Hairy Cell Leukaemia Displaying Multiple Surface Immunoglobulin Isotypes Reveal a Functional B-Cell Receptor In Which Isotype Roles Differ

Abstract 1567

The B-cell receptor (BCR) is critical to survival of normal B-cells, and regulates key aspects of cellular behavior. Of these, response to antigen determines pathways of normal B-cell maturation, including isotype switch events that occur by deletional class switch recombination (CSR), an irrevocable event, to yield IgG/A memory B-cells. Less frequently, CSR via a cryptic site generates IgD+ B-cells whereas IgM+IgD+ antigen experienced B-cells synthesize each isotype by an alternative transcript splicing mechanism. The role of the BCR in survival of malignant B-cells however is less well defined, in particular in response to antigen. Intriguingly, in Hairy cell leukemia (HCL), BCR assembly occurs with multiple surface immunoglobulin (sIg) isotypes (mult-HCL), many co-expressed on individual hairy cells (HCs) in an otherwise monoclonal tumor. Multiple isotypes appear to exclude deletional CSR events, and suggests a RNA processing mechanism of molecular assembly. This phenotype is rare even amongst malignant B-cells, and raises the question of the functional relevance of individual variant isotypes. It also potentially presents a model to dissect roles of multiple isotypes on single B-cells. To examine this, we investigated the BCR in CD19⁺CD11c⁺CD103⁺ mult-HCL cases (n=10), in which 2–4 differing sIg isotypes were present on most HCs, with single or, in 3 cases, dual sIgL expression. In all cases, IGHV genes were mutated, and confirmed monoclonality. Phenotype revealed 2 distinct subsets by sIg isotype co-expression, IgD+ve and IgD-ve. Using Ca²⁺ flux and ERK phosphorylation assays after cross-linking with specific anti-sIg antibodies, we observed a functional BCR in all mult-HCL examined, in both subsets (10/10 cases Ca²⁺, 6/6 cases ERK). However, striking differences emerged between the two subsets. In sIgD+ve mult-HCL, IgD mediated persistent Ca²⁺ flux, with flux also evident via >1 sIgH isotype. In marked contrast, in sIgD-ve mult-HCL Ca²⁺ flux was restricted to a single sIgH isotype, but not via IgM. Flux signals in this subset were transient. In most cases only a single sIgL transduced flux. We next evaluated BCR endocytosis after cross-linking individual isotypes and IgL. In 2 sIgD+ve cases, anti-IgD and anti-Igλ stimulation led to endocytosis of both sIgD and sIgλ, and in 1 case, where examined, anti-IgM stimulation endocytosed both sIgM and sIgλ. In 3 sIgD-ve cases, functional sIgH and sIgL induced endocytosis of the stimulated isotype, but again sIgM was dysfunctional, remaining immobilized on the cell surface. Ca²⁺ flux through endocytosed isotypes was correspondingly either significantly reduced or ablated in both subsets. In HCs, BCR endocytosis is clearly dependent on functional isotypes and IgL, and parallels events in normal B-cells. Lastly, we examined downstream effects of BCR signalling on cell viability, using soluble (sAb) and bound (bAb) anti-sIg antibodies. In a single IgD+ve mult-HCL case, both sAb and bAb anti-IgM yielded a significant level of apoptosis compared to control antibodies, whereas anti-IgD sAb resulted in no appreciable difference to level of spontaneous apoptosis, suggesting a disengagement of signals from this pathway. This disengagement was also observed in a separate HCL case expressing only IgD, and not in the mult-HCL cohort initially selected, where anti-IgD signals again did not increase levels of apoptosis. In IgD-ve mult-HCL (n=4), sAb and bAb specific cross-linking of IgG/A triggered significant apoptosis. These data demonstrate, for the first time, that mult-HCL retains a functional responsiveness via the BCR, suggesting an absence of anergic effects that may follow chronic antigen exposure in-vivo to self-antigen. Signals via sIgM/G/A isotypes, where functional, induce apoptosis in mult-HCL, whereas sIgD opposes such effects. Despite an apparently unique molecular mechanism of IgD expression in mult-HCL, this isotype appears to be hardwired in B-cells to mediate responses that differ from IgM. The persistent flux observed here indicates a more sustained and robust IgD signaling cascade, as also observed in B-cell models. These data reveal distinctive and opposing effects of individual isotypes on BCR mediated behavior in mult-HCL. While apoptotic responses appear to negate a role for antigen in tumor drive in-vivo, potential antigen engagement via IgD, if dominant leaves this question open.