Genetic control of DH reading frame and its effect on B-cell development and antigen-specifc antibody production

Abstract

The power of the adaptive immune system to identify novel antigens depends on the ability of lymphocytes to create antigen receptors with diverse antigen-binding sites. For immunoglobulins, CDR (complementarity-determining region)-H3 lies at the center of the antigen-binding site, where it often plays a key role in antigen binding. It is created de novo by VDJ rearrangement and is thus the focus for rearrangement-dependent diversity. CDR-H3 is biased for the inclusion of tyrosine. In seeking to identify the mechanisms controlling CDR-H3 amino acid content, we observed that the coding sequence of DH gene segments demonstrate conservation of reading frame (RF)-specific sequence motifs, with RF1 enriched for tyrosine and depleted of hydrophobic and charged amino acids. Use of DH RF1 in functional VDJ transcripts is preferred from the earliest stages of B-cell development, "pushing" CDR-H3 to include specific categories of tyrosine-enriched antigen-binding sites. With development and maturation, the composition of the CDR-H3 repertoire appears to be pulled into a more refined specific range. Forcing the use of alternative DH RFs by means of gene targeting alters the expressed repertoire, enriching alternative sequence categories. This change in the repertoire variably affects antibody production and the development of specific B-cell subsets.

Figure 1. CDR-H3 plays a key role in antigen binding site diversity

Top. The variable domains of the L and H chains are created by VJ joining, and by VDJ joining and N addition, respectively. Due to the inclusion of a D gene segment, the opportunity to introduce two sets of N nucleotide additions, and the greater flexibility in length and sequence composition, the CDR-H3 interval is the most diverse portion of the pre-immune repertoire [reviewed in (25)]. Bottom. A cartoon of the classic antigen-binding site as seen head-on. Due its central location, most bound antigens will interact with CDR-H3, including its D_H component.

Figure 2. Deconstruction and analysis of CDR-H3

In this hypothetical sequence, the location of CDR-H3, the CDR-H3 loop, and boundaries of FRs 3 and 4 are shown. Kabat and IMGT (5,76) number designations for the TGT codon which marks the terminus of framework 3 and the TGG which marks the beginning of framework 4 are identified. Here the CDR-3 loop has been evaluated for the distribution of individual amino acids and average Kyte-Doolittle hydrophobicity (77,78). Amino acids at the extreme (arginine and isoleucine) have been included to demonstrate the range of the hydrophobicity index. The normalized average hydrophobicity of this CDR-H3 loop is −0.24. This CDR-H3 has also been evaluated for V_H, D_H, and J_H usage, P junctions, N addition, and the length of CDR-H3 in codons. A single palindromic (P) nucleotide flanks the V_H sequence. D_H DFL16.1 sequence is flanked by three nucleotides of N addition on each side. To facilitate analysis, we have color-coded our data in this and other figures in this application to report relative hydrophobicity. Blue reflects hydrophobicity, green represents neutrality with or without hydrophilicity, and red is used for charge.

Figure 3

Distribution of CDR-H3 charge in VDJCμ transcripts of WT BALB/c mice isolated from phenotypically defined bone marrow and spleen B cell populations as assessed 30 by reference to a normalized Kyte-Doolittle scale (79,80). Prevalence is reported as the percent of the sequenced population of unique, in-frame, open transcripts from each B lineage subset. To facilitate visualization of the change in variance of the distribution, the vertical lines mark the apparent normal boundaries beyond which it appears to be difficult to transition into fraction F.

Figure 4. D-limited mice express polyclonal, altered CDR-H3 repertoires

Prevalence is reported as the percent of the sequenced population of unique, in-frame, open transcripts from each B lineage fraction. (Right) Distribution of CDR-H3 average hydrophobicity in VDJCμ transcripts from CD19⁺IgM⁺IgD⁺ mature, recirculating bone marrow B cells from homozygous ΔD-DFL, ΔD-DμFS, ΔD-iD, and wild type (wt) mice. The normalized Kyte-Doolittle hydrophobicity scale (78) has been used to calculate average hydrophobicity. To facilitate visualization of the change in distribution, the vertical lines mark the preferred range average hydrophobicity observed in wild-type fraction F [Figure 3, (27)]. (Left) Distribution of amino acids in the CDR-H3 loop as a function of B cell development in the same strains of mice. Amino acids are arranged by polarity from arginine (left) to isoleucine (right). The number of sequences per B cell fraction is shown on the far left.

Figure 5

BALB/c DH RF1 amino acid sequences.

Figure 6

Generation of a D-limited IgH allele.

Figure 7

Four D-limited D_H alleles.

Figure 8. Mutant and Control D_H sequences

(Top) Germline DFL16.1. (Middle) A Dμ frameshift in DFL16.1 replaces RF1 Y+G amino acids with RF2 V, T, I, and F (DμFS). (Bottom) Replacement of central RF1 Y+G codons with inverted DSP2.2 sequence introduces codons for positively charged R and H as well as polar N in a new RF1.

Figure 9. Divergence in the absolute numbers of B lineage subpopulations from the bone marrow, spleen, and peritoneal cavity of homozygous ΔD-DFL, ΔD-DμFS, and ΔD-iD mice relative to their littermate controls

A. Percent loss or gain relative to wild type littermate controls in bone marrow fractions B through F; splenic transitional T1 [T(1A)], T2 [T2(A)], and T3 [T3(A)] per Allman et al (81); and splenic mature follicular (FO) B cells. B. Percent loss or gain relative to wild type littermate controls in splenic transitional T1 (T1L) per Loder et al (28); splenic marginal zone (MZ) B cells; CD19 and in both panels the standard peritoneal cavity B1a, B1b, and B2. For the littermate controls, the standard error of the mean of each B lineage subpopulation averaged approximately 11% of the absolute number of cells in each subpopulation (gray area). For ΔD-DFL, ΔD-DμFS, and ΔD-iD, the standard error of the mean is shown as an error bar. ‘*’, p < 0.05; ‘**’, p < 0.01; ‘***’, p < 0.001; and ‘****’, p < 0.0001.

Figure 10. Mortality after challenge with mouse-adapted heterologous influenza virus

Mice were immunized with influenza strain A/Udorn (H3N2) and then challenged with the heterologous strain A/pr/8/34 (H1N1) at day 0. Values are the percent of mice that remained alive in each group of thirty immunized homozygous wild-type (wt/wt), ten immunized homozygous ΔD-DFL (ΔD-DFL/ΔD-DFL) ten immunized homozygous ΔD-iD (ΔD-iD/ΔD-iD), and ten naïve homozygous wild-type wt/wt mice, respectively, on the given day after challenge.