B-cell Receptor In Chronic Lymphocytic Leukemia Research Articles

B-Cell Receptor Function and Gene Expression Profiling at Single-Cell Level across the Spectrum from Normal to Neoplastic CD5-Positive B-Cell Compartments in Humans

Antigen-independent, autonomous signalling of the B-cell receptor (BCR) is an oncogenic driver common to all cases and subtypes of chronic lymphocytic leukemia (CLL). We have recently shown that the dominant clonal population of low-count (<500 cells/µL) monoclonal B lymphocytosis (MBL) with CD5 +CD20 lowCD79B low CLL phenotype identified prospectively in healthy siblings of CLL patients also exhibit autonomous BCR signalling, albeit with lower signalling strength than CLL BCR (Quinten et al., Haematologica 2023). To gain further insight into the mechanisms of clonal expansion within the CD5 + B-cell compartment as the possible origin of CLL, we performed single-cell gene expression profiling and exemplary analyses of BCR function in normal CD5 + B cells (CD20 hiCD79B hi) and in CLL-phenotype B cells (CD5 +CD20 lowCD79B low) of healthy siblings of CLL patients in comparison to CLL and high-count MBL cells. Screening of blood samples of 16 sibling stem cell donors of CLL patients by flow cytometry revealed the presence of CD5 +CD20 lowCD79B low B cells at low counts in 5 donors. These cells and normal CD5 +CD20 hiCD79B hi B cells were sorted in parallel, pooled at equal ratios, and analyzed together with CLL B cells from their sibling stem cell recipients and 2 unrelated high-count MBL B cells on two 10X Genomics chips for single-cell 5‘ gene expression and BCR sequencing. Data deconvolution per individual was performed based on expressed SNPs and on the individuals’ HLA types. In a combined 2-dimensional t-SNE plot based on the 2000 most variably expressed genes, the normal CD5 +CD20 hiCD79B hi B cells from all 5 donors clustered distinctly from CLL-phenotype cells from all sources (dotted lines, Figure) and indeed showed significantly higher expression of CD20 and CD79B. Gene set enrichment analysis revealed significant upregulation of the hallmark oxidative phosphorylation gene set in all CLL samples compared to non-CLL B-cell populations. BCR analysis demonstrated clonal dominance of >99% for both Ig heavy and light chains in the 5 CLL samples. In accordance with the indication for allogeneic stem cell transplantation, these CLL BCR were unmutated. In marked contrast, normal CD5 +CD20 hiCD79B hi B cells from the healthy SC donors were also unmutated but completely polyclonal, i.e. no identical BCR was detected in any two cells of this compartment. In 4/5 donors, the CLL-phenotype CD5 +CD20 lowCD79B low compartment contained multiple clonal expansions of various degrees. In one high-count MBL, 17 other B-cell clones with various degrees of low-level expansion coexisted with the >90% dominant MBL clone. BCR from the most dominant and one or more of the less expanded CD5 +CD20 lowCD79B low clones of the healthy donors were tested for autonomous signaling as quantified by BCR-induced calcium flux without experimental cross-linking in murine TKO pre-B cells and compared to BCR from nonexpanded CD5 +CD20 hiCD79B hi cells and from their CLL siblings. Autonomous BCR signaling strength was calculated from the fraction of cells with spontaneous calcium flux above test background and calibrated against calcium flux after BCR crosslinking. CLL and high-count MBL BCR had robust median autonomous BCR signaling of 0.326. Expanded CD5 +CD20 lowCD79B low clones of the healthy donors showed significantly weaker BCR signalling strength of 0.163 (p<0.01; Scatter Plot). BCR signalling in nonexpanded CD5 + B cells appeared weaker than in expanded clones but was clearly above the background of BCR from mature B cells isolated from lymph node biopsies expressing a mutated IgG. In conclusion, the low-count CD5 +CD20 lowCD79B low CLL-phenotype B-cell compartment in healthy adults is composed of multiple, predominantly mutated B-cell clones. Therefore, the term “monoclonal B lymphocytosis” is highly misleading for these B-cell populations and should be avoided. Normal CD5 +CD20 hiCD79B hi B cells are exclusively unexpanded and express unmutated BCR, indicating a naïve status. Unexpectedly, these nonexpanded CD5 + B cells express BCR with relatively weak but quantifiable autonomous signalling. Globally, autonomous BCR signalling strength across the CD5 + B-cell compartment appears to be correlated to the degree of clonal expansion. Fully malignant CLL cells overexpress genes of the oxidative phosphorylation pathway, presumably to meet increased energy demand from alternative sources.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/μL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

Old and New Facts and Speculations on the Role of the B Cell Receptor in the Origin of Chronic Lymphocytic Leukemia.

