Monoclonal B-cell lymphocytosis in a hospital-based UK population and a rural Ugandan population : a cross-sectional study

BACKGROUND: Reported incidence of B-cell malignancies shows substantial geographical variation, being more common in the Americas and Europe than in Africa. This variation might reflect differences in diagnostic capability, inherited susceptibility, and infectious exposures. Monoclonal B-cell lymphocytosis (MBL) is a precursor lesion that can be screened for in apparently healthy people, allowing comparison of prevalence across different populations independently of health-care provision. We aimed to compare the prevalence and phenotypic characteristics of MBL in age-and-sex-matched populations from rural Uganda and the UK. METHODS: In this cross-sectional study, we recruited volunteers aged at least 45 years who were seronegative for HIV-1 from the established Ugandan General Population Cohort and obtained their whole-blood samples. We also obtained blood samples from anonymised waste material of age-and-sex-matched individuals (aged >45 years, with a normal blood count and no history of cancer) in the UK. We used flow cytometry to determine the presence of MBL, defined according to standard diagnostic criteria, in the samples and compared differences in the proportion of cases with chronic lymphocytic leukaemia (CLL)-phenotype MBL and CD5-negative MBL, as well as differences in absolute monoclonal B-cell count between the two cohorts. FINDINGS: Between Jan 15 and Dec 18, 2012, we obtained samples from 302 Ugandan volunteers and 302 UK individuals who were matched by age and sex to the Ugandan population. Overall MBL prevalence was higher in the Ugandan participants (42 [14%] individuals) than in the UK cohort (25 [8%]; p=0·038). CLL-phenotype MBL was detected in three (1%) Ugandan participants and 21 (7%) UK participants (p=0·00021); all three Ugandan participants had absolute monoclonal B-cell count below one cell per μL, whereas the 21 UK participants had a median absolute number of circulating neoplastic cells of 4·6 (IQR 2-12) cells per μL. The prevalence of CD5-negative MBL was higher in the Ugandan cohort (41 [14%], of whom two [5%] also had CLL-phenotype MBL) than in the UK cohort (six [2%], of whom two [33%] also had CLL-phenotype MBL; p<0·0001), but the median absolute B-cell count was similar (227 [IQR 152-345] cells per μL in the Ugandan cohort vs 135 [105-177] cells per μL in the UK cohort; p=0·13). INTERPRETATION: MBL is common in both Uganda and the UK, but the substantial phenotypic differences might reflect fundamental differences in the pathogenesis of B-cell lymphoproliferative disorders. FUNDING: UK Medical Research Council and UK Department for International Development

Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors

PI3K Signaling in Normal B Cells and Chronic Lymphocytic Leukemia (CLL).

B cells provide immunity to extracellular pathogens by secreting a diverse repertoire of antibodies with high affinity and specificity for exposed antigens. The B cell receptor (BCR) is a transmembrane antibody, which facilitates the clonal selection of B cells producing secreted antibodies of the same specificity. The diverse antibody repertoire is generated by V(D)J recombination of heavy and light chain genes, whereas affinity maturation is mediated by activation-induced cytidine deaminase (AID)-mediated mutagenesis. These processes, which are essential for the generation of adaptive humoral immunity, also render B cells susceptible to chromosomal rearrangements and point mutations that in some cases lead to cancer. In this chapter, we will review the central role of PI3K s in mediating signals from the B cell receptor that not only facilitate the development of functional B cell repertoire, but also support the growth and survival of neoplastic B cells, focusing on chronic lymphocytic leukemia (CLL) B cells. Perhaps because of the central role played by PI3K in BCR signaling, B cell leukemia and lymphomas are the first diseases for which a PI3K inhibitor has been approved for clinical use

B Cell Receptors Of Chlamydophila Psittaci negative MALT Lymphomas Of The Ocular Adnexa Recognize Common Self-Antigens

