Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone’s antigen receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of Toll-like receptor 9 (TLR-9), occasional MYD88 mutations, and BCR specificity for DNA or antigens physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis following B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN + interleukin-15 (IL-15), a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in-vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone’s BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated (ATM) anomaly + del13q14), and negatively linked to a very high proportion of CD38+ cells within the blood-derived B-CLL population. Furthermore, a clone’s intrinsic potential for in-vitro growth correlated directly with doubling time in blood, in the case of B-CLL with IGHV-unmutated BCR and <30% CD38+ cells in blood. Finally, in-vitro high-proliferator status was statistically linked to diminished patient survival. The above findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in-vivo B-CLL growth.

The binding of non-specific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Here we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped antibodies, IgM mutants and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM heavy chain and requires the IgM Cμ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cμ4 domain are equivalent to those regions of IgG and IgA recognised by Fc-binding proteins from bacteria, suggesting that this region of immunoglobulin molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and shows functional similarities with Fc-binding proteins from pathogenic bacteria.

Foci of autoantigen-specific B lymphocytes in nonlymphoid tissues have been associated with development of autoimmune disease. To better understand the genesis of such ectopic lymphoid tissue, this study investigated whether several B cell-tropic innate immune system molecules, known to be elevated in response to inflammatory stimuli, can cooperate in fostering the T cell-independent clonal expansion of mature human B2 cells under conditions of limiting BCR engagement. Notable synergy was observed between BCR coligation with the C3dg-binding CD21/CD19 costimulatory complex, B cell-activating factor belonging to the TNF family (BAFF), and IL-4 in generating B cell progeny with sustained CD86 and DR expression. The synergy was observed over a wide range of BCR:ligand affinities and involved: 1) cooperative effects at promoting early cell cycle progression and viability; 2) BCR:CD21 coligation-promoted increases in BAFF receptors that were highly regulated by IL-4; 3) reciprocal effects of IL-4 and BAFF at dampening daughter cell apoptosis typical of stimulation by BCR:CD21 and either cytokine alone; and 4) BAFF-sustained expression of antiapoptotic Mcl-1 within replicating lymphoblasts. The results suggest that significant clonal proliferation of recirculating B2 cells occurs upon limited binding to C3dg-coated Ag in an inflammatory in vivo milieu containing both BAFF and IL-4. When rare autoantigen-presenting B cells undergo such expansions, both B cell and T cell autoimmunity may be promoted.

IntroductionChronic lymphocytic leukemia (CLL) follows either an indolent or an aggressive course 1 and clinical decompensation is often accompanied by the appearance of new or increasing numbers of genetic aberrations associated with shorter survival, "clonal evolution." 2 The mechanism(s) responsible for the generation of these genetic abnormalities are not defined in CLL, which is not the case in certain other human cancers, especially lymphoid malignancies of germinal center (GC) origin, in which activation-induced deaminase (AID) appears to be pathogenic. [3][4][5] AID is required for the beneficial generation of Ab diversity in normal B lymphocytes by inducing IGV somatic hypermutation (SHM) and helps in the development of protective effector mechanisms by mediating IGH class-switch recombination (CSR). 6,7 These beneficial on-target AID activities occur primarily during a GC reaction and involve conversion of cytidine to uridine on single-stranded DNA at the IG locus. Such on-target actions in CLL B cells have been a matter of interest for several years, primarily because the presence or absence of IGHV mutations (which require AID) in CLL cells is closely linked to clinical outcome. Patients with leukemic clones with minimal (Ͻ 2% difference from germline) or no mutation in the IGHV (unmutated CLL [U-CLL]) have a far worse prognosis than patients with IGHV-mutated CLL (M-CLL). 8,9 Despite this SHM-based subcategorization of CLL cases, some clones exhibit ongoing IGHV diversification in vivo and in vitro, [10][11][12] with an antigen-driven pattern present in some cases, 13 and up to 50% of patients exhibit molecular evidence for intraclonal isotype CSR. [14][15][16][17][18] AID activity focused elsewhere 19 ) can lead to mutations, deletions, or translocations outside of the IG locus, as in GC-derived lymphomas. [3][4][5] However, such a role for AID in CLL has been questioned for several reasons: (1) although circulating CLL cells can express AID mRNA, [20][21][22] the number of such cells is exceedingly low (0.01%-0.2%) 22 ; (2) AID protein synthesis by these same cells has not been demonstrated 18,[20][21][22][23] ; (3) demonstration of the full range of AID functions is lacking in CLL, for example, by failure of cells to demonstrate SHM, especially for U-CLL clones, even on stimulation and induction of AID mRNA, 21 thereby creating the apparent paradox that U-CLL patients express more AID mRNA than M-CLL patients yet exhibit no or minimal SHM; and (4) despite association with several prognostic markers, 20,21,[24][25][26] no prospective analysis linking AID expression and disease severity has been performed.In the present study, we aimed to address these issues as a means of determining whether AID could be involved in the evolution of CLL to a more aggressive disease. We report that CLL cells are able to produce AID protein, but synthesis is The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and...

