Antibody bivalency improves antiviral efficacy by inhibiting virion release independently of Fc gamma receptors.

Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.

Fc-Receptor Targeted Therapies for the Treatment of Myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease in which immunoglobulin G (IgG) antibodies (Abs) bind to acetylcholine receptors (AChR) or to functionally related molecules in the postsynaptic membrane at the neuromuscular junction. IgG crystallizable fragment (Fc)-mediated effector functions, such as antibody-dependent complement deposition, contribute to disease development and progression. Despite progress in understanding Ab-mediated disease mechanisms, immunotherapy of MG remained rather unspecific with corticosteroids and maintenance with immunosuppressants as first choice drugs for most patients. More specific therapeutic IgG Fc-based platforms that reduce serum half-life or effector functions of pathogenic MG-related Abs are currently being developed, tested in clinical trials or have recently been successfully translated into the clinic. In this review, we illustrate mechanisms of action and clinical efficacies of emerging Fc-mediated therapeutics such as neonatal Fc receptor (FcRn)-targeting agents. Furthermore, we evaluate prospects of therapies targeting classical Fc receptors that have shown promising therapeutic efficacy in other antibody-mediated conditions. Increased availability of Fc- and Fc receptor-targeting biologics might foster the development of personalized immunotherapies with the potential to induce sustained disease remission in patients with MG.

Inhibition of Maternal-to-Fetal Transfer of IgG Antibodies by FcRn Blockade in a Mouse Model of Arthrogryposis Multiplex Congenita.

OBJECTIVE: To determine whether blocking the neonatal Fc receptor (FcRn) during gestation with an anti-FcRn monoclonal antibody (mAb) reduces transfer of pathogenic maternal antibodies in utero and decreases the likelihood of maternal antibody-mediated neonatal disease in the offspring. METHODS: Using a previously established maternal-to-fetal transfer mouse model of arthrogryposis multiplex congenita (AMC), we assessed the effect of 4470, an anti-FcRn mAb, on the transfer of total human immunoglobulin G (IgG) and specific acetylcholine receptor (AChR)-antibodies from mother to fetus, as well as its effect on the prevention of neurodevelopmental abnormalities in the offspring. RESULTS: Offspring of pregnant dams treated with 4470 during gestation showed a substantial reduction in total human IgG and AChR antibody levels compared with those treated with the isotype mAb control. Treatment with 4470 was also associated with a significant reduction in AMC-IgG-induced deformities (limb or spinal curve malformations) when compared with mAb control-exposed embryos and a nonsignificant increase in the percentage of fetuses showing spontaneous movements. 4470 exposure during pregnancy was not associated with changes in general parameters of maternal well-being or fetal development; indeed, male neonates showed faster weight gain and shorter time to reach developmental milestones. CONCLUSIONS: FcRn blockade is a promising therapeutic strategy to prevent the occurrence of AMC and other human maternal autoantibody-related diseases in the offspring.

Prevention of diabetes-associated fibrosis: Strategies in FcRn-targeted nanosystems for oral drug delivery.

Diabetes mellitus is a chronic disease with an elevated risk of micro- and macrovascular complications, such as fibrosis. To prevent diabetes-associated fibrosis, the symptomatology of diabetes must be controlled, which is commonly done by subcutaneous injection of antidiabetic peptides. To minimize the pain and distress associated with such injections, there is an urgent need for non-invasive oral transmucosal drug delivery strategies. However, orally administered peptide-based drugs are exposed to harsh conditions in the gastrointestinal tract and poorly cross the selective intestinal epithelium. Thus, targeting of drugs to receptors expressed in epithelial cells, such as the neonatal Fc receptor (FcRn), may therefore enhance uptake and transport through mucosal barriers. This review compiles how in-depth studies of FcRn biology and engineering of receptor-binding molecules may pave the way for design of new classes of FcRn-targeted nanosystems. Tailored strategies may open new avenues for oral drug delivery and provide better treatment options for diabetes and, consequently, fibrosis prevention.

Highlights in Autoimmunity: 2020.

BACKGROUND: Innate and adaptive immune response dysregulations are equally involved in the induction of autoimmunity. Toll-like receptors play a leading role in the activation of innate immune cells, thus priming auto-reactive T cells. Th17 cells and related cytokines are widely involved in many immune-mediated diseases such as rheumatoid arthritis. Thus, the recent introduction of anti-IL-17 therapies should be further evaluated. Janus kinase inhibitors and Fc receptor-targeting drugs are some of the new therapeutic strategies that are being implemented when old classical therapies lack sufficient beneficial outcomes.

