IgD/FcδR is involved in T-cell acute lymphoblastic leukemia and regulated by IgD-Fc-Ig fusion protein.

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis as a result of severe immunosuppression and rapid tumor progression with resistance to conventional chemotherapy. Excessive IgD may play a role in T cell activation via IgD Fc receptor (FcδR). Here we aimed to investigate the effects of IgD in T-ALL and demonstrated the potential benefit by targeting IgD/FcδR in T-ALL patients with IgD-Fc-Ig fusion protein. In T-ALL patients' blood samples and cell lines, the level of IgD, the percentage of FcδR expressing cells and the binding affinity were determined by flow cytometry. T cell viability, proliferation and apoptosis were analyzed. A mouse xenograft model was used to evaluate the in vivo effect of IgD-Fc-Ig, an IgD-FcδR blocker. The levels of serum IgD and FcδR were abnormally increased in part of T-ALL patients and IgD could induce over-proliferation and inhibit apoptosis of T-ALL cells in vitro. FcδR was constitutively expressed on T-ALL cells. IgD-Fc-Ig showed similar binding affinity to FcδR and selectively blocked the stimulation effect of IgD on T-ALL cells in vitro. In vivo study exhibited that IgD-Fc-Ig may also have therapeutic benefit. IgD-Fc-Ig administration inhibited human T-ALL growth and extended survival in xenograft T-ALL mice. In conclusion, this work supports the idea of targeting IgD/FcδR in T-ALL patients with excessive IgD. IgD-Fc-Ig fusion protein might be a potential biological drug with high selectivity for T-ALL treatment.

Clinical characteristics and outcomes of IgD myeloma: experience across UK national trials.

Immunoglobulin D (IgD) myeloma is a subtype often considered to have adverse features and inferior survival, but there is a paucity of data from large clinical studies. We compare the clinical characteristics and outcomes of patients with IgD myeloma from UK phase 3 myeloma trials analyzed in 2 groups: old (1980-2002) and recent (2002-2016) clinical trials, based on the time of adoption of novel myeloma therapies. Patients with IgD myeloma comprised 44 of 2789 (1.6%) and 70 of 5773 (1.2%) of the old and recent trials, respectively. Overall, IgD myeloma was associated with male predominance, low-level paraproteinemia (<10g/L), and λ light chain preference. The frequency of ultra-high-risk cytogenetics was similar in IgD myeloma compared with other subtypes (4.3% vs 5.3%, P > .99). Despite the old trial series being a younger group (median age: 59 vs 63 years, P = .015), there was a higher frequency of bone lesions, advanced stage at diagnosis, worse performance status, and severe renal impairment compared with the recent trials. Furthermore, the early mortality rate was significantly higher for the old trial series (20% vs 4%, P = .01). The overall response rate following induction therapy was significantly higher in the recent trials (89% vs 43%, P < .0001), and this was consistent with improved median overall survival (48 months; 95% confidence interval [CI] 35-67 months vs 22 months; 95% CI, 16-29 months). Survival outcomes for IgD myeloma have significantly improved and are now comparable to other myeloma types because of earlier diagnosis, novel therapies, and improved supportive care. This trial was registered at clinicaltrials.gov as # NCT01554852.

Immunoglobulin D and cardiac amyloidosis: development and a case illustration.

Amyloidosis results from extra-cellular deposition of proteins which interfere with tissue function. We report the case of a patient with pathological heart involvement which is caused by immunoglobulin D amyloidosis, and review current data on the amyloidois diagnosis and management.

Abrogation of nontypeable Haemophilus influenzae protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media.

The pneumococcal polysaccharide conjugate vaccine which includes a nonacylated protein D carrier from Haemophilus influenzae has been recently licensed for use in many countries. While this vaccine is protective against nontypeable Haemophilus influenzae (NTHI)-induced acute otitis media (OM), the mechanism underlying this protective efficacy is not yet fully understood. Protein D/glycerophosphodiester phosphodiesterase (PD/GlpQ) is an outer membrane lipoprotein expressed by NTHI that has been ascribed several functions, including host cell adherence and phosphorylcholine (PCho) acquisition. We found that a pd/glpQ NTHI mutant exhibited reduced adherence to airway epithelial cells, diminished phosphorylcholine (PCho) decoration of biofilms, and compromised fitness during experimental acute OM compared to the parent strain. We also found that exposure of NTHI to antibodies directed against the vaccine formulation recapitulated the PCho decoration and NTHI adherence phenotypes exhibited by PD/GlpQ-deficient NTHI, providing at least two likely mechanisms by which the pneumococcal polysaccharide-PD/GlpQ conjugate vaccine induces protection from NTHI-induced OM.