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.

Spatial organization and early signaling of the B-cell receptor in CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%–10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.

P621: SUBCLONAL EVOLUTIONARY TRAJECTORIES IN IGLV3-21R110 CHRONIC LYMPHOCYTIC LEUKEMIA

Background: There is a large body of evidence supporting the importance of antigenic interactions of the B cell receptor (BCR) with self or non-self antigens that make this receptor and its signaling machinery one of the most important drivers in many types of non-Hodgkin lymphoma. This has led to a new pathobiological understanding of these diseases as well as novel therapeutic strategies. Chronic lymphocytic leukemia (CLL) is a prototype disease in which BCR-BCR self-interactions induce autonomous signaling. While virtually all CLL BCR are prone to autonomous signaling induced by such contacts, the IGLV3-21R110 molecular interaction found in 10% of CLL patients defines a distinct clinical subset of patients that show a very aggressive clinical course despite novel treatments. In these cases, a single amino acid replacement from G to R located at the boundary of variable and constant region of the BCR is an important tumor promotor driving neoplastic B cell growth. Aims: It has been unclear how the R110 point mutation is generated. We set out to investigate the trajectory of the malignant CLL B cell clone and subclonal heterogeneity in IGLV3-21R110 cases in order to gain insight into the molecular pathogenesis of this unique amino acid exchange. Methods: We screened an initial CLL cohort of 127 cases by flow cytometry using an anti-IGLV3-21R110 antibody. Positive cases were subjected to immunosequencing of the light chain locus via amplicon based next-generation sequencing and characterized for typical chromosomal deletions and driver gene mutations. Results: We identified twelve CLL cases with IGLV3-21R110. Of these, we found a higher frequency of mutations in ATM (42%) and SF3B1 (25%) than in unselected CLL cohorts, which is in line with findings reported by others. As expected, the repertoire of healthy B cells in the blood of IGLV3-21R110 CLL patients contained IGLV3-21 light chains with the wild-type G110 residue, indicating that the R110 exchange is of acquired nature. Interestingly, in half of the IGLV3-21R110 CLL cases we found additional clones with IGLV3-21 and R110, including cases with divergent J gene cassettes originating from different pre-B cells. This indicates that the R110 exchange may occur multiple times within independent B cell clones in the same patient. Due to the unique location at the variable-constant boundary and the high correlation of the IGLV3-21R110 patient subset with mutations of genes involved in DNA repair (ATM/SF3B1), R110 is likely generated as a result of incorrect V-J recombination facilitated by the error-prone-environment. Summary/Conclusion: Our work indicates that the G110R point mutation results from incorrect V-J rearrangements in the error-prone environment that may be induced by SF3B1 and ATM mutations associated with this CLL subset. We found evidence for a branching light chain evolution that leads to a striking convergence of separate clones on the identical light chain amino acid exchange in the same patient. This strongly emphasizes the role of this mutation as a tumor promotor involved in malignant transformation of this subset of CLL.

Comparison with Private and Public Clonotypes Reveals That, While CLL Type Stereotyped BCR Are Produced in 90% of Healthy Subjects from 18 to 78 Years, They Are Not Accumulated and Are Mostly Found in Immature and Naïve B-Cells