Drug-Disease Interaction and Time-Dependent Population Pharmacokinetics of Isatuximab in Relapsed/Refractory Multiple Myeloma Patients.

Isatuximab, a monoclonal antibody (mAb) of immunoglobulin G (IgG) isotype, specifically targets the cluster of differentiation 38 antigen overexpressed in malignant plasma cells. Isatuximab is used to treat multiple myeloma (MM), characterized by the excessive production of abnormal "myeloma proteins" (M-proteins) that may interact with therapeutic IgG mAb on the neonatal Fc receptor (FcRn)-mediated recycling pathway. The clinical pharmacology profile of isatuximab was investigated by population pharmacokinetics (PKs) modeling in 476 patients with MM who received 1-20 mg/kg isatuximab either as single agent or in combination with pomalidomide-dexamethasone in 4 clinical trials. Isatuximab PKs were characterized by a two-compartment model with parallel time-varying linear clearance (CL) and nonlinear elimination. Due to a mechanism-based drug-disease interaction, patients secreting IgG M-protein exhibited a twofold lower drug exposure compared with patients with non-IgG MM. No dose adjustment was required based on MM immunoglobulin type because efficacy and safety profiles were comparable between IgG and non-IgG MM subpopulations. ß2-microglobulin, body weight, sex, drug material, and race have a limited effect on drug exposure and do not require any dose adjustment. A typical 50% decrease in linear CL from initial treatment to steady-state was predicted, and this decrease correlated with the best overall response rate and was slower for patients with IgG MM. These findings suggest that the time-dependent effect of isatuximab is likely mediated by a combined factor of both disease state evolution and the perturbation of the FcRn-mediated recycling pathway.

A Physiologically-Based Pharmacokinetic Model for the Prediction of "Half-Life Extension" and "Catch and Release" Monoclonal Antibody Pharmacokinetics.

Monoclonal antibodies (mAbs) can be engineered to have "extended half-life" and "catch and release" properties to improve target coverage. We have developed a mAb physiologically-based pharmacokinetic model that describes intracellular trafficking, neonatal Fc receptor (FcRn) recycling, and nonspecific clearance of mAbs. We extended this model to capture target binding as a function of target affinity, expression, and turnover. For mAbs engineered to have an extended half-life, the model was able to accurately predict the terminal half-life (82% within 2-fold error of the observed value) in the human FcRn transgenic (Tg32) homozygous mouse and human. The model also accurately captures the trend in pharmacokinetic and target coverage data for a set of mAbs with differing catch and release properties in the Tg32 mouse. The mechanistic nature of this model allows us to explore different engineering techniques early in drug discovery, potentially expanding the number of "druggable" targets.

The neonatal Fc receptor: Key to homeostasic control of IgG and IgG-related biopharmaceuticals.

IgG and albumin are the most abundant proteins in the circulation and have the longest half-lives. These properties are due to a unique receptor, the neonatal Fc receptor (FcRn). Although FcRn is named for its function of transferring IgG across the placenta from maternal to fetal circulation, FcRn functions throughout life to maintain IgG and albumin concentrations. FcRn protects IgG and albumin from intracellular degradation and recycles them back into the circulation. Clinical trials have confirmed that pathogenic antibodies can be depleted by blocking this homeostatic function of FcRn. Moreover, understanding the molecular interactions between IgG and FcRn has resulted in the design of therapeutic monoclonal antibodies with more efficacious pharmacokinetics. As a result of genetic engineering these monoclonals can be delivered at lower doses and at longer intervals. More recent findings have demonstrated that FcRn enhances phagocytosis by neutrophils, immune complex clearance by podocytes and antigen presentation by dendritic cells, macrophages, and B cells. This minireview highlights the relevance of FcRn to transplantation.

Directed evolution of super-secreted variants from phage-displayed human Interleukin-2.

Selection from a phage display library derived from human Interleukin-2 (IL-2) yielded mutated variants with greatly enhanced display levels of the functional cytokine on filamentous phages. Introduction of a single amino acid replacement selected that way (K35E) increased the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity- the major manufacturability issue limiting IL-2 usefulness up to now. Improvement of secretion was also shown for a panel of IL-2-engineered variants with altered receptor binding properties, including a selective agonist and a super agonist that kept their unique properties. Our findings will improve developability of the growing family of IL-2-derived immunotherapeutic agents and could have a broader impact on the engineering of structurally related four-alpha-helix bundle cytokines.

The immunoglobulin D Fc receptor expressed on fibroblast-like synoviocytes from patients with rheumatoid arthritis contributes to the cell activation.

