Analysis of Single B Cells Reveals a Double Drive of Endocytosis and Transcriptional Downregulation Acting on the CLL B-Cell Receptor In Vivo

Abstract

The cells of virtually all CLL cases, including those with unmutated (U) Ig gene heavy-chain variable regions ( IGHV) of poorer prognosis (U-CLL), exhibit variable degree of anergy. This is defined by (auto)antigen-mediated low surface immunoglobulin M (sIgM), but not sIgD, expression and signaling capacity, both reversible in vitro. In U-CLL there is also a tumor-related highly asymmetric use of IGHV1-69 ( 51p1 allele) (~30% of all U-CLL vs ~1% of the normal B-cell repertoire), claiming selection by (auto)antigen. While the effect of (auto)antigen on sIgM down-modulation by endocytosis is well known, less is known about the biosynthetic mechanisms regulating sIgM expression. We investigated the phenotypic, functional, and transcriptional consequences of sIgM engagement in bulk and single cells of IGHV1-69+ve U-CLL, compared to IGHV1-69-ve U-CLL and non-tumor IGHV1-69+ve B cells (identified by G6 monoclonal antibody F(ab)‘). The functional consequences were measured by intracellular calcium [iCa 2+] mobilization assay following anti-IgM F(ab‘)2 engagement. Phenotypic and functional analyses of bulk IGHV1-69+ve U-CLL populations (n=32) documented homogeneously lower sIgM, but not sIgD, levels and signaling capacity compared to IGHV1-69-ve U-CLL (n=49). Also, sIgM engagement mobilized iCa 2+ more slowly than IGHV1-69-ve U-CLL. However, following in vitro culture in ‘antigen-free’ medium for 48 hours, sIgM levels and signaling capacity/speed recovered more rapidly in IGHV1-69+ve than IGHV1-69-ve U-CLL, indicating a more profound and dynamic control of Ig expression in the IGHV1-69+ve CLL cells. Single cell t-SNE plots of IGHV1-69+ve U-CLL (3 patients) and normal B cells (2 donors) exposed ±anti-IgM F(ab')2 identified 2 distinct unstimulated or anti-IgM stimulated B cell clusters, in which IGHM transcripts could also be compared. We found that the basal (unstimulated) IGHM transcript levels were downmodulated in CLL cells compared to normal B cells. However, while IGHM transcript levels reduced dramatically following 2 hours anti-IgM engagement in the stimulated normal B cells, further downmodulation was not visible in the stimulated CLL cells, suggesting that (auto)antigen induced IGHM transcript downmodulation had likely already occurred in those CLL cells in vivo. Combined kinetics studies of the IGHM transcripts by qPCR and sIgM levels in flow cytometry revealed that IGHM transcripts recovered in the CLL cells within 48 hours of culture in vitro. The increase of IGHM transcripts anticipated sIgM protein recovery of expression on the CLL cells in vitro. Instead, in the normal B cells there was no transcript or sIgM protein increase. These data in IGHV1-69+ve cells provide additional fundamental evidence of chronic (auto)antigen drive in CLL and a novel mechanism of antigen-mediated transcriptional control of sIgM levels. The antiapoptotic machinery provides valuable time to the circulating leukemic cells to resynthesize sIgM glycoprotein following downmodulation by endocytosis. Therefore, there is a danger of interrupting sIgM signaling by therapeutic approaches with BCR inhibitors alone. A coordinated inhibition of BCR signaling and cell survival is required to prevent sIgM recovery in CLL. We thank Dr Roy Jefferis, University of Birmingham, UK, for kindly providing the G6 hybridoma. Stuart Lanham and Luis Del Rio Fernandez should be considered joint first authors.