Introduction:One third of Chronic Lymphocytic Leukemia (CLL) patients harbor stereotyped B-Cell receptor (BCR). This is in contrast with the 1012 potential different BCRs that can be produced by the immune system. Some stereotyped BCR are also highly associated with prognosis. For example, the stereotyped subset #2 identifies aggressive CLLs with frequent SF3B1 and ATM alterations. This underlies an important role of BCR selection for emergence of CLL clones.Aim and methods:We raised the question of the existence and quantification of B-cells with stereotyped BCR of the CLL-type in a series of 69 healthy subjects (HS) from 18 to 78 years old, without any past of hematological history nor any detectable monotypic B-cells by flow cytometry.HS were divided in age three groups, adults (18 - 55 years, n= 31), senior-1 (55 - 65 years, n = 14) and senior-2 (> 65 years, n = 24).The variable region of the immunoglobulin heavy chain gene (IGHV) was amplified from genomic DNA of purified B-cells sequenced on an Illumina MiSeq and annotated with the online IMGT HighV-Quest tool.Clonotypes were defined as IGHV genes with the same rearranged V and J segment and with the same CDR3 amino acid sequence with one mismatch tolerance, and if a minimal threshold read number was reached (ranging 5 to 15 according to the DNA input and sequencing depth).Public clonotypes were those shared in between at least 4 (5%) HS.Detection of the 19 major stereotyped CLL's BCRs was performed with an in house R script. And was confirmed with the ARResT/AssignSubsets online tool (V Bistry et al, Bioinformatics, 2015).Exploration of the IGHV repertoire of the different B-cell subpopulations was done from public data (Mitsunaga, Mol Cell Proteomics, 2020).Results:To be independent of the number of reads and to compare HS to each other, clonotypic frequencies were ranked in 150 increasing intervals. In each case, the mean rank of clonotypic frequency ranged 3 to 7 with an SD in between 2 and 6 and was independent of the age category. A Z-score was calculated for each clonotype. Less than 1% of private clonotypes had a Z-score>1.96, while 10% to 30% of public clonotypes had a significant Z-score>1.96 (figure 1). This indicates that public clonotypes are often accumulated when compared to their private counterpart.A stereotyped CLL's BCR was found in 90% HS all stereotypes together. Subsets #2 and #5 were found in more than 50% of cases (figure 2). Frequencies of subsets #3, #59, #7C, #202, #14, #64B, decreased from 23% to 6% cases. Subsets #1, #4, #6, #8, #12, #16, #99 and #201 were never found.Frequencies of stereotyped BCRs ranged 5.2x10 -5 to 1.2x10 -4, being similar in the different HS age categories. Ranks of stereotyped BCR frequencies were almost always below the mean rank of private clonotypes, that indicates a very low abundance (figure 3). Junction analyses suggest that B-cells with stereotyped BCRs were polyclonal since they were very likely to derive from different B-cell precursors. Exploring the longitudinal repertoires of Mitsunaga and Snyder(Mol Cell Proteomics 2020), showed that stereotyped BCRs were almost restricted to immature and naïve B-cells and were fugace while public clonotypes were recurrently and frequently found in memory B-cells and could be stable over time for many of them.Conclusion:B-cells with stereotyped CLL BCRs were easily detected in healthy people whatever their age category. Remarkably, subsets #2 and #5 that are associated with aggressive CLL were found in more than 50% healthy donors at any age. Comparison with private and public clonotypic repertoire reveals that abundance of stereotyped BCR was almost always very low and was restricted to immature and naïve B-cells.Therefore, B-cells with stereotyped BCRs are produced throughout life in almost everybody. But these B-cells do not accumulate and do not seem to undergo to B-cell maturation.This suggests that stereotyped BCRs are not intrinsically prone to transformation and that clonal selection of CLL cells would involve additional events such as antigenic encounter or oncogenic alterations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IgM and IgD Differential CLL BCRs Organization and Interaction with Co-Receptors before and during Ibrutinib Therapy