Immunoglobulin IgD might play an important role in autoimmune diseases, but the function of IgD has remained elusive, despite multiple attempts to define its biological function. Fibroblast-like synoviocytes (FLSs) are specialized cells of the synovium that play a key role in the pathogenesis of rheumatoid arthritis (RA). In this study we explored the possible roles of excessive IgD expression on the function of FLSs from RA patients (RA-FLSs). We showed that IgD Fc receptor (IgDR) was constitutively expressed on FLSs, and was significantly elevated in RA-FLSs compared with FLSs prepared from synovial tissues of healthy controls (HC-FLSs). Furthermore, IgDR was mainly detected on the cell surface and in the cytoplasm. We further detected the intrinsic binding affinity of IgD to IgDR on HC-FLSs with an equilibrium dissociation constant (KD) of 0.067 nmol/L. Incubation of RA-FLSs with IgD (1-10 µg/mL) for 48 h dose-dependently promoted the expression of IgDR, and stimulated the production of inflammatory cytokines and chemokines, such as IL-1ß, IL-6, monocyte chemotactic protein (MCP)-1, TNF-α and receptor activator of nuclear factor-κB ligand (RANKL), thus potentially contributing to IgD-IgDR crosslinking. Moreover, incubation with IgD (0.1-10 µg/mL) for 48 h dose-dependently enhanced viability for both HC-FLSs and RA-FLSs. Our results demonstrate that IgDR is expressed on RA-FLSs and contributes to the activation of FLSs, and suggest that IgD-IgDR is a potential novel immunotherapeutic target for the management of RA.

Development of Romiplostim for Treatment of Primary Immune Thrombocytopenia From a Pharmacokinetic and Pharmacodynamic Perspective.

Romiplostim is a novel thrombopoiesis-stimulating peptibody consisting of a carrier Fc domain and a peptide domain that binds to the thrombopoietin receptor (TPOR) on platelets and platelet precursors. Similar to endogenous thrombopoietin, romiplostim activates the TPOR to stimulate the growth and maturation of megakaryocytes, resulting in increased production of platelets in the circulation. Binding of romiplostim to TPOR on the platelets and megakaryocytes presumably triggers subsequent internalization and degradation. Therefore, increased platelet counts following romiplostim treatment results in increased elimination of the drug. The TPOR target-mediated process is saturable, resulting in nonlinear volume of distribution and clearance of romiplostim. Therefore, target-mediated disposition plays a decreasing role in drug elimination with increasing romiplostim serum concentration. Conversely, nonspecific elimination processes such as renal clearance play an increasing role with increasing romiplostim serum concentration. Limited pharmacokinetics data demonstrated that the exposure to romiplostim was lower after multiple dose administrations than after the first dose, although large inter-subject variability was observed. Large inter- and intra-subject variability in the platelet response was also observed at a given dose. These findings suggest considerable heterogeneity of disease in patients with primary immune thrombocytopenia and support the need for individual dose adjustments based on platelet counts.

Fc Receptor-mediated Effector Function Contributes to the Therapeutic Response of Anti-TNF Monoclonal Antibodies in a Mouse Model of Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Anti-tumour necrosis factor [TNF] monoclonal antibodies [infliximab, adalimumab] induce complete mucosal healing in a proportion of patients with Crohn's disease whereas a TNF receptor fusion protein [etanercept] is not effective and the anti-TNF F[ab']2 fragment [certolizumab] shows a very low rate of complete mucosal healing. In contrast, all four TNF-neutralising drugs have demonstrated efficacy in the treatment of rheumatoid arthritis. These observations suggest that factors other than neutralisation of TNF may contribute to clinical outcomes in Crohn's disease. Here we tested the hypothesis that Fc receptor [FcR]-mediated effects may contribute to the therapeutic response of anti-TNF antibodies in inflammatory bowel disease. METHODS: We modified an IgG2c mouse anti-TNF antibody that binds the high-affinity FcRs to generate an IgG1 isotype with strongly diminished binding. We examined the therapeutic effects of both antibodies in the T cell transfer model of inflammatory bowel disease and the collagen-induced arthritis model. RESULTS: The IgG2c anti-TNF antibody prevented colonic inflammation in the T cell transfer model of colitis, whereas the IgG1 anti-TNF did not. Conversely, both the IgG2c and IgG1 anti-TNFs were similarly effective in reducing the severity of articular inflammation in mouse collagen-induced arthritis. CONCLUSION: These data support the concept that the mechanism of action for TNF-neutralising drugs may differ across immune-mediated diseases and, potentially, between therapeutics within a particular disease. Our data suggest a specific role of Fc-mediated immune regulation in the resolution of intestinal inflammation by anti-TNF monoclonal antibodies.