Neisseria lactamica selectively induces mitogenic proliferation of the naive B cell pool via cell surface Ig.

Neisseria lactamica is a commensal bacteria that colonizes the human upper respiratory tract mucosa during early childhood. In contrast to the closely related opportunistic pathogen Neisseria meningitidis, there is an absence of adaptive cell-mediated immunity to N. lactamica during the peak age of carriage. Instead, outer membrane vesicles derived from N. lactamica mediate a B cell-dependent proliferative response in mucosal mononuclear cells that is associated with the production of polyclonal IgM. We demonstrate in this study that this is a mitogenic human B cell response that occurs independently of T cell help and any other accessory cell population. The ability to drive B cell proliferation is a highly conserved property and is present in N. lactamica strains derived from diverse clonal complexes. CFSE staining of purified human tonsillar B cells demonstrated that naive IgD(+) and CD27(-) B cells are selectively induced to proliferate by outer membrane vesicles, including the innate CD5(+) subset. Neither purified lipooligosaccharide nor PorB from N. lactamica is likely to be responsible for this activity. Prior treatment of B cells with pronase to remove cell-surface Ig or treatment with BCR-specific Abs abrogated the proliferative response to N. lactamica outer membrane vesicles, suggesting that this mitogenic response is dependent upon the BCR.

CD20 deficiency in humans results in impaired T cell-independent antibody responses.

CD20 was the first B cell differentiation antigen identified, and CD20-specific mAbs are commonly used for the treatment of B cell malignancies and autoantibody-mediated autoimmune diseases. Despite this the role of CD20 in human B cell physiology has remained elusive. We describe here a juvenile patient with CD20 deficiency due to a homozygous mutation in a splice junction of the CD20 gene (also known as MS4A1) that results in "cryptic" splicing and nonfunctional mRNA species. Analysis of this patient has led us to conclude that CD20 has a central role in the generation of T cell-independent (TI) antibody responses. Key evidence to support this conclusion was provided by the observation that although antigen-independent B cells developed normally in the absence of CD20 expression, antibody formation, particularly after vaccination with TI antigens, was strongly impaired in the patient. Consistent with this, TI antipolysaccharide B cell responses were severely impeded in CD20-deficient mice. Our study therefore identifies what we believe to be a novel type of humoral immunodeficiency caused by CD20 deficiency and characterized by normal development of antigen-independent B cells, along with a reduced capacity to mount proper antibody responses.

K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity.

Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD(+)CD27(+) memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca(2+)-activated intermediate-conductance IKCa1 channel when quiescent, and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast, quiescent class-switched memory B cells express high levels of Kv1.3 ( approximately 2000 channels/cell) and maintain their Kv1.3(high) expression after activation. Consistent with their channel phenotypes, proliferation of naive and IgD(+)CD27(+) memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore, specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders.

BCR mutants deficient in ligand-independent and more sensitive for ligand-dependent signaling.

Signal transduction by the B cell antigen receptor (BCR) regulates development, survival and clonal expansion of B cells. The BCR complex comprises the membrane-bound immunoglobulin molecule (mIg) and the Ig-alpha/Ig-beta heterodimer, and was shown to form oligomeric structures. In pervanadate (PV)-treated B cells, multiple proteins are tyrosine phosphorylated upon expression of the BCR, indicating that the BCR can signal in an antigen-independent fashion. We analyzed the signal transduction from BCR mutants which either have an altered heavy chain transmembrane region or lack the Ig-alpha cytoplasmic tail. In comparison to cells expressing the wild-type receptors, those with a mutant BCR respond to PV treatment with reduced and retarded tyrosine phosphorylation of substrate proteins. Conversely, the cells with mutant BCR are more sensitive to stimulation with low doses of antigen. These data suggest that a correctly assembled BCR complex is important for antigen-independent signaling and setting the threshold for antigen-dependent BCR activation.

