontal lines represent averages for each group. Significance values were determined using a two-tailed student test. NS: P>0.05, *P≤0.05, **P ≤0.01, ***p ≤0.001.