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B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response. However, B1 cell-associated antibodies often cross-react with self-antigens, which leads to autoimmunity, and B1 cells have been implicated in cancer. How B1 cell activity is regulated remains unclear. We show that the Ikaros transcription factor is a major negative regulator of B1 cell development and function. Using conditional knock-out mouse models to delete Ikaros at different locations, we show that Ikaros-deficient mice exhibit specific and significant increases in splenic and bone marrow B1 cell numbers, and that the B1 progenitor cell pool is increased ∼10-fold in the bone marrow. Ikaros-null B1 cells resemble WT B1 cells at the molecular and cellular levels, but show a down-regulation of signaling components important for inhibiting proliferation and immunoglobulin production. Ikaros-null B1 cells hyper-react to TLR4 stimulation and secrete high amounts of IgM autoantibodies. These results indicate that Ikaros is required to limit B1 cell homeostasis in the adult., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC., Methods: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC., Results: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1β and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes., Conclusions: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Mixed cryoglobulinemia is usually considered to be a nonmalignant human B cell proliferation that produces a monoclonal IgM rheumatoid factor (RF). Important immunologic similarities and differences were described between the monoclonal B cells during mixed cryoglobulinemia and during malignant chronic lymphocytic leukemia (CLL):high frequency of the same VH and V kappa gene usage by both types of monoclonal B cells producing IgM with RF activity, apparent intraclonal homogeneity, but different expression of the pan T cell CD5 Ag. The description of an unusual CD5-negative B cell CLL case secreting a mutated IgM RF led the authors to suggest that the usage of non-mutated germline Ig genes is a property of cells derived from the CD5 lineage or stage of differentiation, rather than an intrinsic property of CLL or of IgM RF-producing cells in general. Because mixed cryoglobulinemia cells are usually CD5-negative, it was of interest to test for the existence of mutations in the VH and V kappa regions, as well as for the intraclonal homogeneity of the expressed Ig genes. In this study, we used the PCR technique to analyze the monoclonal rheumatoid factor (mRF) V genes from a patient with mixed cryoglobulinemia. We show that the CD5-negative monoclonal B cells express a slightly mutated V kappa 3 gene, but a more mutated VH1 gene whose genomic counterpart was shown to be the 51p1 germline gene. The sequence analysis of several independent clones shows some degree of intraclonal diversity, suggesting the existence of a clonal filiation. These results are discussed in terms of the origin of the monoclonal B cell during mixed cryoglobulinemia and CLL.

IgM-RF B cell precursors are abnormally overrepresented in "well differentiated" lymphoid monoclonal proliferations while data on less mature lymphoid malignancies are still awaited. This nevertheless suggests that RF activity plays a role in the transforming process perhaps by inducing constant stimulation of the precursor B cells. Despite the preferential use of similar VH and VL genes with little or no somatic hypermutations in both malignant B-cell CLL and nonmalignant mixed cryoglobulinemia, these proliferations do differ in CD5 membrane expression and in their clinical evolution. One possibility could be that CD5 glycoprotein is lost during maturation of the lymphocyte into a secreting cell as suggested by data on Waldenström's disease and the LES-CLL and by in vitro studies. Alternatively, CD5 expression could play an additional direct role in malignant transformation as suggested by recent data on the CD5 receptor ligand. Further data on the proliferating cells in both situations as well as on the genetic control of CD5 expression in B cells and its physiology should shed additional light on the mechanisms of B-cell malignancy.

The mixed cryoglobulinemia is considered to be a nonmalignant human B-cell proliferation that frequently produces a monoclonal IgM with anti-IgG activity (rheumatoid factor). Using murine monoclonal anti-idiotypic antibodies specific for private or minor idiotopes on monoclonal IgM from three patients suffering from nonmalignant mixed cryoglobulinemia, we investigated the presence of the CD5 antigen on the monoclonal IgM producing cells in these patients. It is shown by two-color cytofluorometric analysis that the majority of the peripheral blood monoclonal IgM rheumatoid factor secreting cells is CD5 negative in these three patients. One of the monoclonal rheumatoid factor K variable regions was sequenced at the protein level and belongs to the human VK III group, as a high proportion of monoclonal rheumatoid factors and some B-cell chronic lymphocytic leukemia (CLL) membrane bound Igs. Thus, despite the preferential use of similar VK genes and the absence of somatic mutation affecting these variable regions in both malignant B-cell CLL and nonmalignant mixed cryoglobulinemia, these proliferating B cells differ in the CD5 membrane expression.

Using dithiothreitol (DTT) technique, IgM anti-HLA anti-idiotypic antibodies were detected in a multiparous multitransfused woman. These antibodies were able to inhibit the binding of specific IgG anti-HLA antibodies on their corresponding antigen. The recognized determinants were cross-reactive determinants since they were partially found on anti-HLA antibodies from unrelated individuals. By studying the patient's sera over a period of 2 years, no IgM-IgG switch was observed but the presence of these antibodies was stable in time, despite the disappearance of the idiotypes (anti-HLA antibodies). However, when looking at the patient's earlier serum, it was shown that these IgM anti-idiotypic antibodies were absent from the first available serum. Thus, these anti-idiotypic antibodies seem to behave both like natural and immune antibodies. The incidence of such antibodies in pretransplant patients is discussed.

The idiotypic determinants on IgM rheumatoid factor (RF) from a single family have been analyzed. Rabbit Fab'2 antiidiotypic antibody was prepared against purified IgM-RF from a patient with rheumatoid arthritis. As measured by radioimmunoassay, the antiidiotype reacted with at least 90% of the patient's RF, but not with non-RF immunoglobulins from the same serum, nor with 10 of 11 polyclonal and monoclonal RF from unrelated individuals. Cross-reacting idiotypes were detected on RF in four of the patients' first degree relatives, spanning three generations, without apparent relation of HLA type or clinical rheumatoid arthritis. These results suggest that IgM-RF associated idiotypes were inherited in this family.

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenströms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.

The effects of DL-alpha-difluoromethylornithine (DFMeOrn), an irreversible inhibitor of L-ornithine decarboxylase, on immunoglobulin production were studied in vitro using human peripheral blood lymphocytes stimulated with pokeweed mitogen. DFMeOrn inhibits in a concentration-dependent manner the usual pokeweed mitogen-induced increases of polyamine contents (putrescine, spermidine, spermine) and of [3H]thymidine incorporation. In parallel with the reduction of polyamine content and of thymidine incorporation, IgG and IgM productions are diminished, a 70% decrease being observed at 5 mM DFMeOrn concentration. Therefore, it appears that inhibition of polyamine biosynthesis may ultimately interfere with the cellular immunologic response by blocking cell proliferation. These findings certainly deserve further consideration both under in vitro and in vivo conditions.