The novel IgD binding protein from Moraxella catarrhalis induces human B lymphocyte activation and Ig secretion in the presence of Th2 cytokines.

Moraxella IgD binding protein (MID) is a novel bacterial outer membrane protein with IgD-binding properties. MID was purified from the respiratory pathogen Moraxella catarrhalis and is here shown to have B cell stimulatory properties. Purified MID in the range of 0.01-0.1 microg/ml was optimal to induce a proliferative response in human PBL. MID coupled to Sepharose and formalin-fixed M. catarrhalis preparations induced similar proliferative responses in PBL cultures. MID or MID-Sepharose stimulated purified human peripheral B cells as measured by proliferation. In contrast, MID or MID-Sepharose did not activate T cells. Preincubation of purified B cells with anti-IgD Abs inhibited MID-Sepharose-induced B cell proliferation. The addition of IL-4 specifically induced IL-6 production in MID-Sepharose-activated B cells. IgM secretion was detected in B cell cultures stimulated with MID or MID-Sepharose and IL-2 for 10 days. Secretion of IgG and IgA was efficiently induced in cultures from purified B cells stimulated with the combination of MID or MID-Sepharose and IL-4, IL-10, and soluble CD40 ligand, suggesting that Th2-derived cytokines were required for optimal plasma cell generation. Taken together, MID has properties that make it an important tool to study IgD-targeted activation of B cells.

Involvement of Bik, a proapoptotic member of the Bcl-2 family, in surface IgM-mediated B cell apoptosis.

Apoptosis plays a central role in shaping the repertoire of circulating mature B lymphocytes, but the underlying molecular mechanisms regulating B cell fate are not well understood. Human B104 B lymphoma cells undergo apoptosis after surface Ig (sIg)M, but not sIgD, ligation; sIgM-mediated apoptosis of B104 cells apparently requires new gene transcription because actinomycin D can inhibit the apoptotic response. Here we report that expression of Bik, a proapoptotic member of the Bcl-2 family, is greatly increased after sIgM ligation. Bik expression was tightly controlled at both transcriptional and post-transcriptional levels. Whereas a calcineurin-dependent pathway was essential for Bik mRNA induction, both the phosphatidylinositol 3-kinase (PI3K)- and the calcineurin-dependent pathways were required for the sustained production of Bik protein. Consistent with these findings, sIgD ligation, which leads to the similar calcium mobilization and increases in Bik mRNA, induced only a transient activation of PI3K and did not lead to sustained Bik protein expression. Furthermore, sustained Bik protein expression correlated with B cell apoptosis, as treatment with either a calcineurin inhibitor or PI3K inhibitors blocked both sIgM-mediated sustained Bik protein induction and apoptosis. In addition, sIgM ligation strongly increased the amount of Bik associated with endogenous Bcl-x, but sIgD ligation did not. Studies with caspase inhibitors also revealed that Bik and Bcl-x interacted upstream of caspases in the B cell apoptosis cascade. Thus, Bik protein induction and, subsequently, sequestering of antiapoptotic Bcl-x by Bik may play an important role in regulating B cell apoptosis.

Structural and functional properties of membrane and secreted IgD.

More than 35 years ago, study of an unknown immunoglobulin (Ig) in the serum from a myeloma patient led to the discovery of IgD. Subsequently, the finding that it also exists as a membrane-bound Ig stimulated a large number of studies during the 70s. Then, the interest on IgD shrank, largely because of the lack of known function of secretory IgD (secIgD) and of a stagnating knowledge of the functions of surface IgD. In the recent years, very significant advances followed the tremendous accumulation of data on the physiology of the B cell receptor, of which IgD is the major component, on the role of secIgD in normal and diseased individuals. This review, which is focused on human IgD but integrates data in the mouse and other species when needed, summarizes present data on the structure, synthesis and functions of both membrane and secIgD, IgD receptors and the involvement of IgD in various diseases, especially the hyperIgD syndrome.