Studies of the B cell receptor (BCR) on normal B lymphocytes indicate that membrane IgM and IgD (respectively mIgM and mIgD) are organized in independent protein islands containing different BCR co-receptors that then reorganize as B cells transition from resting (CD19 and CD20 associated with IgD) to activated (CD19 and CD20 associated with IgM). In this way the signaling balance between IgM and IgD regulates B cell fate. Since BCR signaling is a key factor for chronic lymphocytic leukemia (CLL), as evinced by the efficacy of the signaling inhibitory drug ibrutinib, we decided to evaluate the membrane organization of the BCR-complex on primary CLL cells in an isotype dependent fashion.We analyzed 4 U-CLL and 4 M-CLL samples, taken before and during (4-6 weeks of) ibrutinib treatment, by imaging flow cytometry, to evaluate the tendency of mIgM or mIgD to appear in high density regions (clusters). CLL cells displayed more IgM- than IgD-containing clusters, and the spot diameters for mIgM clusters were twice as broad as those for mIgD. However, while mIgD density correlated with the number of clusters, mIgM density did not. Taken together, these data indicate that mIgM and mIgD are organized differently on CLL cell membranes.Next, we assessed the association of activating (CD19, CD20) and inhibitory (CD22, Siglec10) co-receptors with the two mIG isotypes. These findings were then correlated with in vivo birth rates measured after patients drank 2H2O. Co-localization of CD19 and CD20 with mIgM directly correlated with increased proliferation in vivo . Conversely, those patients whose CLL B cells had more CD22 co-localized with IgM than IgD had lower in vivo birth rates. Notably, there was no such correlation for co-localization of Siglec10. Thus, there was differential co-localization of IgM with CD19 and CD20 versus with CD22 and these correlated respectively positively or negatively with CLL cell growth in vivo .The mIGs of CLL cells can spontaneously self-associate and transmit a signal to the leukemic cells. This property could influence membrane organization of the two isotypes, either directly by self-binding or indirectly by repetitive signaling and subsequent cytoskeleton rearrangement. To test this, we expressed 13 different CLL BCRs in triple knockout (TKO) cells as IgMs or IgDs while maintaining their original IGHVDJ and IGLVJ structure. For BCR receptors expressed as IgM, Ca++ mobilization occurred spontaneously, without the need for an external ligand (“autonomous signaling”). In contrast, BCR receptors expressed as IgD required ligand crosslinking for signaling. Therefore, while both mIgM and mIgD BCRs are capable of signaling upon antigen engagement, only mIgM BCRs naturally self-associate and transduce a signal. This implies a constitutive, self-reorganization of the BCR clusters and co-receptor localization that differs from normal resting B cells and primes CLL activation. The self-binding strength and the quality and reorganization capacities of the induced signaling likely lead to different cellular consequences.Finally, we studied patients undergoing ibrutinib treatment. Ibrutinib significantly upregulated the sm density of IgM and downregulated IgD and CD20; however, there was little change for CD19, CD22 and Siglec10. Ibrutinib also led to even more and bigger IgM and less and smaller IgD clusters. In addition, CD22 no longer co-localized with IgM, while the level of association with IgD did not change. Finally, co-localization of CD19 and CD20 with the IgM and IgD BCRs was not significantly altered indicating: i) their organization is not dependent on Btk signaling; ii) Localization changes take place in subpopulations, but the clonal median is invariant; or iii) Changes occur at levels below our detection method.In conclusion, IgM and IgD are organized differently on CLL B cells. IgM forms bigger and more clusters than IgD, and higher CLL birth rates are found for cells with more CD19 and CD20 in proximity to IgM than IgD. Also, autonomous signaling occurs only through IgM. Thus, CLL clones exhibit a membrane organization like that of an activated B cell. Conversely, increased association of CD22 with IgM correlates with slower birth rates and a less activated cell status. Ibrutinib treatment disregulates BCR isotype organization increasing IgM cluster size and number, decreasing those with IgD and changing the relative proximity of co-receptors such as CD22. DisclosuresBarrientos:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

CLL Stereotyped Subset #4 Igs Acquire Binding to Viable B Lymphocyte Surfaces By Somatic Mutations, Isotype Class Switching, and with the Prerequisite of IG Self-Association