Reversal of Arthritis by Human Monomeric IgA Through the Receptor-Mediated SH2 Domain-Containing Phosphatase 1 Inhibitory Pathway.

OBJECTIVE: Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) configuration through SH2 domain-containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling. METHODS: The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase. RESULTS: Treatment with mIgA impaired cell activation in an FcαRI-FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration. CONCLUSION: These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

Inhibitory effects of areca nut extract on expression of complement receptors and fc receptors in human neutrophils.

BACKGROUND: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement- and antibody-opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F-actin in ANE-treated neutrophils is also analyzed. METHODS: The viability of ANE-treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G-opsonized fluorescent beads was analyzed using flow cytometry. Expression of F-actin was determined using confocal laser scanning microscopy. RESULTS: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration-dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F-actin was inhibited after ANE treatment. CONCLUSIONS: ANE inhibits the complement- and IgG-mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F-actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.

Serum-derived immunoglobulins alter amyloid beta transport across a blood-brain barrier in vitro model.

Since passive immunization with serum-derived immunoglobulins (intravenous immunoglobulins) showed several positive effects in some patients with Alzheimer's disease (AD), intravenous immunoglobulins (IVIG) are discussed as a possible treatment option. IVIG, an antibody product derived from human plasma, contains natural antibodies against amyloid beta(Abeta) peptide. Until now it is not known, how IVIG interferes with pathogenesis in AD, but several proposed mechanisms are in discussion. Receptor types which are involved in transport processes at the BBB are LRP, RAGE and hFcRn. We were looking for an in vitro BBB model expressing these receptors and studied the alteration of transport of Abeta peptides across this model under the influence of immunoglobulins. Cell line ECV304 was found to be suitable for our experiments. We found evidence for involvement of an improved clearance of Abeta across the BBB as well as a decreased Abeta influx from blood to the brain probably following complex formation of immunoglobulins with free Abeta in the periphery. Furthermore, we were able to confirm the activity of IVIG preparations which acted the same way but showed slightly less efficacy in comparison to monoclonal anti-Abeta antibodies. Based on these results we suggest multiple mechanisms responsible for the efficacy of immunotherapy in Alzheimer's disease.

Induction of interleukin-10 expression through Fcalpha receptor in human monocytes and monocyte-derived dendritic cells: role of p38 MAPKinase.

As previously reported by others for immunoglobulin (Ig)G, we observed that IgA can induce interleukin (IL)-10 expression in human monocytes. In this study, we explored the molecular mechanisms of IL-10 induction by IgA in monocytes and monocyte-derived dendritic cells (MD-DCs). Monomeric IgA induced IL-10 production in monocytes and this production was further increased upon IgA cross-linking. Similar IL-10 responses were observed in monocytes and autologous MD-DCs, and were inhibited (by approximately 77%) by preincubation with a blocking mAb to FcalphaRI. IL-10 induction by IgA correlated with activation of MAPKinases ERK1/2, p38 and JNK, whereas only p38-inhibitor SB-203580 inhibited IL-10 induction. Upon IgA stimulation, AP-1, NFkappaB and Sp1 transcription factors were activated and inhibitors of NFkappaB and of Sp1 suppressed IgA-driven transcriptional activation of IL-10. In addition, p38 MAPK activation appeared that it was required to control nuclear translocation of NFkappaB and Sp1 upon IgA stimulation. Therefore, in human monocytes and MD-DCs the mechanisms of IL-10 induction by IgA involve p38 MAPK-dependent recruitment of both NFkappaB and Sp1.

Ex vivo TCR-induced leukocyte gene expression of inflammatory mediators is increased in type 1 diabetic patients but not in overweight children.

