Abstract

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 VH gene mutations by using Cmu downstream primers. We examined VH3 and VH5 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 VH3 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 VH5 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 VH5 donor germline gene exists in the IgH repertoire, but 4 allelic variants which differ by 1 nucleotide are known. We mapped the VH5 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 VH5 transcripts in the IgM+D+CD27 population were generated by SHM. In contrast, between 74–90% of VH3 and VH5 transcripts from the IgM+DCD27+ 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.

Author notes

Disclosure: No relevant conflicts of interest to declare.