The selection and transformation of a normal B cell into the leukemic state in chronic lymphocytic leukemia (CLL) is mediated at least in part by the structure of the B cell receptor (BCR) immunoglobulin (IG) antigen binding site. A dramatic indication of the role played by BCR is demonstrated by those CLL cases expressing “stereotyped” BCR IGs, characterized by very similar VH CDR3 amino acid sequences often encoded by specific IGHV, IGHD, and IGHJ gene segments. CLL stereotyped subset #4 BCR IGs expressing IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements are prime examples of this. Moreover, these clones are always IgG isotype class switched, and contain IGHV and IGLVκ somatic mutations. Moreover, some of these mutations are shared among patients, and are almost invariably similar physicochemically and located at specific positions in the antigen binding site. In the following study, we evaluated the effects of these unique, acquired structural changes on the (auto)antigenic specificities of subset #4, using 3 mAbs belonging to the subset.Gamma-irradiated Ramos B and Jurkat T cell line cells were incubated with subset #4 IGs under non-permeabilizing conditions, and binding to the B and T cell surfaces was detected by flow cytometry. Unlike most other CLL BCR IGs, the average binding percentage to apoptotic Ramos B cells of subset #4 mAbs was low. Conversely, each subset #4 IG bound live B cells well (CLL 183 - 44.8%; CLL 240 - 55.6%; CLL 342 - 52.0%). Moreover, all 3 IGs exhibited minimum binding to live and apoptotic Jurkat T cells. Next we exposed the subset #4 IGs to viable human B lymphocytes from tonsillectomy samples to confirm that binding was not limited to transformed B cells, and indeed the IGs bound well to CD19+ while minimally to CD3+ tonsil cells. When murine mAbs reactive with appropriate B-cell surface antigens were used to define subpopulations of normal B cells with which the subset #4 IGs interacted, we found that the subset #4 IGs and a non-subset #4 IGHV4-34-expressing CLL case (CLL 141) bound viable, naïve human B cells as expected from other studies. However, it became clear that the subset #4 IGs also interacted with viable memory B cells (CLL 183 - 28.1%; CLL 240 - 34.0%; CLL 342 - 42.7%; CLL 141 - 11.8%), a subpopulation that CLL 141 do not bind; the latter similar to IGHV4-34-expressing IGs in other disease states. Furthermore, although DNA and the “i” and “I” carbohydrate antigens are common targets of IGHV4-34 utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively, the subset #4 Igs did not bind to either of these autoantigens when tested by ELISA or glycan binding arrays, respectively.Strikingly, subset #4 IG binding to viable Ramos B cells and memory B lymphocytes depended on an asparagine (N) to aspartic acid (D) mutation at position 66 of FR3 in the rearranged IGKV2-30 gene (N vs. D: 5.26% vs. 55.6%); this amino acid residue is shared by many other clones in this subset and comes about by somatic hypermutation (SHM). Interestingly and consistent with the previous finding, another focused mutation of this subset, glutamic acid (E) at position 28 of FR1 in the rearranged IGHV4-34 (G28E), was responsible for the loss of DNA binding and for the diminished I/i glycan binding.Finally, binding to viable Ramos B cells required the presence of the IgG isotype characteristic of this subset, since reactivity with Ramos B lymphocytes disappeared when the stereotyped antigen-binding site was linked to IgM (183, 240, 342 IgG vs. IgM: 44.8%, 55.6%, 51.2% vs. 24.5%, 1.85%, 7.02%, respectively). This requirement for IgG isotype binding to live Ramos B cells is likely due to the documented subset #4 self-association which requires the gamma isotype, since the change of a single amino acid at VH FR1 (E16HA), which disrupts self-association, eliminated binding to Ramos B lymphocytes.Together our findings indicate that distinct autoantigens mediate the positive and negative selection of the structural elements acquired by SHM and isotype class switching that are characteristic of this unique subset of stereotyped BCR IGs. Moreover, our data show that the reactivity of subset #4 IGs with viable B-cell surface requires the self-association of these IGs and, therefore, strongly suggests that viable B-cell surface binding is mediated by a non-conventional binding site created by self-associated surface membrane bound IGs. DisclosuresKolitz:Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Gilead, Magellan, and Novartis: Honoraria; Celgene, Jazz: Equity Ownership. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

The multi-kinase inhibitor TG02 induces apoptosis and blocks B-cell receptor signaling in chronic lymphocytic leukemia through dual mechanisms of action

The constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

Molecular modelling of epitopes recognized by neoplastic B lymphocytes in Chronic Lymphocytic Leukemia

Identification of epitopes recognized by tumour B cells could provide insights into the molecular mechanisms of B cell tumorigenesis through aberrant B cell receptor (BCR) signalling. Here, we analysed the structure of eleven peptides binders of BCRs expressed in Chronic Lymphocytic Leukemia (CLL) patients in order to identify the chemical features required for cross-reactive binding to different CLL clonotypes. Four cross-reactive (CR) and seven no-cross-reactive (NCR) peptides were analysed by means of GRID molecular interaction fields, ligand-based pharmacophore and 3D-QSAR approaches. Based on pharmacophore model, two peptides were generated by specific amino acids substitutions of the parental NCR peptides; these new peptides resumed the common chemical features of CR peptides and bound the CLL BCR clonotypes recognized by CR peptides and parental NCR peptides. Thus, our computational approach guided the pharmacophore modelling of CR peptides. In perspective, peptide binders of CLL BCR clonotypes could represent a powerful tool for computational modelling of epitopes recognized by tumour B cells clones.