IgD can largely substitute for loss of IgM function in B cells.

The mu and delta heavy chains of IgM and IgD, the first antibody isotypes expressed during bone-marrow B-cell development, are encoded by a common transcription unit. Expression of the mu chain on the surface of late pre-B cells allows their further development to immature B cells. Coexpression of the delta chain and emigration of the immature B cells to the periphery eventually leads to the development of naive mature IgM/IgD double-positive cells. Although IgM is important in driving B-cell development, the contribution of IgD is not clear. Here we investigate the function of IgD. We generated mice deficient in IgM (IgM-/- mice) by deleting the mu region in embryonic stem cells. IgM-/- mice showed normal B-cell development and maturation, with IgD replacing membrane-bound and secretory IgM. Moreover, specific B-cell responses and isotype class switches occurred during immunization or infection. In contrast to mice deficient in B cells, IgM-/- mice survived infection with vesicular stomatitis virus by developing neutralizing immunoglobulins, but they were more susceptible than wild-type controls with delayed specific immunoglobulin responses. These data lead us to conclude that IgD is largely able to substitute for IgM functions.

Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes.

Despite intensive efforts, the intracellular signaling pathways that mediate apoptosis remain unclear. The human B lymphoma cell line, B104, possesses characteristics that make it an attractive model for analysis of receptor-mediated apoptosis. Although these cells express both membrane IgM (mIgM) and membrane IgD (mIgD) crosslinking mIgM results in significant apoptosis while crosslinking mIgD does not. Our results show that crosslinking mIgM but not mIgD induced a delayed and sustained activation of the mitogen-activated protein kinase (MAPK) family members stress-activated protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin, which also induces apoptosis in B104 cells, stimulated a similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor of apoptosis induced by either mIgM or ionomycin, inhibited activation of both SAPK and p38 MAPK, suggesting that stimulation of these kinases may be required for induction of apoptosis. Collectively, our results indicate that SAPK and p38 MAPK may be downstream targets during mIgM-induced, calcium-mediated, apoptosis in human B lymphocytes.

Phosphatidylinositol-3-kinase activity is required for the anti-ig-mediated growth inhibition of a human B-lymphoma cell line.

Stimulation of B lymphocytes through the Ig receptor initiates a cascade of biochemical changes, which can ultimately lead to either activation and growth, or cell-cycle arrest and cell death. One of the critical events that occurs in both cases is the activation of tyrosine kinases, and the resulting phosphorylation of a variety of proteins on tyrosine residues. In this report we identify one of the substrates of phosphorylation as the 85-kD subunit of the enzyme phosphatidylinositol-3 kinase (PI3K), and show that both anti-IgM and anti-IgD stimulation results in an increase in the anti-phosphotyrosine-precipitable PI3K activity. Furthermore, we show that the potent and specific inhibitor of PI3K, Wortmannin, can completely abrogate anti-Ig-mediated growth inhibition without affecting tyrosine kinase induction or protein kinase C (PKC) activation. Treatment of intact cells with Wortmannin results in an irreversible decrease in anti-Ig-induced PI3K activity, suggesting that the effect of Wortmannin on anti-Ig-mediated growth inhibition is caused by its inactivation of PI3K activity. Taken together, these data show that activation of PI3K is a critical component of the anti-Ig-initiated signaling cascade that leads to growth inhibition of human B lymphoma cells.

Antigen receptor-triggered apoptosis in immature B cell lines is associated with the binding of a 44-kDa phosphoprotein to the PTP1C tyrosine phosphatase.

