Promiscuous Antigen Reactivity May Underlie Clinical Aggressiveness and Increased Risk for Richter's Syndrome in Chronic Lymphocytic Leukemia with Stereotyped IGHV4-39/IGKV1(D)-39 B Cell Receptors

Abstract 561

Mounting evidence suggests that the molecular classification of CLL into subsets with stereotyped B cell receptors (BcRs) is functionally and clinically relevant. In fact, cases of the same subset can share not only BcR sequence motifs but also biological and clinical features as well. This suggests that the functional antigen reactivity profile of the BcR can be critical in determining the clinical features and outcome even independently of IGHV gene mutational status, at least for selected subsets. A distinctive CLL stereotyped subset, known as subset #8, is defined by the expression of unmutated, IgG-switched IGHV4-39/IGKV1(D)-39 BcRs. Subset #8 patients experience aggressive clinical courses and exhibit the highest risk for Richter′s transformation (RT) among all CLL. In order to obtain biological insight into the underlying reasons for this behavior, we profiled the antigen reactivity of subset #8 vs. other stereotyped subsets, in particular: (1) subset #1: unmutated IGHV1/5/7-IGKV1(D)-39 IgM BcRs, the largest unmutated CLL subset, with bad prognosis; (2) Subset #2: mostly borderline-mutated IGHV3-21/IGLV3-21 IgM BcRs, with bad prognosis; (3) subset #4: mutated IGHV4-34/IGKV2-30 IgG BcRs, the largest mutated CLL subset, with indolent disease. Twenty-five monoclonal antibodies (mAbs) from CLL cells were prepared as recombinant human IgG1: 11 subset #1, 6 subset #2, 3 subset #4, and 5 subset #8. The CLL mAbs were used as primary antibodies in ELISA assays against antigens which are representatives of the major classes of established antigenic targets for CLL, namely molecular structures on microbial pathogens, autoantigens and neo-epitopes created by chemical modifications during apoptosis. In particular, we tested the reactivity against lipopolysaccharides (LPS) from E. coli 055:B5, dsDNA, native BSA, malondialdehyde (MDA)-BSA, 4-hydroxynonenal (HNE)-BSA and Advanced Oxidation Protein Products (AOPP)-HSA. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, showing in particular strong reactivity to BSA and MDA-BSA, E. coli LPS and dsDNA, but also against the other oxidation markers tested (HNE-BSA and AOPP-HSA), albeit to a lesser extent. This high binding was in clear contrast with the mAbs from all other stereotyped subsets. Indeed, subset #1 mAbs exhibited only medium to low reactivity against MDA-BSA and dsDNA and very low reactivity against E.coli LPS; subset #2 mAbs did not react against any of the tested antigens; and, subset #4 showed low-level reactivity only against MDA-BSA and E. coli LPS. A propos these findings, we previously demonstrated that subset #8 mAbs exhibited the strongest binding also to myosin-exposed apoptotic cells as compared to all CLL mAbs, both unmutated and mutated, including subset #1 and #2 mAbs. In addition, through in vitro functional studies of immune signaling in CLL, we observed an unrestricted and intense response of subset #8 CLL cells to ligands for Toll-like receptors (TLR) 1/2, 2/6, 7 and 9, thus differing significantly from other subsets that respond to ligands for selected TLRs only. Alltogether, these findings help to draw a scenario that may explain the particular aggressiveness of subset#8 and its increased propensity to transform. An unlimited capacity to respond to multiple immune/inflammatory stimuli present in the microenvironment may elicit unabated stimulation thoughout the natural history of these patients, leading to progressive selection of the more aggressive clonal variants. Finally, our work further indicates that immunogenetic information can be used for the rational categorization of CLL, with implications for both research and management of CLL.