IGHV Gene Replacement: A Potential Mechanism for Establishing Stereotypy in Certain Cases of Chronic Lymphocytic Leukemia

Immunoglobulin heavy chain variable gene (IGHV) replacement or “VH replacement” (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible “footprint” at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL.We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences.The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions.Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to “short” (5 - ≤13), “medium” (13.1 - ≤20) and “long” (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above.Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5‘ portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5‘ end of the subset #6 VH CDR3.Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic. DisclosuresAgathangelidis:Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

Chronic Lymphocytic Leukemia B Cells Display IgM and IgD Isotype-Restricted Features That Affect Association with Co-Receptors, BCR Signaling, and Leukemic B-Cell Growth In Vivo

The leukemic cells in patients with chronic lymphocytic leukemia (CLL) are highly dependent on B-cell receptor (BCR) mediated signaling. Despite this and the fact that >90% of CLL clones co-express IgM and IgD, the composition and molecular mechanisms regulating BCR signaling regarding the two isotypes and the co-receptors with which they associate is lacking. Here we have addressed these issues.First, using Imaging Flow Cytometry, we evaluated BCR organization on the surface membrane of CLL cells from 11 patients who had participated in a 2H2O-labeling study that determined in vivoCLL B-cell birth rates (BR). We found that in all cases mIgM resided in more and larger surface clusters than mIgD. Also, a statistically significant, direct correlation was observed for IgM density and in vivoCLL-cell BR, with patients exhibiting more recently-divided cells having the highest expression of IgM. This was not the case for IgD.BCR signaling requires co-receptors that can co-localize differently with the two isotypes. Thus, we tested co-localization of stimulatory (CD20) and inhibitory (CD22) co-receptors with mIgM and mIgD, using the proximity ligation assay technique that discriminates 10 to 40 nm distances. Higher IgM:CD20 and lower IgD:CD20 co-localization ratios directly associated with in vivo BR. Conversely, patients whose CLL B cells showed greater IgM to CD22 co-localization ratios had lower BRs. Thus, association of IgM with stimulatory versus inhibitory co-receptors correlated with positive or negative regulation of CLL growth in vivo.Next, we questioned the extent that the observed differences in BCR organization affected the entire clone by measuring a marker of single cell metabolic activity - cell size. IgM and BR associated with entire clonal populations that were skewed toward larger, more active cells. Similarly, high BR CLLs displayed an increased mitochondrial maximal respiration and glycolytic activity and capacity, based on measurements of oxygen consumption rate and extracellular acidification rate, respectively.Since our findings supported a link between IgM- but not IgD-BCRs, growth rate in vivoand clonal metabolic activity, we questioned whether intrinsic, constitutive CLL BCR autonomous signaling differed for these two isotypes. To address this, we examined the signaling capacities of CLL-derived BCRs expressed as IgM or IgD isotypes, while maintaining the original IGHV-D-J and IGLV-J rearrangements. We used B cells that do not express endogenous BCR-related molecules but do express an inducible ERT2- SLP-65 fusion protein which enables examining Ca++influx. All BCRs expressed as IgM effectively mobilized Ca++ without need for an external ligand, indicating autonomous signaling. In contrast, BCRs expressed as IgD did not signal autonomously but required crosslinking with anti-BCR. Thus, only mIgM BCRs naturally transduce a signal in the absence of antigen.To determine the extent that BCR signaling influences clonal activity and in vivoBR, we compared cell size of CLL B cells taken from patients before and after 4 weeks of treatment with the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib (iBTK). Ibrutinib had a strong treatment effect on cell activity, reducing overall cell size in 10/11 patients. A comparison of single cell areas for patients with lower (BR = 0.54%) and higher (BR = 1.42%) BRs showed an overall reduction of the median cell size for both cases. Thus, iBTK treatment leads to an equilibration of the cell size profile among the cases differing in BR, indicating that ibrutinib acts proportionally more potently on more metabolically active CLL B cells. Likewise, these findings are consistent with BCR signaling, transduced through BTK, being responsible for the increased cellular activity of aggressive CLL clones.In conclusion, increased mIgM density and proximity of mIgM to stimulatory receptors is linked to greater metabolic activity clones and increased rate of proliferationin vivo. Conversely, proximity of mIgM to inhibitory receptors has the opposite correlations.Moreover, only mIgM carries out autonomous signaling, providing another biologic trait linking all these features. Thus, our data support a tight, isotype-dependent regulation of BCR signaling and its consequences for CLL B cells. Further understanding these mechanisms should help generate novel therapies to modify the quality of BCR-transduced signaling and thus cell fate. DisclosuresBarrientos:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency

