IgM+ B-Cells Lacking CD27 Expression Display Somatically Mutated Ig VH Genes and Define a New Memory Population.
Authors: Weston-Bell Nicola J. , Townsend Mark R. , Forconi Francesco , Stevenson Freda K. , Sahota Surinder S. 
Published: October 2019

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+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.