Although thymus-matured T cells are not necessary for in vivo B cell responses to a class of antigens designated T-independent (TI) 1 (1, 2), we wished to determine whether T cells could influence the nature of the antibody produced. It was our hypothesis that in a TI antigen system in which there is no obligate need for carrierspecific helper T cell triggering signals, T cells may still exert subtle regulatory effects on the B cell antibody response via a distinct population of Ig-specific regulatory cells (3-9). If this were indeed the case, such a system might be ideal for studying the regulatory effects of Ig-speeific T cells independently of carrier-specific T cells. The characteristics of Ig-specific T cell interactions with B cells could thus be elucidated and compared with the known characteristics of carrier-specific T cell-B cell interactions.In testing for T cell influences on B cell responses to TI antigens, our emphasis has been to search for possible effects of isotype-specific T cells. Our approach has been to determine whether T cells could differentially regulate the levels of the various isotypes of antibody made by the B cell population responding to a subset of TI antigens, designated type 2 TI (TI-2) antigens. These antigens are characterized by their lack of polyclonal activating activity and their inability to stimulate B cells from neonatal mice or mice with the CBA/N immune defect (10). We here demonstrate that the in vivo B cell response to TI-2 antigens can be influenced by T lymphocytes and that this influence is restricted to only some of the Ig isotypes produced, namely the IgG2a, and, to a lesser extent, the IgG2b subclasses. Materials and MethodsAnimals. C57BLI0/ScN nu/nu, C57BL10/ScN nu/+, C57BL/KaLwN, and (CBA/N × C57BL/6)F1 mice were obtained from the Small Animal Section, Division of Research Services, National Institutes of Health, Bethesda, Md. Age matched nu/nu and nu/+ male and female mice from 2-4 mo of age were used in each experiment.Antigens and lmmunizations. Trinitrophenyl-aminoethylcarbamylmethyl (TNP-AECM)-Ficoll (mol wt = 400,000; 55 mol TNP/mol Ficoll) was purchased from Biosearch (San Rafael, Calif.).

Of relevance to both protective and pathogenic responses to Ag is the recent finding that soluble molecules of the innate immune system, i.e., IL-4, B cell-activation factor of the TNF family (BAFF), and C3, exhibit significant synergy in promoting the clonal expansion of human B2 cells following low-level BCR ligation. Although IL-4, BAFF, and C3dg each contribute to early cell cycle entry and progression to S phase, only BAFF promotes later sustained viability of progeny needed for continued cycling. The present study sought to further clarify the mechanisms for BAFF’s multiple functions. By comparing BAFF and a proliferation-inducing ligand (APRIL) efficacy at different stages in the response (only BAFF binds BR3; both bind transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag, the early role was attributed to BR3, while the later role was attributed to TACI/B cell maturation Ag. Importantly, BAFF- and APRIL-promoted viability of cycling lymphoblasts was associated with sustained expression of cyclooxygenase 2 (COX-2), the rate-limiting enzyme for PGE2 synthesis, within replicating cells. Supernatants of cultures with BAFF and APRIL contained elevated PGE2. Although COX-2 inhibitors diminished daughter cell viability, exogenous PGE2 (1–1000 nM) increased the viability and recovery of lymphoblasts. Increased yield of viable progeny was associated with elevated Mcl-1, suggesting that a BAFF/APRIL → TACI → COX-2 → PGE2 → Mcl-1 pathway reduces activation-related, mitochondrial apoptosis in replicating human B2 cell clones.

