The peritoneal cavity B-2 antibody repertoire appears to reflect many of the same selective pressures that shape the B-1a and B-1b repertoires

Abstract

To assess the extent and nature of somatic categorical selection of CDR-3 of the Ig H chain (CDR-H3) content in peritoneal cavity (PerC) B cells, we analyzed the composition of V(H)7183DJCμ transcripts derived from sorted PerC B-1a, B-1b, and B-2 cells. We divided these sequences into those that contained N nucleotides (N(+)) and those that did not (N(-)) and then compared them with sequences cloned from sorted IgM(+)IgD(+) B cells from neonatal liver and both wild-type and TdT-deficient adult bone marrow. We found that the PerC B-1a N(-) repertoire is enriched for the signatures of CDR-H3 sequences present in neonatal liver and shares many features with the B-1b N(-) repertoire, whereas the PerC B-1a N(+), B-1b N(+), and B-2 N(+) repertoires are enriched for adult bone marrow sequence signatures. However, we also found several sequence signatures that were not shared with other mature perinatal or adult B cell subsets but were either unique or variably shared between the two or even among all three of the PerC subsets that we examined. These signatures included more sequences lacking N nucleotides in the B-2 population and an increased use of D(H) reading frame 2, which created CDR-H3s of greater average hydrophobicity. These findings provide support for both ontogenetic origin and shared Ag receptor-influenced selection as the mechanisms that shape the unique composition of the B-1a, B-1b, and B-2 repertoires. The PerC may thus serve as a general reservoir for B cells with Ag binding specificities that are uncommon in other mature compartments.

Figure 1. Frequency of N addition at both V_H-D and D-J_H junctions among V_H7183DJCμ transcripts isolated from 1-day liver, bone marrow, peritoneal cavity and splenic B cells

The frequency of sequences containing N additions is reported as the percent of the sequenced CDR-H3 (containing identifiable D gene) transcripts from 1-day liver Hardy fraction F (NL F) (15), adult bone marrow Hardy fraction F (BM F) (6, 27), peritoneal cavity (PerC) B-1a, B-1b and B-2 (Supplementary Figure 1), and spleen follicular (FO) and marginal zone (MZ) B cells (14). χ² analyses demonstrate that the frequency of B-1a N− sequences differ significantly from B-1b (p=0.005) and all other B cell subsets (p<0.0001). The B-1b population differs from BM F, FO (p<0.0001) and MZ (p=0.007) but does not differ from B-2 (p=0.152). The B-2 population differs from BM F (p<0.0001) and FO (p=0.001), but does not differ from MZ (p=0.247).

Figure 2. V_H usage of V_H7183DJCμ transcripts isolated from sorted 1-day liver, peritoneal cavity and bone marrow mature B cells

V_H usage is reported as the percent of the sequenced population of unique, in-frame, open transcripts from neonatal liver fraction F (NL F - mature), peritoneal cavity (PerC) B-1a, B-1b and B-2 (Supplementary Figure 1), and bone marrow fraction F (BM F - mature, recirculating) CDR-H3s from either wild type or TdT^−/− mice (BM F TdT^−/−). The V_H segments are arranged in germline order with the most D_H proximal sequences to the right. The number of sequences analyzed is shown. (top) all V_H7183 transcripts, (middle) V_H7183 transcripts from PerC B cells CDR-H3 without N nucleotide addition (N−), (bottom) V_H7183 transcripts from PerC B cell CDR-H3 containing N nucleotide addition (N+). All comparisons were made to fraction F, either from 1 day liver (NL F) or bone marrow (BM F) using χ²-test or Fisher’s exact test as appropriate. Significant differences between PerC B cell subsets and both fraction F are labelled with asterisks with *p≤0.05, **p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 3. D_H, D_H reading frame and J_H usage of V_H7183DJCμ transcripts isolated from sorted 1-day liver, peritoneal cavity and bone marrow mature B cells

D_H and J_H usage is reported as the percent of the sequenced population of unique, in-frame, open transcripts from neonatal liver fraction F (NL F - mature), peritoneal cavity (PerC) B-1a, B-1b and B-2, and bone marrow fraction F (BM F - mature, recirculating) CDR-H3 from either wild type or TdT^−/− mice (BM F TdT^−/−) which use the particular D_H or J_H. D_H reading frame usage is reported as the percent of the sequenced population of DFL- and DSP-containing transcripts from each B cell population that use the specified reading frame. (top) all V_H7183 transcripts; (middle) V_H7183 transcripts from PerC B cells CDR-H3 without N nucleotide addition (N−); (bottom) V_H7183 transcripts from PerC B cell CDR-H3 containing N nucleotide addition (N+). All comparisons were made to fraction F, either from 1 day liver (NL F) or bone marrow (BM F) using χ²-test or Fisher’s exact test as appropriate. Significant differences between PerC B cell subsets and both fraction F are designated labelled with asterisks, with *p≤0.05, **p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 4. Distribution of amino acids in the CDR-H3 loops of the V_H7183DJCμ transcripts isolated from sorted 1-day liver, peritoneal cavity and bone marrow mature B cells

