IgM⁺ B-Cells Lacking CD27 Expression Display Somatically Mutated Ig V_H Genes and Define a New Memory Population.

Somatic hypermutation (SHM) and isotype class switch recombination are critical mechanisms that diversify normal B-cells in response to antigen, and are generally confined to the germinal center (GC). Post-GC B-cells differentiate either into memory B-cells or antibody forming cells maturing as plasma cells. Memory B-cells which exit the GC have been regarded as invariably expressing CD27 and this population comprises both IgM⁺ and switched B-cells. It is, however, the presence of SHM that remains the defining feature of memory status in B-cells. Recently, it has been reported that dye extruding membrane ATP-binding transporters expressed by naive B-cells can distinguish them from memory B-cells which lack transporters and this, notably, led to the identification of IgG⁺ memory B-cells that did not express CD27. However, these observations were not correlated with SHM status. Here, we have examined these findings further and focused specifically on IgM⁺D⁺ B-cells in relation to our interest in mapping origins of B-cell tumors. Using dye-based transporter assays, we identified a substantial IgM⁺CD27⁻ population in PBMNC from healthy individuals which lacked transporter activity. These cells were next isolated using a different strategy, employing IgD and CD27 expression, and a highly purified (>99%) IgD⁺CD27⁻ fraction obtained by FACS sorting. There was virtually nil contamination by CD27⁺ B-cells. IgM transcripts were then specifically analyzed in this CD27⁻ population for V_H gene mutations by using Cmu downstream primers. We examined V_H3 and V_H5 gene transcripts from 2 separate sorts from a healthy donor following amplification by primary and nested RT-PCR and cloning products for sequence analysis. Of 104 V_H3 clones from this CD27⁻ fraction, 38 (37%) were germline and the remainder mutated (55% with 98–99.9% homology to germline, 9% <97.9%). Of these, 27% displayed 3–5 or more nucleotide mutations. Analysis of 124 V_H5 clones from the same fraction showed 50% to be germline and the remainder mutated (45% with 98–99.9% homology, 5% <97.9%), of which 16% exhibited 3–5 or more mutations. Only 1 V_H5 donor germline gene exists in the IgH repertoire, but 4 allelic variants which differ by 1 nucleotide are known. We mapped the V_H5 germline gene in our donor using recombination sequence primers and only 1 allele was identified, identical to the donor gene mapped by database alignment, eliminating polymorphisms as a source of apparent base changes. Mutational load was also over and above a low level of PCR error which was quantified in the analysis. This confirmed that the levels of mutation detected in the rearranged V_H5 transcripts in the IgM⁺D⁺CD27⁻ population were generated by SHM. In contrast, between 74–90% of V_H3 and V_H5 transcripts from the IgM⁺D⁻CD27⁺ population were mutated, with a higher frequency of sequences (65–87%) displaying 3–5 or more mutations. Our data reveal the existence of a novel IgM⁺D⁺ population of normal B-cells which are somatically mutated but lack CD27. Unmutated IgM⁺D⁺ B-cells in this population point to heterogeneity in the CD27⁻ B-cell pool. As yet, the clonal derivation of these mutated CD27⁻ cells is undefined. Importantly, they have relevance for understanding the cell of origin in Hairy Cell leukaemia and Waldenstrom’s macroglobulinemia, where tumor cells are mutated yet lack CD27 expression.