Antibodies in a heavy chain knock-in mouse exhibit characteristics of early heavy chain rearrangement

Abstract

Studies in autoantibody transgenic mice have demonstrated receptor editing rearrangements at Ab H and L chain loci. However, the physiologic role of H chain editing (V(H) replacement and rearrangement on the second allele) has been called into question. It is unclear if additional rounds of H chain rearrangement are driven by BCR specificity. In this study, we analyze the manner in which B cells undergo additional H chain rearrangements in an anti-DNA H chain knock-in mouse, B6.56R. We find that rearrangements in 56R(+) B cells tend to involve the D gene locus on both alleles and the most J(H)-proximal V(H) gene segments on the endogenous allele. As a result, some B cells exhibit V(D)J rearrangements on both H chain alleles, yet allelic exclusion is tightly maintained in mature 56R B cells. As B cells mature, a higher proportion expresses the nontransgenic H chain allele. Rearrangements on both H chain alleles exhibit junctional diversity consistent with TdT-mediated N-addition, and TdT RNA is expressed exclusively at the pro-B cell stage in B6.56R. Collectively, these findings favor a single, early window of H chain rearrangement in B6.56R that precedes the expression of a functional BCR. B cells that happen to successfully rearrange another H chain may be favored in the periphery.

FIGURE 1. Rearrangements at the H chain loci in B6.56R B cells

A. The 56R H chain replaces the J_H locus and most proximal D gene segment on one allele (top), which contains Cμ of the “a” allotype. The endogenous locus (bottom) is in germline configuration and contains Cμ of the “b” allotype. The 56R transgene consists of a J558 family V_H gene that contains a cryptic recombination sequence in its 3′ end (depicted by a dashed triangle), a D gene, and a J_H4 gene, which can be differentiated from the endogenous J_H4 gene due to two base pair changes (denoted by vertical arrow heads). The transgenic allele also carries a neomycin resistance gene upstream of 56R (denoted by an asterisk). B. Three rearrangements of upstream Ig genes to the cryptic heptamer of 56R. Gene segments are represented as boxes, recombination signal sequences are denoted with triangles (filled triangles have a 23 bp spacer and open triangles have a 12 bp spacer). Rearrangements are denoted by arrows under the corresponding gene segments.

FIGURE 2. IgM expression in 56R B cell populations

A. Bone marrow and spleen cells from a 3.5 month-old 56R mouse and a 4.5 month old B6 mouse were co-stained for IgM^a and IgM^b and markers to differentiate several stages of B cell development. Plots shown are live, B220+ cells. 56R plots are representative of 7 mice from 4 separate experiments. 56R plots are gated for IgM negative, IgM^a low, IgM^a high, and IgM^b+ cells. Percentages are shown within the gated regions. B. The percentage of IgM^a, IgM^a dim, IgM^b and lack of IgM are shown for cells in different B cell subsets. Each stacked bar graph corresponds to the cells of a single mouse. C. The IgM expression profile for 7 individual 56R mice from the gates shown in A. Ages range from 3.5 months to 13 months. The dashed line represents the average frequency of IgM^b cells within each B cell subset for the seven mice.

FIGURE 3. Rearrangements on the 56R allele in IgM^b+ hybridomas

A. 38 IgM^b secreting hybridomas were screened for 56R DNA and rearrangements of upstream V_H and D genes to 56R using a degenerate V_H primer (MH1) that recognizes approximately 90% of V_H genes, a primer that recognizes a sequence upstream of several D genes, a 56R specific J_H primer, and the 56R genotyping primers (see materials and methods for details). The number of hybridomas testing positive for each type of rearrangement is shown to the right. Arrows denote the PCR primers. B. Sequences of V_H rearrangements on the 56R allele from IgM^b+ hybridomas. All sequences are out-of-frame (OF).

FIGURE 4. Rearrangement of the endogenous allele in 56R+ cells

A. 56R+ and IgM^b+ hybridomas were screened for endogenous H chain rearrangements by PCR with a degenerate V_H primer that recognizes 90% of V_H genes (MH1) and multiple J_H specific oligos (see materials and methods). V_H and D genes present in each sequence are indicated. B. Rearrangements of 7183.2.3, 7183.9.15, and J606 to J_H2 (endogenous allele) were PCR amplified from IgM^a+ and IgM^b+ sorted B cells using V_H specific primers and a FAM labeled J_H2 primer. PCR products were analyzed by capillary electrophoresis and the number of products per 1,000 cells was calculated. Representative electropherograms for each PCR are shown in Supplemental Figure 2 and the numerical data are provided in supplementary table 1. RF= reading frame, IF- in-frame, OF= out-of-frame

FIGURE 5. TdT protein and RNA expression in B cell subsets from B6.56R

A. TdT protein levels were detected in 56R and B6 lymphocytes by intracellular staining. Bone marrow from one three month old 56R mouse and an age-matched B6 control was resolved into Fractions B-C′, D, E, and F (see materials and methods). Data are representative of 9 B6 and 8 B6.56R mice analyzed in 7 individual experiments. Splenic B cells from one 5 month-old 56R mouse and an age-matched B6 control were resolved into transitional, marginal zone, and follicular subsets based on the expression of CD93, IgM, CD21 and CD23 (as shown in Supplemental fig. 3A). Data are representative of 3 B6 and 2 B6.56R mice analyzed in 3 individual experiments (see Supplemental fig. 3B for additional data). thick black line-no primary antibody, solid gray-B6, thin black line-56R, IgM^a+, red line-56R, IgM^b+. B. Expression of TdT mRNA was determined by quantitative RT-PCR in cells sorted from B6 and B6.56R mice; expression levels are shown relative to expression in thymus (dashed horizontal line) and normalized to β-actin expression. Two mice were analyzed in the same experiment. Error bars indicate SD. Additional TdT data are shown in Supplemental Fig. 4. C. Rag1 RNA levels in B6 and 56R B cell subsets. Rag1 RNA transcript abundance in B cells sorted from B6 and B6.56R mice is shown relative to abundance in thymus (dashed horizontal line), normalized to β-actin expression. B6 and B6.56R data are combined for all subsets because no significant difference was found between these mice. The number of mice analyzed for each subset are: D (56R n= 3; B6 n =3), E (56R n = 2; B6 n = 1), F (56R n = 4; B6 n = 3), spleen CD93+IgM+ (56R n = 9; B6 n = 5), spleen CD93+IgM− (56R n = 2; B6 n = 1), spleen CD93− (56R n = 3; B6 n = 3).