Amino acids use is reported as the percent of the sequenced population from neonatal liver Fraction F (NL F - mature); peritoneal cavity (PerC) B-1a, B-1b and B-2; and bone marrow fraction F (BM F - mature, recirculating) from either wild type or TdT^−/− mice (BM F TdT^−/−). The amino acids are arranged by relative hydrophobicity, as assessed by a normalized Kyte-Doolittle scale (39, 40). The number of loops analyzed is shown. (top) all V_H7183 transcripts; (middle) V_H7183 transcripts from PerC B cells CDR-H3 without N nucleotide addition (N−); (bottom) V_H7183 transcripts from PerC B cell CDR-H3 containing N nucleotide addition (N+). All comparisons were made to fraction F, either from 1 day liver (NL F) or bone marrow (BM F) using χ²-test or Fisher’s exact test as appropriate. Significant differences between PerC B cell subsets and both fraction Fs are labelled with asterisks, with *p≤0.05, **p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 5. Average CDR-H3 length of V_H7183DJCμ transcripts isolated from sorted 1-day liver, peritoneal cavity and bone marrow mature B cells

Average CDR-H3 codon lengths of the sequenced population from neonatal liver fraction F (NL F - mature); peritoneal cavity (PerC) B-1a, B-1b and B-2; and bone marrow fraction F (BM F - mature, recirculating) from either wild type or TdT^−/− mice (BM F TdT^−/−). Error bars depict the standard error of each mean. (left) all V_H7183 transcripts; V_H7183 transcripts from PerC B cells CDR-H3 without N nucleotide addition (N−); (right) V_H7183 transcripts from PerC B cell CDR-H3 containing N nucleotide addition (N+). Statistical analyzes were made comparing to PerC B cell subsets using Student’s t-test, two tailed; the extent of statistical significance (p<0.05) is indicated with (^a) compared to B-1a, (^b) compared to B-1b and (²) compared to B-2.

Figure 6. Average CDR-H3 hydrophobicity of V_H7183DJCμ transcripts isolated from sorted 1-day liver, peritoneal cavity and bone marrow mature B cells

Average CDR-H3 loop hydrophobicity as assessed by a normalized Kyte-Doolittle scale (39, 40) from neonatal liver fraction F (NL F - mature); peritoneal cavity (PerC) B-1a, B-1b and B-2; and bone marrow fraction F (BM F - mature, recirculating) from either wild type or TdT^−/− mice (BM F TdT^−/−). Error bars depict the standard error of each mean. (left) all V_H7183 transcripts; V_H7183 transcripts from PerC B cells CDR-H3 without N nucleotide addition (N−); (right) V_H7183 transcripts from PerC B cell CDR-H3 containing N nucleotide addition (N+). Statistical analyzes were made comparing to PerC B cell subsets using Student’s t-test, two tailed; the extent of statistical significance (p≤0.05) is indicated with (^a) compared to B-1a, (^b) compared to B-1b and (²) compared to B-2.

Figure 7. Distribution of the predicted structures of the base and the loop of CDR-H3 from V_H7183DJCμ transcripts isolated from sorted 1-day liver, peritoneal cavity and bone marrow mature B cells

Structural properties were predicted according to Shirai’s (23) “H3-rules” deduced from the analysis of well-determined CDR-H3 crystal structures. These rules allow prediction of structural features from the primary CDR-H3 sequence based upon the location and hydrophobicity of amino acids, and size of the side chain. Frequencies are reported as percentage of all sequences analyzed. (A) Frequency of kinked (k−), extra- kinked (k+) and extended (E) CDR-H3 bases from neonatal liver fraction F (NL F - mature); peritoneal cavity (PerC) B-1a, B-1b and B-2; and bone marrow fraction F (BM F - mature, recirculating) from either wild type or TdT^−/− mice (BM F TdT^−/−). (B) Frequency of broken and intact hydrogen bond ladders within the CDR-H3 loop for those H chains that contain kinked or extra-kinked bases, depicted in A. (top) all V_H7183 transcripts; (middle) V_H7183 transcripts from NL F and PerC B cells CDR-H3 without N nucleotide addition (N−) and bone marrow fraction F from TdT deficient mice; (bottom) V_H7183 transcripts from NL F, PerC B and BM F cells CDR-H3 containing N nucleotide addition (N+). Statistical analyzes were made comparing to PerC B cell subsets, using χ²-test or Fisher’s exact test as appropriate. The extent of statistical significance (p≤0.05) is indicated with (a) compared to B-1a, (b) compared to B-1b and (2) compared to B-2.

Figure 8. Schematic representation of the shared and unique features of the PerC B cell CDR-H3 repertoire

The key signatures shared between the PerC subsets and mature B cells within the neonatal liver and the adult bone marrow with (N+) or without (N−) N addition are represented. Also represented are the key signatures that appeared to be specific for each individual B cell subset as well as those that were shared between two or all three (PerC) of these subsets. Note that the CDR-H3 repertoire overlap is greater among the PerC N+ transcripts than in PerC N− transcripts. ( ) NL F: features shared with neonatal liver fraction F; ( ) BM F: features shared with adult bone marrow from WT; ( ) TdT: features shared with adult bone marrow from TdT deficient mice; ( ) B-1a: unique features of B-1a cells; ( ) B-1b: unique features of B-1b cells; ( ) B-2: unique features of B-2 cells. (Bold black) features shared between two or all three PerC B cell subsets.