Chronic lymphocytic leukemia (CLL) can be divided into prognostically distinct subsets with stereotyped or non-stereotyped, mutated or unmutated B cell receptors (BCRs). Individual subsets vary in antigen specificity and origin, but the impact of antigenic pressure on the CLL BCR repertoire remains unknown. Here, we employed IgH.TEμ mice that spontaneously develop CLL, expressing mostly unmutated BCRs of which ~35% harbor VH11-2/Vκ14-126 and recognize phosphatidylcholine. Proportions of VH11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEμ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of VH11/Vκ14-expressing CLL. Furthermore, CLL onset was accelerated by enhanced BCR signaling in Siglec-G−/− mice or in mice expressing constitutively active Bruton's tyrosine kinase. Transcriptional profiling revealed that VH11 and non-VH11 CLL differed in the upregulation of specific pathways implicated in cell signaling and metabolism. Interestingly, principal component analyses using the 148 differentially expressed genes revealed that VH11 and non-VH11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively. We identified an expression signature consisting of 13 genes that were differentially expressed in a larger panel of T cell-dependent non-VH11 CLL compared with T cell-independent VH11/Vκ14 or mutated IgH.TEμ CLL. Parallel differences in the expression of these 13 signature genes were observed between heterogeneous and stereotypic human unmutated CLL. Our findings provide evidence for two distinct unmutated CLL subsets with a specific transcriptional signature: one is T cell-independent and B-1 cell-derived while the other arises upon antigen stimulation in the context of T-cell help.

Combined Influence of B-Cell Receptor Rearrangement and Somatic Hypermutation on B-Cell Class-Switch Fate in Health and in Chronic Lymphocytic Leukemia.

A diverse B-cell receptor (BCR) repertoire is required to bind a wide range of antigens. BCRs are generated through genetic recombination and can be diversified through somatic hypermutation (SHM) or class-switch recombination (CSR). Patterns of repertoire diversity can vary substantially between different health conditions. We use isotype-resolved BCR sequencing to compare B-cell evolution and class-switch fate in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). We show that the patterns of SHM and CSR in B-cells from healthy individuals are distinct from CLL. We identify distinct properties of clonal expansion that lead to the generation of antibodies of different classes in healthy, malignant, and non-malignant CLL BCR repertoires. We further demonstrate that BCR diversity is affected by relationships between antibody variable and constant regions leading to isotype-specific signatures of variable gene usage. This study provides powerful insights into the mechanisms underlying the evolution of the adaptive immune responses in health and their aberration during disease.

Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.

Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment

Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined.Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells.Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands.Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases.Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment. DisclosuresWierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

IgM and IgD Receptors Differentially Contribute to CLL Survival and Chemokine Secretion: Implications for CLL Biology and Treatment

