High-Throughput Ig Sequencing of Paired Blood and Spleen Samples Allows a Redefinition of Memory IgM Subsets in Humans

In humans, whether B cells with the IgM+IgD+CD27+ phenotype represent an independent lineage involved in T-independent responses, similar to mouse marginal zone B cells, or whether they are part of the germinal center-derived memory B-cell pool generated during responses to T-dependent antigens, is still a debated issue.

To address this question, we performed high-throughput Ig sequencing of B-cell subsets from paired blood and spleen samples and analyzed the clonal relationships between them.

We isolated and analyzed 3 different B cell subsets based on CD27 and IgD staining from both blood and spleen:

IgD+CD27+ (MZ) - amplified with Cmu primers

IgD-CD27+ (switched and IgM-only) with Cmu, Cgamma and Calpha primers

IgD-CD27- (CD27- memory or double-negative DN) with the same three primers

We obtained 95729 unique sequences that clustered in 49199 different clones: 1125 clones were shared between blood and spleen of the same B-cell subset, and 1681 clones were shared between different subsets, allowing us to trace their relationships. We analyzed these clones that share sequences from different subsets/tissues for their mutation frequency distribution, CDR3-length, and VH/JH family usage, and compared these different characteristics with the bulk of sequences from their respective subset of origin.

The analysis of clones shared between blood and spleen for switched IgG/IgA and for MZ subsets suggests different recirculation dynamics. For switched cells, the blood appears to be a mixture of splenic and other lymphoid tissues B cells. For MZ B cells in contrast, the blood appear to be only composed of a subgroup of the splenic repertoire, in agreement with the observation that marginal zone B cells recirculate and are mainly generated in the spleen.

Clonal relationships between the IgM clones (originating from the MZ, IgM-only and double negative compartments) show that the clones involved display the characteristics of IgM-only B cells whatever their subset of origin, even in the case of the paired MZ/double-negative sequences that were not supposed to include IgM-only sequences. We therefore conclude that the clones shared between the various IgM subsets do not represent b between them, but rather correspond to a heterogeneous phenotype of the IgM-only population that concerns both IgD and CD27 expression, leading to a partial overlap with the MZ and double-negative gates.

Clones shared between the MZ and the switched IgG and IgA compartment also show, for their IgM part, the mutation and repertoire characteristics of IgM-only cells and not of MZ B cells, reinforcing the conclusion that IgM-only are true memory B cells, and constitute the only subset showing clonal relationships with switched memory B cells.

In summary, we report that MZ B cells have different recirculation characteristics and do not show real clonal relationships with IgM-only and switched memory B cells, in agreement with the notion that they represent a distinct differentiation pathway. In contrast, the only precursor-product relationship between IgM memory and switched B cells appear to concern a B cell subset that has been described as "IgM-only", but appears to have a more heterogeneous expression of IgD than previously reported and therefore contribute to 3-15% of the MZ compartment. Searching for markers that would permit to discriminate between marginal zone and germinal center-derived IgM memory B cells is obviously required to further delineate their respective function.