Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity

Abstract

Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs))/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PK(cs) activity.

Figure 1

Somatic mutation of the Vκ transgene in MD4 mice. (A) Sequences of the four distinct transgenic rearranged Vκ domains that are deduced to exist in the germline of MD4 mice. The transgenic rearranged Vκ was PCR amplified from tail DNA and cloned into Bluescript. Of 33 clones sequenced, 5 derived from copy 0, 7 from copy 1, and 11 each from copies 2 and 3. The copies are named according to the number of nucleotide substitutions by which each diverges from the consensus sequence (copy 0). Nucleotide 1 corresponds to the first nucleotide of Kabat codon 1. (B) Prevalence of somatic mutations in the four transgenic Vκ genes PCR amplified from sorted GC B cells from Peyer's patches of MD4 mice. The number in the center of the pie chart indicates the number of sequences contributing to each database, with the segments of the pie indicating the number of sequences carrying 0, 1, 2, etc. mutations.

Figure 3

Somatic mutation of the Vκ transgene in MD4/DO-TCR(Rag1 ^−/−) mice. (A) Sequence diversity due to somatic mutation of the transgenic Vκ PCR amplified from sorted GC B cells from Peyer's patches of MD4/DO-TCR(Rag1 ^−/−) mice. The mutations identified in samples obtained from five mice (analyzed in two pools) have been combined. (B) Comparison of the distribution of mutations across the Vκ domain in MD4 (above the line) and MD4/DO-TCR(Rag1 ^−/−) mice (below the line).

Figure 2

Reconstitution of peripheral lymphocyte compartments in MD4/DO-TCR(Rag1 ^−/−) and MD4/DO-TCR(Scid/Scid) mice. (A and B) Flow cytometric analyses of splenic lymphocytes double stained for CD45R(B220) and IgM, CD4 and CD8, and CD45R(B220) and the HyHEL10 idiotype as indicated. (C) Reconstitution of Peyer's patches. Peyer's patches were classified as very small, small, or normal (light gray, dark gray, and black bars, respectively), and the histogram depicts the average number of each size (based on four to six mice per group). (D) Immunohistological examination of Peyer's patches from an MD4/DO-TCR(Rag1 ^−/−) mouse (left) and an MD4(Rag1 ^−/−) mouse (right). Sequential sections have been stained for CD45R(B220), PNA, and Ki67 (a marker of proliferating cells). GCs (which were also found to stain for FDC markers; not shown) can be seen as small PNA⁺Ki67⁺ clusters within the B cell follicles (marked B) of MD4/DO-TCR(Rag1 ^−/−) but not MD4(Rag1 ^−/−) mice, as indicated by arrowheads.

Figure 4

Somatic mutation of the Vκ transgene in MD4/DO-TCR(Scid/Scid) mice. (A) Sequence diversity due to somatic mutation of the transgenic Vκ PCR amplified from sorted GC B cells from Peyer's patches of MD4/DO-TCR(Scid/Scid) mice. The mutations identified in samples obtained from five mice (analyzed in pools of two mice, two mice, and one mouse) have been combined. (B) Mutations other than nucleotide substitutions identified in the transgenic Vκ in MD4/DO-TCR(Scid/Scid) mice. Numbers indicate the first nucleotide in the sequence string (see Fig. 1 A). Deleted nucleotides are shown above the line, and single nucleotide substitutions are circled, with the novel base being specified. The deletion in sequence string 409 forms part of a mutational dynasty, indicating that it did not arise from a PCR artefact. Deletions were also identified among transgenic Vκ sequences from GC B cells of normal MD4 mice. (C) Comparison of the distribution of mutations across the Vκ domain in MD4 (above the line) and MD4/DO-TCR(Scid/Scid) mice (below the line).