A Reassessment of IgM Memory Subsets in Humans

Abstract

From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities.

Figure 1. Isolation of naive, marginal zone, memory and double-negative B cell subsets

Human peripheral blood and splenic CD19⁺ B cells were stained with CD38 and CD24 to exclude transitional, germinal center, and plasma cells. The four gated populations were sorted according to CD27 and IgD expression: CD27⁺IgD⁻ memory cells (Mem), CD27⁺IgD⁺ marginal zone B cells (MZ), CD27⁻IgD⁺ naive (N) and CD27⁻IgD⁻ double-negative (DN) B cells. The profile shown corresponds to a spleen sample.

Figure 2. Fraction of clones with single sequences among each subset

Fraction of clones with single sequences among each subset, reported either to the total number of clones or the total number of sequences of this subset. Two different calculations are performed, depending whether clones have single sequence at the level of a given subset (first two bars), or at the level of the total sequence pool of the donor (last two bars).

Figure 3. Clonal relationships between subsets and tissues

(A) Percentage of clones shared between B cell subsets, analyzed for each individual subset and color-coded for the subset sharing these clones. (B) Percentage of clones showing clonal relationships between blood and spleen for the same subset among blood cells (white) and spleen cells (black). The error bars represent the binomial proportion confidence interval (95%). Clonal relationships were analyzed for each donor separately, and the total number of clones belonging to the same category (e.g. clones shared between blood and spleen Mem_A subsets) were pooled from the three donors and reported to the total number of clones obtained in the corresponding subset for the three samples (e.g., total clones from blood Mem_A or spleen Mem_A subsets, first two columns, panel B).

Figure 4. V_h mutation frequencies, V_h family and J_h gene usage of the different B-cell subsets

(A) V_h mutation frequencies were estimated by comparison with the IMGT database. Mutation frequencies are calculated based on the average mutation value obtained for each clone. The mutation frequency observed for the naive B cell pool (0.7 %) is represented by a dotted line, and is taken as an estimate of the contribution of sequencing errors. V_h family (B) and J_h gene (C) usage.

Figure 5. Repertoire profile of clones shared between different subsets

Analysis of clones shared between the different IgM B cell subsets (A), between IgM memory or MZ and IgG/IgA memory B cells (B), and between MZ and double-negative IgG/IgA B cells (C). . Each column represents a specific B cell population, with its name indicated on top of each panel: B cell subset, CD27⁺IgD⁻ memory (Mem), CD27⁺IgD⁺ marginal zone (MZ) and CD27⁻IgD⁻ double-negative B cells (DN); immunoglobulin isotype: IgG (G), IgA (A), IgM (M). MZ are IgM⁺, and only analyzed in (C). Populations indicated in brackets represent the tissue/subset of origin between which the clones analyzed are shared. The repertoire analysis includes 5 rows, with, from the top: i) V_h mutation frequency depicted as fraction of clones within 6 different 2% mutation intervals, from 0-2% (left) to >10% mutation frequency (far right); ii) average V_h mutation frequency; iii) average H-CDR3 amino acid length; iv) V_h family 1 to 6 usage v) J_h 1 to 6 usage. The mutation data for shared clones (first and second row) is plotted separately for the sequences belonging to each of the two populations involved, because, contrary to other parameters, V_h mutations can vary between B cells belonging to the same clone. All analyses are based on clones, not sequences, and take into account, for mutation frequencies, the average mutation frequency of the clone. (D) 3-D plot representation according to mutation frequency, V_h4 and J_h6 usage of clones shared between two IgM-positive subsets (filled blue dots), or between MZ or Mem_M and Mem_A/Mem-G or DN_A/DN_G (open blue dots). In the latter case, data concerning MZ clones shared with DN_A and DN_G subsets were pooled. Parental IgM subsets are represented by diamonds, red for MZ, green for DN_M and black for Mem_M. Statistics are depicted in Table S3 to facilitate reading, with the different pair wise comparisons indicated. Error bars represent the 95% interval of confidence of data distribution and therefore directly reflect the size of the sample analyzed.

Figure 6. Repertoire profile of clones shared between blood and spleen

Analysis of clones shared between blood and spleen for IgG and IgA (A), and IgM subsets (B). Abbreviations and criteria of repertoire analysis are depicted in legend to Figure 5 with the addition of tissue: blood (bl) and spleen (sp). (C) 2D plot representation according to mutation frequency and V_h4 usage of the clones shared between blood and spleen for Mem_G (blue), Mem_A (green) and MZ (red) B cells, together with the profile of the parental blood and spleen subsets. Spleen sequences are represented by squares, blood sequences by dots, and sequences shared between blood and spleen by a hexagon framed in black. Circles mark the closest parental subset of the clones shared between both tissues.

Figure 7. Mutation frequency of DN_M subsets and average H-CDR3 amino acid length in mutated and unmutated splenic clones

A) V_h mutation frequency depicted as fraction of clones within 6 different 2% mutation intervals, from 0-2% to >10% mutation frequency (first row); average CDR3 amino acid (a.a.) length (second row) of blood (bl) and spleen (sp) DN_M subsets. B) Average H-CDR3 length of each splenic B-cell subset, represented according to their mutation frequency (white bars: less than 1% mutation; black bars: more than 2% mutation). Error bars represent the 95% interval of confidence.

Figure 8. Recirculation of spleen MZ and switched memory subsets and clonal relationships between B cell subsets

A. Recirculation between blood and spleen is schematized for MZ and switched memory B cell subsets, as deduced from the repertoire characteristics of clones shared between both tissues (within dotted lines). Brown circles represent splenic and spleen-derived cells, and green circles cells from other lymphoid tissues. B. Clonal relationships between subsets. Each subset is represented with a different color, distributed over a theoretical FACS profile for IgD and CD27 expression. Clonal relationships with switched memory B cells are observed for IgM memory B cells, which present the repertoire profile of IgM-only B cells (IgD⁻CD27⁺) while extending within the MZ and DN gate. MZ B cells only present a few clonal relationships with DN IgG⁺ and IgA⁺ cells. The switched memory and DN pool have many clones in common, which could correspond either to precursor-product relationships or reversible up- and down-modulation of CD27 expression. Subsets are not drawn on scale, since IgM memory B cells represent on average 2-4% of the total B cell pool, switched memory 20% and double-negative IgM less than 1%.