Katerina Hatzi

Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation.

Chronic lymphocytic leukemia (CLL) represents the outgrowth of a CD5+ B cell. Its etiology is unknown. The structure of membrane Ig on CLL cells of unrelated patients can be remarkably similar. Therefore, antigen binding and stimulation could contribute to clonal selection and expansion as well as disease promotion. Initial studies suggest that CLL mAbs bind autoantigens. Since apoptosis can make autoantigens accessible for recognition by antibodies, and also create neo-epitopes by chemical modifications occurring naturally during this process, we sought to determine if CLL mAbs recognize autoantigens associated with apoptosis. In general, ~60% of CLL mAbs bound the surfaces of apoptotic cells, were polyreactive, and expressed unmutated IGHV. mAbs recognized two types of antigens: native molecules located within healthy cells, which relocated to the external cell surface during apoptosis; and/or neoantigens, generated by oxidation during the apoptotic process. Some of the latter epitopes are similar to those on bacteria and other microbes. Although most of the reactive mAbs were not mutated, the use of unmutated IGHV did not bestow autoreactivity automatically, since several such mAbs were not reactive. Particular IGHV and IGHV/D/J rearrangements contributed to autoantigen binding, although the presence and degree of reactivity varied based on specific structural elements. Thus, clonal expansion in CLL may be stimulated by autoantigens occurring naturally during apoptosis. These data suggest that CLL may derive from normal B cells whose function is to remove cellular debris, and also to provide a first line of defense against pathogens.

Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, ‘stereotyped’ B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of ‘stereotyped’ cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first (∼30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells.

B-cell chronic lymphocytic leukemia, a clonal disease of B lymphocytes with receptors that vary in specificity for (auto)antigens.

Abstract: B‐Cell chronic lymphocytic leukemia (B‐CLL) is an incurable disease that is relatively common among aging Caucasians. Patients with this leukemia can be divided into prognostic categories using clinical staging parameters, as well as molecular features [presence or absence of IgVH mutations in rearranged VHDJH segments that code for the leukemic B cells receptor for antigen (BCR)]. In addition, the deduced amino acid structure of the BCRs from patients that fall into different prognostic categories is shared, to varying degrees, within these groups. In this paper, the molecular features of the genes that code for the BCRs of B‐CLL patients are reviewed, and these are comapred to antibodies of known specificity. These comparisons suggest that the BCRs of many cases resemble autoantibodies, and in some cases, antibodies to microbial antigens. Antigen‐binding analyses confirm these impressions, and also indicate that polyreactivity appears to distinguish cases with worse clinical outcomes differ from those with better outcomes. The persistence of autoreactivity and polyreactivity is somewhat surprising, because IgV DNA sequence analyses suggest that many of the B cells that become leukemic have undergone one form or another of receptor editing. Thus, B‐CLL appears to be a disease of B‐cell clones that have undergone various types of receptor reconfiguration and yet retain inappropriate antigen‐binding properties.

Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells

Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL-derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL-derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL-isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL-derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.

Mechanisms of epigenetic deregulation in lymphoid neoplasms

Aberrant lymphoma epigenome is the result of a combinatorial contribution of changes in DNA methylation, histone modifications, and noncoding RNA (ncRNA) expression in lymphoma cells. All of the components of epigenome have been the focus of intense studies in various subtypes of lymphoma, which

Chronic lymphocytic leukemia immunoglobulins display bacterial reactivity that converges and diverges from auto-/poly-reactivity and IGHV mutation status

Chronic lymphocytic leukemia (CLL) is an incurable leukemia of unknown etiology. Multiple studies suggest that the structure of the variable domains of the surface IGs on these cells, and signaling through them, play key roles in developing the disease. Hence, CLL appears to be driven by antigen-BCR interactions, and identifying the selecting antigens involved in this process is an important goal. We studied the antigen-binding characteristics of 23 CLL-derived, recombinantly-expressed IGs with 5 pathogenic bacteria, determining that CLL IGs differ in bacterial reactivity based on IGHV gene use, mutation status, and association with IGHD and IGHJ genes (stereotypy). Although most bacterial-reactive IGs followed the paradigm that IGHV-unmutated IGs were more auto-/poly-reactive, several did not. In addition, some CLL IGs were bacterial mono-reactive, and these displayed IGKV use biases. These findings are consistent with CLL B cells being driven into the leukemogenic process by bacterial as well as auto- antigens.