While cross-linking of the membrane IgM (mIgM) molecules expressed on WEHI 231 lymphoma cells induces these cells to undergo apoptosis, we have previously observed that ligation of the mIgD expressed on IgD-transfected WEHI 231 (W delta) cells is not associated with induction of cell death. Thus mIgM+IgD+ W delta cells provide a valuable reagent for delineating the molecular events which modulate the physiologic outcome of B cell antigen receptor (BCR) engagement. In view of recent data implicating the cytosolic phosphotyrosine phosphatase PTP1C in the regulation of BCR signaling capacity, we used W delta cells to investigate the potential role for PTP1C in modulating the cell response to BCR activation. The results of this analysis revealed PTP1C to undergo rapid tyrosine phosphorylation following mIgM or mIgD cross-linking and to associate with a number of other phosphoproteins in stimulated W delta cells. Among these latter phosphoproteins, one prominent species of about 44 kDa (pp44) which co-precipitated with PTP1C in mIgM-ligated cells was not detected in PTP1C immunoprecipitates from mIgD-ligated cells. The association of PTP1C with this 44-kDa phosphoprotein following mIgM cross-linking was also observed in two additional B cell lines representing an immature state of differentiation, but was not detected after BCR engagement in two representative mature B cell lines or in splenic B cells. Initial data concerning the identity of pp44 indicate that this molecule does not represent the Shc, MAPK or Ig-beta proteins and may, therefore, constitute a previously unidentified signaling effector. While the structural and biochemical properties of pp44 require further definition, the findings suggest that BCR-triggered interactions of PTP1C with pp44 occur only in the context of an immature state of cellular differentiation and the induction of apoptosis. These data therefore suggest that PTP1C interactions with pp44 may be relevant to the transduction of BCR signals which evoke cell death.

Regulation of IgD-receptor expression on murine T cells. II. Upregulation of IgD receptors is obtained after activation of various intracellular second-messenger systems; tyrosine kinase activity is required for the effect of IgD.

The presence of IgD receptors (IgD-R) on T cells during a primary response to antigen causes augmented antibody production and facilitates priming for a secondary response. Cross-linked, but not monomeric IgD leads to a rapid upregulation of these receptors on T cells. As shown in the present study, the rapid upregulation of IgD-specific receptors is also induced by cross-linking of T cell surface molecules known to mediate triggering of T cell activation, such as CD3, CD2, and Thy 1. Furthermore, IgD-R are also upregulated by pharmacologically active compounds that increase intracellular cAMP and by PMA/DiOG plus ionomycin, but not by either PMA or ionomycin alone. The upregulation of IgD-R by anti-CD3 is inhibited by both calphostin C and herbimycin A, while that due to DiOG plus ionomycin is only inhibited by calphostin C. Upregulation of IgD-R by increased cAMP is blocked by HA1004, but not by low concentrations of staurosporine or herbimycin A. IgD itself does not cause an increase in intracellular cAMP, protein kinase C translocation, influx of extracellular Ca2+, or a change in membrane potential. Relatively specific inhibitors of these activation pathways, HA1004, calphostin C, and neomycin, also fail to interfere with IgD-receptor upregulation by IgD itself. However, tyrosine kinase inhibitors, including herbimycin A, tyrphostin C11, and genistein, completely prevent the effect of IgD on IgD-R expression. Although an influx of Ca2+ is apparently not involved, a role for intracellular Ca2+ in the upregulation of IgD-R by IgD on T cells is indicated by the susceptibility to inhibition by BAPTA, W7, and FK520. We conclude that activation of at least three different second-messenger systems can cause IgD-R upregulation, but that the effect of IgD itself requires tyrosine kinase activity, perhaps in an intracellular Ca(2+)-dependent manner.

Membrane region of surface IgM is not sufficient for transducing growth inhibitory signals in an immature B cell line WEHI-231.

The murine B lymphoma line WEHI-231 is representative of immature B cells. Like normal immature B cells, WEHI-231 is susceptible to growth arrest following cross-linking of surface IgM (sIgM). Previously, we have shown using a WEHI-231 immunoglobulin (Ig) delta-transfectant that sIgD cross-linking failed to initiate growth arrest, in contrast to sIgM. In this report, we extend our research to investigate the structural requirement of Ig mu chain for regulating growth inhibition. Recombinant, chimeric Ig molecules delta/mu m and mu/delta m consisting of exons encoding extracellular delta and mu domains and membrane regions of different isotypes were constructed and introduced into WEHI-231 cells. A similar approach was used for sIgG2b-expressing transfectants. Our findings indicate that the mu m region is not sufficient for regulation of growth inhibition in WEHI-231 cells and suggest that additional extracellular region(s) of mu chain may be required for this response.