Abstract Ocular adnexal mucosa associated lymphoid tissue lymphomas (OAMALTLs) are the most common lymphomas in the ocular adnexa. The etiology and pathogenesis of OAMALTLs are still controversial. Association with Chlamydophila (C.) psittaci infection demonstrates marked geographic differences. We have previously reported biased IGHV (IGHV4 family and IGHV4-34 gene) and IGKV (IGKV3-20) usage in C. psittaci-negative OAMALTLs (PLoS One. 2011;6(12):e29114; Am J Hematol.2013; 88:379). However, the identity and role of potential antigens (Ags) in OAMALTL pathogenesis are unknown. Herein we evaluated the reactivity of OAMALTLs derived B-cell receptors (BCRs) for bacterial and human antigens. IGHV and IGKV genes derived from 5 OAMALTLs (tumors 4726 and 4438 expressing IGHV4-34 with and without somatic mutations, respectively, both paired with IGKV3-20; tumor 4968 expressing IGHV3-30 and IGKV1-33; tumor 11274 expressing IGHV3-23 and IGKV3-20; and tumor 5334 expressing IGHV4-59 and IGKV3-1) were cloned into pAH6180 and pAN6714 plasmids, respectively. Irrespective of the original isotype, all recombinant antibodies (rAbs) representing tumor BCRs were expressed on a common IgG3 heavy chain backbone to facilitate the detection and comparability in reactivity assays and to ensure that differences in binding activity could be attributed to specific variable regions. rAbs were produced in HEK293T cells adapted to serum-free suspension cultures. rAbs did not react with bacterial proteins derived from lysates of Staphylococcus aureus, Salmonella enteridis and C. psittaci infected HeLa cells. The rAbs also did not react with I/i Ags. Rheumatoid factor (RF) enzyme-linked immunosorbent assay (ELISA) suggested that rAbs derived from tumors 4726, 4968, 5334, and 11274 exhibit rheumatoid factor activity. However, a competitive inhibition assay using increasing quantities of purified human IgG Fc fragment failed to block the reactivity, indicating non-specific RF reactivity. To test the rAbs for reactivity with self-antigens, the standard indirect immunofluorescence assay (IFA) using permeabilized human HEp-2 cells was performed. All tumor derived rAbs were reactive with HEp-2 cells and exhibited diverse staining patterns; predominantly cytoplasmic staining with rAbs 5334 and 4968 and combined nuclear and cytoplasmic staining with the others. We next examined tumor derived rAbs for polyreactivity based on the commonly used definition of reactivity with at least two of the following diverse self and non-self antigens: ssDNA, dsDNA, insulin, and LPS. Only rAb 4726 showed polyreactivity based on these standard criteria. To identify the self-antigens recognized by the other OAMALTLs derived rAbs, antibody specificity profiling was performed using ProtoArray Human Protein Microarrays v5.0 containing more than 9,000 proteins (Life Technologies), using 0.1 μg/mL and 1.0 μg/mL concentrations of each rAb. The data were processed using Invitrogen’s proprietary ProtoArray Prospector software. These analyses identified 17, 11, 9 and 25 candidate Ags for rAb4438, rAb4726, rAb5334, and rAb11274, respectively. Some of the candidate Ags were unique for specific rAbs (e.g. methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 (MTHFD2) reactive with rAb4438) while others were recognized by more than one rAb (e.g. galectin-3/LGALS3 was reactive with rAb4438, rAb4726 and rAb11274). Follow up bidirectional co-immunoprecipitation with commercial and tumor derived rAbs confirmed recognition of these, as well as several additional protein microarray identified Ags. We specifically focused on galectin-3, which was recognized by 3 of the tumor BCRs. ELISAs performed with and without lactose confirmed rAbs reactivity with galectin-3. IFA using rAbs and a commercial galectin-3 Ab confirmed colocalization; siRNA-induced galectin-3 knockdown eliminated staining with both commercial and tumor derived rAbs. Immunohistochemistry of primary OAMALTLs showed that galectin-3 is expressed by tumor microenvironment cells but not by the tumor lymphocytes. Our findings demonstrate that the BCR of C. psittaci negative OAMALTLs exhibit oligo-self-reactivity and identified galectin-3 as a common antigen. The role of these antigens in pathogenesis of OAMALTLs is under investigation. Disclosures: No relevant conflicts of interest to declare

Responsiveness of B cell is regulated by hinge region of IgD

Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function