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, 163 (5), 1124-1137

Sequence-Intrinsic Mechanisms That Target AID Mutational Outcomes on Antibody Genes

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Sequence-Intrinsic Mechanisms That Target AID Mutational Outcomes on Antibody Genes

Leng-Siew Yeap et al. Cell.

Abstract

In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss the implications of these findings for harnessing antibody diversification mechanisms.

Figures

Figure 1
Figure 1. V(D)J Replacement Passenger Allele System
(A) Top: RDBC generates chimeric mice whose mature B cells carry a fixed VB1–8 productive allele and test sequences in the V(D)J passenger allele. Bottom: assay of SHM of GC B cells from spleen and PP of immunized mice. Pro, pas, V pro and L represent productive allele, passenger allele, V promoter and leader sequence, respectively. Red lines across the leader sequence and VB1–8 in the passenger allele represents a termination codon and two nucleotide changes from productive VB1–8, respectively. (B) Pie charts showing the proportion of sequences that have the indicated number of mutations per sequence in productive and passenger VB1–8 alleles from splenic GC B cells (top) and PP GC B cells (middle) of six independent mice. Total number of sequence reads from each mouse is indicated below each pie chart. Bottom: Ratios of mutation frequencies of PP GC B cells to mutation frequencies of splenic GC B cells (calculated within each mouse), for productive VB1–8 (left) and passenger VB1–8 alleles (right). Ratios were calculated for each of the six mice separately and displayed as the mean ± standard deviation (SD) of the six mice. One-sample student t-test was performed to test for significance of difference of the mean from a hypothetical mean of 1.0 (labeled in blue dotted line). One-tailed p value is shown. See also Figure S1 and Table S1.
Figure 2
Figure 2. SHM Profiles of VB1–8 Productive and Passenger Alleles
(A and B) Map of mutations (SHM Profile) on the VB1–8 productive and passenger allele sequences in (A) splenic and (B) PP GC B cells. The y-axis indicates the mutation frequency at each nucleotide plotted as the mean % of sequences in the indicated strata that contain a mutation at the indicated nucleotide ± standard error of the mean (SEM) (green shading indicates top error bar) from 6 independent mice. Orange and yellow bars mark the positions of AGCT and other RGYW motifs, respectively. See also Figure S2.
Figure 3
Figure 3. Deletions in VB1–8 Passenger Allele
(A and B) Deletion frequency, calculated as the % of all mutated sequences that contain deletions of VB1–8 productive and passenger alleles in (A) splenic and (B) PP GC B cells. Data are represented as mean ± SD from 6 mice. Two-tailed, paired t test p values are indicated. (C and D) Map of unique deletions in VB1–8 passenger allele from (C) splenic and (D) PP GC B cells. Deletions are represented by lines whose start and end indicate the start and end of the deletion. Deletions from each of the 6 mice are displayed with a line of a different color. (E and F) The location of SHMs compared to the location of deletion endpoints. Pearson correlation coefficient (r) between SHM frequency and deletion (endpoint) frequency of each bin in (E) splenic and (F) PP GC B cells are indicated. See also Figure S3 and Table S2.
Figure 4
Figure 4. E.coli gpt Sequence Mutates as Frequently as VB1–8 Sequence
(A–B) Left: Schematic of productive VB1–8 and passenger gpt alleles. Middle: Pie charts showing proportion of sequences that have the indicated (see legend, right) number of mutations per sequence in productive VB1–8 and passenger gpt allele from (A) splenic and (B) PP GC B cells of 6 independent mice. (C–D) SHM profiles of productive VB1–8 and passenger gpt allele from (C) splenic and (D) PP GC B cells from 6 mice. The y-axis and other details are as described for Figure 2. Data from mutation strata 3–10 mutation/sequence is shown. See also Figure S4.
Figure 5
Figure 5. Human β-globin Sequence Mutates as Frequently as VB1–8 Sequence
(A–B) Left: Schematic of productive VB1–8 and passenger β-globin alleles. Middle: Pie charts showing proportion of sequences that have the indicated (see legend, right) number of mutations per sequence in productive VB1–8 and passenger β-globin allele from (A) splenic and (B) PP GC B cells of 5 independent mice. (C–D) SHM profiles of productive VB1–8 and passenger β-globin allele from (C) splenic and (D) PP GC B cells from 5 mice. The y-axis and other details are as described for Figure 2. Data from mutation strata 3–10 mutation/sequence is shown. See also Figure S5.
Figure 6
Figure 6. SHM Profiles and Deletion Maps of Switch Regions
(A) Schematic of passenger core Sμ (cSμ) (left) and inverted Sμ (inSμ) (right) alleles. (B) Excerpt of SHM pattern of cSμ (top) and inSμ (bottom) passengers from PP GC B cells. The y-axis indicates the total number of mutations. Green open boxes show the positions of G-stretches and C-stretches on cSμ and inSμ alleles, respectively. (C) Location of unique deletions in passenger cSμ (left) and inSμ alleles (right). Deletions from splenic GC B cells are depicted with red lines and PP GC B cells with black lines. For (B) and (C), data are pooled from 6 mice. See also Figure S6.
Figure 7
Figure 7. SHM of the V exon and S region in B cells Activated in Culture
(A) Naïve B cells of VB1–8 passenger mice were stimulated in culture for 4 days with αCD40 and IL4. (B) Mutation frequency of total nucleotides, nucleotide 454 and 455 of VB1–8 productive and passenger alleles at day 4. All sequences that have 0–2 mutations per sequence were included in the analysis. Unpaired t-test was performed. (C–D) SHM profiles of matched VB1–8 productive and passenger alleles in sequences that contain 1–2 mutations/sequence from (C) day 4 CSR-activated B cells and (D) splenic GC B cells shown in Figure 2A. Peaks that have a SD greater than the mean were excluded. (E) Naïve B cells of cSμ passenger mice were stimulated in culture for 4 days with αCD40 and IL4. The 5’ region immediately upstream of the core in passenger Sμ (Pas 5’ Sμ) and endogenous Sμ (Endo 5’ Sμ), indicated in blue lines, were analysed. (F) Mutation frequency over entire indicated allele/region (left), and at individual nucleotides indicated (right). All sequences that have 0–3 mutations/sequence were included in the analysis. The mean values and fold change between each mean are indicated. (G) SHM profiles of VB1–8 productive allele, Pas 5’ Sμ and Endo 5’ Sμ of data shown in (F). For (B and F) data represent mean ± SD of 6 independent stimulation of cells from 6 independent mice. For (C, D and G) data represent mean frequency ± SEM of 6 independent mice. The y-axis and other details are as described for Figure 2. For (B, C F, G), data shown are mutation frequency at day 4 after subtraction of mutation frequency at day 0. See also Figure S7 and Supplemental Experimental Procedures.

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