Background: B-cell receptor (BCR) signaling is a central pathway activated in the tissue microenvironment of patients with Chronic Lymphocytic Leukemia (CLL). CLL cells express IgM and IgD receptors, but the contribution of these isoforms to signaling and functional responses remains incompletely defined. The mutational status of CLL BCRs, on the other hand, has clear correlation with clinical outcome: patients with unmutated IGHV genes (U-CLL) characteristically have more aggressive disease and an inferior prognosis when compared to patients with mutated IGHV (M-CLL).Aim: The aim of this present study is to characterize functional differences between IgM and IgD signaling, in the context of the CLL microenvironment and of ibrutinib treatment.Methods: Freshly isolated, purified CLL cells from 80 patients (40 M-CLL and 40 U-CLL) were stimulated with soluble or bead-bound anti-IgM or anti-IgD; IgM, IgD expression, phosphorylation of the BCR-related molecules HS1 and ERK, F-actin polymerization and cell viability were analyzed by flow cytometry; HS1 and ERK phosphorylation, and BCL6 protein expression were also analyzed by Western Blot. CCL3 and CCL4 chemokine secretion was measured by ELISA. BCL6 binding to the CCL3 gene promoter region was assessed by ChIP-PCR. We utilized nurselike cell (NLC) co-cultures to activate BCR signaling and to induce CCL3 and CCL4 production in this model system that resembles the lymph node microenvironment. Effects of ibrutinib treatment on CLL-cell viability and BCR activation were also tested.Results: Surface IgM and IgD levels were significantly higher in U-CLL (P<0.0001), ZAP70 positive (P<0.01), and/or del17p or del11q CLL cells (P<0.05). Early BCR signaling with activation of the cytoskeletal protein HS1 and F-actin polymerization were similar after anti-IgM and anti-IgD stimulation. Downstream activation of ERK kinases was more prolonged after anti-IgM than after anti-IgD stimulation. Transient activation of IgD signaling was paralleled by rapid IgD receptor endocytosis, as compared to IgM. Furthermore, anti-IgM increased CLL viability to a significantly greater extent than anti-IgD, and exclusively induced secretion of CCL3 and CCL4 chemokines. This effect was consistently observed after stimulation with either soluble or bead-bound anti-IgM and anti-IgD. Expression of BCL6, which functions as a transcriptional repressor for CCL3 gene, was down-regulated following anti-IgM, but not after anti-IgD stimulation. In addition, we observed BCL6 binding to CCL3 promoter regions and noted that BCL6 down-regulation coincided with the onset of CCL3 secretion. In all experimental settings, U-CLL cells were more responsive to BCR stimulation than M-CLL. NLC co-culture induced down-modulation of surface IgM and IgD on CLL cells, increased HS1 and ERK phosphorylation and induced CCL3 chemokine secretion, along with BCL6 down-regulation. When CLL cells were removed from NLCs, recovery of surface IgM and IgD, baseline BCL6 expression, and HS1 and ERK phosphorylation was observed after 72 hours. Ibrutinib treatment preferentially interfered with anti-IgM induced survival signals and BCR signaling activation, in particular of U-CLL cells.Conclusion: Compared to IgD, IgM stimulation induces more durable signaling responses, CCL3 and CCL4 chemokine secretion and increases CLL-cell survival, in particular in U-CLL cells. BCL6 binding to CCL3 promoter and its down-regulation concomitant to CCL3 production suggests that BCL6 represses CCL3 gene expression in CLL cells. Co-culture of CLL cells with NLCs triggers BCR signaling, with functional outcomes that mainly recapitulate the features of IgM stimulation in vitro. Ibrutinib treatment preferentially interferes with IgM-mediated signals, and reduces CCL3 and CCL4 levels in CLL-cell supernatants and in the plasma of ibrutinib-treated patients. Taken together, these results support the relevance of IgM signaling in the pathogenesis of CLL, and suggest that IgM is the main receptor isotype targeted in vivo during ibrutinib treatment. DisclosuresEstrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Perturbation of the normal immune system in patients with CLL

AbstractImmune dysregulation is a cardinal feature of chronic lymphocytic leukemia (CLL) from its early stage and worsens during clinical observation, even in absence of disease progression. Although the mechanisms remain unclear, new insights are emerging into the complex relationship between the CLL clone and its immune environment. T cells are increased in early-stage disease and show progressive accumulation and exhaustion. The mechanisms that drive this expansion may include auto-antigens involved in the original clonal expansion. In addition, chronic viral infections such as cytomegalovirus generate huge virus-specific immune responses, which are further expanded in CLL. Attention is now focused largely on the direct immunosuppressive properties of the tumor. Remarkably, CLL clones often have features of the recently described regulatory B cells producing immunosuppressive IL-10. Better knowledge of the regulatory properties intrinsic to CLL cells may soon become more important with the switch from chemotherapy-based treatments, which trade control of CLL with further impairment of immune function, to the new agents targeting CLL B-cell receptor–associated signaling. Treatment with these new agents is associated with evidence of immune recovery and reduced infectious complications. As such, they offer the prospect of immunologic rehabilitation and a platform from which to ultimately replace chemotherapy.

Peptides That Bind Specifically to an Antibody from a Chronic Lymphocytic Leukemia Clone Expressing Unmutated Immunoglobulin Variable Region Genes

Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the disease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) engage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined the binding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable domains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected peptide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in families based on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb revealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (micromolar KD), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations (nanomolar KD) and with immunoglobulin heavy chain variable (IGHV)-mutated antibodies selected by environmental antigens. This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthy subjects with monoclonal B lymphocytosis.

Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

AbstractAbstract 2880 Background:The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods:The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results:Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions:These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures:No relevant conflicts of interest to declare.