Differences in the composition of the human antibody repertoire by B cell subsets in the blood

Abstract

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD(+), memory IgD(-), and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse, the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.

Keywords: B cells subsets; CDR-H3; human antibody repertoire.

Figure 1

Flow cytometric gates for the collection of six distinct B cell lineage populations from the peripheral blood of a healthy adult human subject. B cells were separated from the total lymphocytes using CD19⁺ magnetic beads and further separated into CD27^± populations using CD27 magnetic beads. The CD19⁺CD27⁺ B cells were stained with CD19 APC₇₈₀, CD27 PE-Cy7, CD24 APC, and IgD FITC and sorted into IgD+ memory B cells (CD19⁺/CD27⁺/IgD⁺/CD24⁺), IgD⁻ memory B cells (CD19⁺/CD27⁺/IgD⁻/CD24⁺), and plasmacytes (CD19⁺/CD27⁺/CD24⁻) using the high speed sorting cytometer. The CD19⁺/CD27⁻ B cells were stained with CD19, APC 780, CD24 APC, CD38 PE, and IgD FITC, and sorted into mature (CD19⁺/CD27⁻/IgD⁺/CD38⁺/CD24⁺), transitional (CD19⁺/CD27⁻/IgD⁺/CD38⁺⁺⁺/CD24⁺⁺⁺), and immature (CD19⁺/CD27⁻/IgD⁻) B cell subsets.

Figure 2

Deconstruction of the contributing components to CDR-H3 length in Igμ and Igγ reads containing identifiable D_H gene segments as a function of B cell development in the peripheral blood. The contributions of nucleotides provided by the V_H, D_H, and J_H gene segments, by P junctions, and by the extent of N addition at the V_H → D_H and D_H → J_H junctions to the CDR-H3 length are illustrated. The IgAT (42) identified the CDR-H3 as amino acids 105–117, according to the IMGT unique numbering system. The average length was calculated with the components of the CDR-H3, namely the V length, P-nucleotides 3′ of the V, N1 nucleotides, P-nucleotides 5′ of D, D length, P-nucleotides 3′ of D, N2 nucleotides, P-nucleotides 3′ of J, and J length. The deconstructed CDR-H3 segments shown are of CDR-H3 sequences with identifiable D_H gene segments. The reported average length is the average length of all CDR-H3 sequences (with the identifiable D_H and without identifiable D_H gene segments) accompanied by the standard deviation.

Figure 3

V_H gene segment usage in Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. V_H gene segments are arranged according to their position relative to the J_H locus in the genome. Percent of unique, in-frame sequences using the V_H gene segment specified in the peripheral blood from immature, transitional, mature, memory IgD⁺, memory IgD⁻ B cells, and plasmacytes are displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 4

Individual V_H gene segment usage in Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. Percent of unique, in-frame sequences using the individual V_H gene segments specified in the peripheral blood from immature, transitional, mature, memory IgD⁺, memory IgD⁻ B cells, and plasmacytes are displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001.

Figure 5

D_H family usage in Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. The percent of sequences using members of the specified D_H family among in-frame reads obtained from the peripheral blood from immature, transitional, mature, memory IgD⁺, memory IgD⁻ B cells, and plasmacytes are displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 6

Individual D_H gene segment usage in Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. Percent of unique, in-frame reads using the individual D_H gene segments specified in the peripheral blood from immature, transitional, mature, memory IgD⁺, memory IgD⁻ B cells, and plasmacytes are displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001.

Figure 7

J_H usage in Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. The percent of sequences using J_H1 through J_H6 among in-frame reads cloned from the peripheral blood from immature, transitional, mature, memory IgD⁺, memory IgD⁻ B cells, and plasmacytes are displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 8

Amino acid usage in the CDR-H3 loop of Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. The distribution of individual amino acids is displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisk: *p < 0.0001.

Figure 9

The prevalence of highly charged and highly hydrophobic CDR-H3 loops of Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. (A) Prevalence of CDR-H3 loops with an average hydrophobicity of ≤0.7 is displayed. (B) Prevalence of CDR-H3 loops with an average hydrophobicity of >0.7 is displayed. The normalized Kyte–Doolittle hydrophobicity scale (48) and normalized by Eisenberg (49) has been used to calculate average hydrophobicity (23). Prevalence is reported as the percent of the sequenced population of unique, in-frame, open transcripts from each B lineage fraction. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 10

D_H reading frame usage in Igμ and Igγ transcripts from selected B cell populations in the blood of a normal, healthy human. The percent of sequences using members of the specified D_H family members in reading frames 1, 2, or 3 among in-frame sequences cloned from the peripheral blood from immature B cells through plasmacytes are displayed. All comparisons were made using χ²-test or Fisher’s exact test as appropriate. Significant differences among each fraction in the different mice are indicated by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 11

CDR-H3 loop charge and length as a function of B cell development in the peripheral blood of this adult subject. (A) Distribution of CDR-H3 hydrophobicities in Igμ and Igγ transcripts from peripheral blood as a function of B cell development. The Kyte–Doolittle hydrophobicity scale (48) as normalized by Esienberg (49) has been used to calculate average hydrophobicity (30). Although this scale ranges from −1.3 to +1.7, only the range from –1.0 (charged) to +1.0 (hydrophobic) is shown. Prevalence is reported as the percent of the sequenced population of unique, in-frame, open transcripts from each B lineage fraction. (B) Distribution of CDR-H3 lengths in nucleotides of μ and γ H chain transcripts is displayed.