BACKGROUND: Abnormal systemic concentrations of proinflammatory cytokines/chemokines have been implicated in the development of long-term cardiovascular complications in type 1 diabetes (T1DM) and obesity. Whether leukocyte white blood cell (WBC) gene expression of these proinflammatory mediators contributes to their increased systemic levels, however, remains unclear, especially in the pediatric patient populations. This study examines mRNA changes of 9 cytokines and chemokines in WBCs following ex vivo immunostimulation from 9 T1DM (13.4 +/- 0.5 year, 4F/5 M), 23 overweight (OW, 12.3 +/- 0.5 year, 10F/13M, BMI% 97.1 +/- 0.5 and > 90.0), and 21 healthy (CL, 13.8 +/- 0.7 year, 9F/12 M, BMI% 59.6 +/- 4.6 and < 85.0) children. METHODS: All subjects had been maintained in euglycemic conditions for at least 90 min before blood draws. Whole blood was then sampled and incubated with anti-T-cell receptor (TCR) antibody or heat-aggregated IgG (HAG) to stimulate T-cell and Fc receptors (FcR), respectively. After lysis of leukocytes, mRNA levels of six tumor necrosis factor superfamily cytokines (TNFSF2, 5, 6, 7, 9, 14) and three chemokines (CCL8, 20, and CXCL10) were measured using RT-PCR. RESULTS: Following TCR stimulation, T1DM displayed significantly greater mRNA responses than CL for TNFSF5, 7, 9, and CCL8, and CXCL10; TNFSF9, CCL8, and CXCL10 were also significantly higher in T1DM than OW; no difference was observed between OW and CL. FcR stimulation induced similar responses across groups. CONCLUSIONS: Leukocytes of T1DM children displayed exaggerated gene expression in response to ex vivo TCR induction of five key proinflammatory cytokines/chemokines. This elevated leukocyte gene expression may be one of the pathophysiological contributors to the development of vascular complications in T1DM.

Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells.

AIMS: The purpose of this study was to characterize the internalization mechanism of human IgG into the epithelial cells of human small intestine, employing human intestinal epithelial cell line Caco-2 as an in vitro model system. MAIN METHODS: Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells. KEY FINDINGS: Real-time PCR analysis showed that mRNA level of the neonatal Fc receptor (FcRn) was increased during the differentiation process in Caco-2 cells. The binding of FITC-labeled human IgG to the membrane surface of Caco-2 cells increased with a decrease in pH of incubation buffer. The uptake of FITC-IgG was also stimulated at acidic pH and was time-dependent. The binding and uptake of FITC-IgG at pH 6.0 was partially, but significantly, decreased by human gamma-globulin in a concentration-dependent manner. A mixture of metabolic inhibitors (sodium azide and 2-deoxyglucose) significantly inhibited the uptake, but not the binding, of FITC-IgG. In addition, endosomal acidification inhibitors such as bafilomycin A(1) and chloroquine significantly increased the accumulation of FITC-IgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) and caveolin-dependent endocytosis inhibitors (nystatin and indomethacin) did not decrease the uptake of FITC-IgG at pH 6.0. In contrast, macropinocytosis inhibitors such as cytochalasin B and 5-(N-ethyl-N-isopropyl) amiloride significantly decreased the uptake of FITC-IgG at pH 6.0. SIGNIFICANCE: The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis, which could occur by a process other than clathrin- and caveolin-dependent mechanisms.

Delayed acquisition of somatic hypermutations in repopulated IGD+CD27+ memory B cell receptors after rituximab treatment.

OBJECTIVE: Transient B cell depletion by rituximab has been used with clinical efficacy in the treatment of patients with rheumatoid arthritis (RA). Previous studies of B cell repopulation have shown long-term numerical reduction in memory B cells. Non-class-switched IgD+CD27+ memory B cells, in particular, repopulate slowly. This study was undertaken to determine whether mutational acquisition in individual B cell receptors in repopulating class-switched and non-class-switched memory B cells is affected by rituximab. METHODS: Cells obtained from 16 RA patients, 4 healthy donors, and 3 patients who underwent allogeneic stem cell transplantation (ASCT) were analyzed using single B cell sorting followed by nested polymerase chain reaction and Ig V(H)3 sequencing. RESULTS: There was a delayed acquisition of mutations in Ig receptors of IgD+ memory B cells over a period of 6 years after a single course of rituximab. One year after rituximab treatment, 84% of single repopulating IgD+CD27+ B cells were unmutated, and no highly mutated Ig receptors were found (compared with 52% before therapy). Over time, increasing numbers of mutations were detected. Even 6 years after rituximab treatment, however, mutations in IgD+ memory B cells were still significantly reduced. In contrast, class-switched memory B cells repopulated with quantitatively normal mutations. In comparison, in patients undergoing ASCT, IgD+ memory cells repopulated earlier with higher mutations in Ig receptors. CONCLUSION: Our data suggest that IgD+ memory B cells are particularly susceptible to the effects of rituximab, with delayed acquisition of mutations in their Ig receptors still evident 6 years after a single course of rituximab. Our findings indicate that these cells have different requirements for mutational acquisition compared with class-switched memory B cells.