Extended duration of DH-JH rearrangement in immunoglobulin heavy chain transgenic mice: implications for regulation of allelic exclusion

Abstract

Here we show that suppression of VH-DJH rearrangement in mice bearing a mu heavy (H) chain transgene (mu-tg mice) is associated with an extended period of DH-JH rearrangement, the first step of Immunoglobulin H chain gene rearrangement. Whereas DH-JH rearrangement is normally initiated and completed at the pro-B cell stage, in mu-tg mice it continues beyond this stage and occurs most frequently at the small (late) pre-B stage. Despite ongoing DH-JH rearrangement in late pre-B cells of mu-tg mice, VH-DJH rearrangement is not detectable in these cells. We infer that the lack of VH-DJH rearrangement primarily reflects tg-induced acceleration of B cell differentiation past the stage at which rearrangement of VH elements is permissible. In support of this inference, we find that the normal representation of early B lineage subsets is markedly altered in mu-tg mice. We suggest that the effect of a productive VH-DJH rearrangement at an endogenous H chain allele may be similar to that of a mu-tg; i.e., cells that make a productive VH-DJH rearrangement on the first attempt rapidly progress to a developmental stage that precludes VH-DJH rearrangement at the other allele (allelic exclusion).

Figure 1

Model for B cell differentiation in Ig tg scid mice. Early pro-B, late pro-B (pre-BI), pre-BII (large), pre-BII (small), and immature B cells (37) are designated with the letter code of Hardy et al. (17). Members of subsets B, C, and C′ are B220⁺CD43⁺ and are distinguished on the basis of their staining for the markers, BP1 and HSA (see Fig. 2); subsets D and E are B220⁺CD43⁻ and B220⁺IgM⁺, respectively. The periods at which RAG is normally upregulated and DJH, VH–DJH, and VJL rearrangement are ongoing are indicated. In scid (s/s) and μ-tg s/s bone marrow, differentiation is arrested at the C and D stages, respectively. In μ/κ-tg scid bone marrow, differentiation is not arrested and cells reach the immature B cell stage (E). B cell subsets denoted with a small circle are under represented or missing. The horizontal arrow signifies direct or very rapid differentiation between the stages indicated.

Figure 2

In μ-tg scid mice, B cell differentiation from the B to the C′ stage appears to bypass the C stage. Flow cytometry was used to analyze bone marrow of scid (s/s), scid heterozygotes (s/+), M54 s/s, and 3H9 s/s mice for the presence of early B lineage subsets. Contour plots show intensity of BP1 versus HSA staining for B220⁺CD43⁺-gated cells. Cells belonging to subset B (BP1⁻HSA⁺), B′ (BP1⁻HSA²⁺), C (BP1⁺HSA⁺), and C′ (BP1⁺HSA²⁺) are designated in the boxed areas in accordance with the original description of these cell types (17) (B′ is an additional designation as explained in the text).

Figure 3

In μ/κ-tg scid mice, B cell development occurs in the absence of significant pre–B cell development. Flow cytometry was used to analyze bone marrow of scid (s/s), scid heterozygotes (s/+), M54/Vκ8 s/s, and 3H9/Vκ8 s/s mice for the presence of late pre-B cells (subset D) and immature B cells (subset E) in bone marrow of scid (s/s), scid heterozygotes (s/+), M54/Vκ8 s/s, and 3H9/Vκ8 s/s mice. Top row of contour plots shows intensity of B220 versus CD43 staining for IgM⁻-gated cells. The percentage of cells in subset D (B220⁺CD43⁻) is indicated within the boxes. Bottom row of contour plots shows intensity of B220 versus IgM staining for CD43⁻-gated cells. The percentage of cells in subset E (B220⁺IgM⁺) is indicated outside the boxes.

Figure 4

Initiation of DH–JH rearrangement in μ-tg mice occurs predominantly at stage D, the late pre-B (B220⁺CD43⁻) cell stage. Genomic DNA from sorted B220⁺CD43⁻ (CD43⁻) and B220⁺CD43⁺ (CD43⁺) cells of M54 scid (s/s) and M54 scid heterozygote (s/+) bone marrow was analyzed by LM-PCR for JH signal ends (JH1, JH2, JH3, and JH4) and by PCR for DJH coding joints (DJH1, DJH2, and DJH3) and the retention of JH germline [JH (G)] alleles. Non-tg controls included DNA from sorted CD43⁺ cells of s/s bone marrow and DNA from CD43⁺- and CD43⁻-sorted cells of s/+ bone marrow. PCR amplification of the α actin gene served as an internal control for input DNA. In this and subsequent figures, the first and second lane of PCR products under each bracket proceeding from left to right correspond to undiluted and threefold-diluted input DNA, respectively. The primers and probes used for amplification and hybridization of PCR products are described in Materials and Methods.

Figure 5

Initiation of VH–DJH rearrangement is not detectable in late pre-B (B220⁺CD43⁻) cells of μ-tg mice despite ongoing initiation of DH–JH rearrangement. Genomic DNA from sorted B220⁺CD43⁻ (CD43⁻) and B220⁺CD43⁺ (CD43⁺) cells of scid heterozygotes (s/+) and M54 s/+ bone marrow were analyzed by LM-PCR for 3′ and 5′ DHfl signal ends. CD43⁺ cells from scid (s/s) bone marrow served as a positive control for initiation of DH–JH rearrangement. The primers and probes used for amplification and hybridization of PCR products are described in Materials and Methods.

Figure 6

Reduced frequency of initiation of DH–JH rearrangement in developing B cells of μ/κ-tg scid mice. Genomic DNA from sorted B220⁺IgM⁻ bone marrow cells of scid (s/s), 3H9/Vκ8 s/s, and 3H9/Vκ8 s/+ mice was analyzed by LM-PCR for JH signal ends (JH1, JH2, and JH3) and by inverse PCR for signal joints resulting from the joining of JH signal ends with the 3′-DHfl signal. The nonrearranging α actin gene served as an internal control for the amount of DNA (input DNA). The primers and probes used for amplification and hybridization of PCR products are described in Materials and Methods. To compare the level of signal joints in the three groups of mice, we used a Bio-Image Analyzer to measure the amount of ³²P-labeled probe hybridizing to JH2 signal joint product/α actin in 3H9/Vκ8 s/s (or 3H9/Vκ8 s/+) divided by that hybridizing to JH2 signal joint/α actin in the s/s control. The ratios obtained for 3H9/Vκ8 s/s and 3H9/Vκ8 s/+ mice were 0.11 and 0.62, respectively.

Figure 7

Status of H chain loci in splenic B cell hybridomas from (A) 3H9/Vκ8 scid/+ and (B) 3H9/Vκ8 scid mice. Approximately 10 μg of genomic DNA from each hybridoma (designated with even or odd numbers) was EcoR1 digested, Southern blotted, and hybridized to a JH-specific probe. DNA from adult liver (L), the Ag8.563 cell fusion partner (Ag8), and the hybridoma from which the VDJH transgene was derived (3H9) served as controls for the position of the germline (G) H chain fragment, the Ag8.563 H chain allele, and the 3H9 transgene, respectively. The absence of hybridizing fragments in lanes 10 and 15 reflects incomplete DNA digestion.