In the absence of T cells, B cells were found to respond to the type 2 T-independent (TI-2) antigen, trinitrophenyl (TNP)-Ficoll, with a characteristic hierarchy of IgM and IgG subclass Ab production which directly correlated with 5' to 3' Igh-C gene order, i.e., IgM greater tha IgG3 greater than IgG1 greater than IgG2b greater than IgG2a. This was evident when immune serum Ab titers were analyzed, when in vitro secretion of antibody from immune cells was measured and when TNP-Ficoll-stimulated clones in a splenic focus assay were analyzed for isotype production. T cells were found to cause a preferential relative increase in the amount of IgG2a antibody produced to TNP-Ficoll. The T cell responsible was present in anti-IgM neonatally suppressed mice and was needed early in the response, i.e., on the day of immunization or earlier. T cells were found to increase the frequency of TNP-Ficoll-responsive B cell clones that produced IgG2a in the splenic focus assay. The great majority of these IgG2a-positive clones also produced IgM and all or nearly all of the IgG isotypes whose genes are encoded 5' to the Igh-gamma 2a gene. The data are discussed in terms to T cell enhancement of IgG2a Ab synthesis being mediated through T cell enhancement of the Igh-C gene switching mechanism within TNP-Ficoll-responsive B cell clones. Thus, isotypes encoded by genes on the 3' end of the Igh-gamma gene complex, which in the absence of T cells have a low probability of being switched to, are the most influenced by T cell help.

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fca/l receptor (hFca/lR). Ligand polymerization status was crucial for the interaction, because hFca/lR binding did not occur with monomeric Ab of either class. hFca/lR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFca/lR binding. IgM binding required contributions from both Cl3 and Cl4 Fc domains, whereas for dIgA, an exposed loop in the Ca3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFca/lR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFca/lR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFca/lR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.Key words: Human Fca/m receptor . IgA . IgM IntroductionA receptor for IgM and IgA, termed Fca/m receptor (Fca/mR), was first described in the mouse where it is constitutively expressed on the majority of B cells and macrophages [1]. Mouse Fca/mR (mFca/mR) can mediate endocytosis of IgM-coated targets, and may contribute to the primary stages of the immune response to microbes. It has been suggested that mFca/mR, due to its high affinity for IgM and intermediate affinity for IgA, may assist also in the maintenance of plasma concentrations of IgM and IgA [2]. Although there is no significant homology between Fca/mR and other known proteins, the N-terminal Ig-like domain of Fca/mR Ã These authors contributed equally to this work. Certain mAb raised against mFca/mR have been found to bind to a peptide encompassing the region from mFca/mR's Ig-like domain that corresponds to the Ig-binding motif in domain 1 of pIgR [6]. Interestingly, these mAb can block binding of IgA and IgM to mFca/mR. The fact that these same mAb cross-react with hFca/mR, which carries the conserved motif in its Ig-like domain, suggests that the site for IgM and IgA on hFca/mR lies in this same region. In contrast, the site(s) on human IgM and IgA that interacts with hFca/mR is unknown. To gain a better understanding of the structural requirements for the interaction of human IgA and IgM with hFca/mR, we have produced recombinant dIgA and pentameric IgM and have adopted a mutational approach to identify individual residues on the Fc involved in interaction with hFca/mR. We demonstrate that both the Cm3 and Cm4 domains of IgM are required for binding to the receptor, and that residues at the Fc inter-domain region of dIgA are critical for hFca...