CD20-specific Monoclonal Antibody Research Articles

Persistence of Virus-Specific Antibody after Depletion of Memory B Cells.

Humoral immunity is a major component of the adaptive immune response against viruses and other pathogens with pathogen-specific antibody acting as the first line of defense against infection. Virus-specific antibody levels are maintained by continual secretion of antibody by plasma cells residing in the bone marrow. This raises the important question of how the virus-specific plasma cell population is stably maintained and whether memory B cells are required to replenish plasma cells, balancing their loss arising from their intrinsic death rate. In this study, we examined the longevity of virus-specific antibody responses in the serum of mice following acute viral infection with three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza virus, and vesicular stomatitis virus (VSV). To investigate the contribution of memory B cells to the maintenance of virus-specific antibody levels, we employed human CD20 transgenic mice, which allow for the efficient depletion of B cells with rituximab, a human CD20-specific monoclonal antibody. Mice that had resolved an acute infection with LCMV, influenza virus, or VSV were treated with rituximab starting at 2 months after infection, and the treatment was continued for up to a year postinfection. This treatment regimen with rituximab resulted in efficient depletion of B cells (>95%), with virus-specific memory B cells being undetectable. There was an early transient drop in the antibody levels after rituximab treatment followed by a plateauing of the curve with virus-specific antibody levels remaining relatively stable (half-life of 372 days) for up to a year after infection in the absence of memory B cells. The number of virus-specific plasma cells in the bone marrow were consistent with the changes seen in serum antibody levels. Overall, our data show that virus-specific plasma cells in the bone marrow are intrinsically long-lived and can maintain serum antibody titers for extended periods of time without requiring significant replenishment from memory B cells. These results provide insight into plasma cell longevity and have implications for B cell depletion regimens in cancer and autoimmune patients in the context of vaccination in general and especially for COVID-19 vaccines. IMPORTANCE Following vaccination or primary virus infection, virus-specific antibodies provide the first line of defense against reinfection. Plasma cells residing in the bone marrow constitutively secrete antibodies, are long-lived, and can thus maintain serum antibody levels over extended periods of time in the absence of antigen. Our data, in the murine model system, show that virus-specific plasma cells are intrinsically long-lived but that some reseeding by memory B cells might occur. Our findings demonstrate that, due to the longevity of plasma cells, virus-specific antibody levels remain relatively stable in the absence of memory B cells and have implications for vaccination.

Development of a T Cell-targeted siRNA Delivery System Against HIV-1 Using Modified Superparamagnetic Iron Oxide Nanoparticles: An In Vitro Study

In spite of the promising properties of small interfering RNAs (siRNAs) in the treatment of infectious diseases, safe and efficient siRNA delivery to target cells is still a challenge. In this research, an effective siRNA delivery approach (against HIV-1) has been reported using targeted modified superparamagnetic iron oxide nanoparticles (SPIONs). Trimethyl chitosan-coated SPION (TMC-SPION) containing siRNA was synthesized and chemically conjugated to a CD4-specific monoclonal antibody (as a targeting moiety). The prepared nanoparticles exhibited a high siRNA loading efficiency with a diameter of about 85 nm and a zeta potential of +28 mV. The results of the cell viability assay revealed the low cytotoxicity of the optimized nanoparticles. The cellular delivery of the targeted nanoparticles (into T cells) and the gene silencing efficiency of the nanoparticles (containing anti-nef siRNA) were dramatically improved compared to those of nontargeted nanoparticles. In conclusion, this study offers a promising targeted delivery platform to induce gene silencing in target cells. Our approach may find potential use in the design of effective vehicles for many therapeutic applications, particularly for HIV treatment.

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function.

The first-in-human clinical trial of the CD28-specific monoclonal antibody (mAb) TGN1412 resulted in a life-threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγreceptor (FcγR) interactions, these interactions contributed to TGN1412-induced T-cell activation. Using cell lines (TFs) expressing human FcγRI, -IIa, -IIb, or -III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412-decorated Tcells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest Tcell proliferation, while high-affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T-cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T-cell proliferation. In line with this, FcγRI- FcγRII+ but not FcγRI+ FcγRII+ monocytes mediate TGN1412-induced T-cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR-mediated effector function, which has major implications for the design of therapeutic mAbs.

Cancer Immunotherapy and the Immune Response in Follicular Lymphoma.

Follicular lymphoma (FL) is the most frequent indolent lymphoma in the Western world and is characterized in almost all cases by the t(14;18) translocation that results in overexpression of BCL2, an anti-apoptotic protein. The entity includes a spectrum of subentities that differ from an indolent to a very aggressive growth pattern. As a consequence, treatment can include watch & wait up to intensive chemotherapy including allogeneic stem cell transplantation. The immune cell microenvironment has been recognized as a major driver of outcome of FL patients and gene expression profiling has identified a clinically relevant gene expression signature that classifies an immune response to the lymphoma cells. It is known for some time that the immune cell composition of the lymphoma microenvironment is important because high numbers of tissue-infiltrating macrophages correlate with poor outcome in patients receiving chemotherapy but not in patients receiving the combination of chemotherapy and CD20-specific monoclonal antibody rituximab. In addition, TCR signaling of tumor-infiltrating lymphocytes is dysfunctional leading to an impaired capacity to form an intact immunologic synapse. Approaches restoring local T cell function, e.g., by usage of checkpoint inhibitors has demonstrated clinical activity (ORR 40%) and can achieve long-term remissions. Ongoing trials with re-programmed autologous CART cells achieve response rates in approximately 50% of FL patients with relapsed and even refractory disease. Responses lasting for more than 6 months might be durable, indicative for a successful restoration of a functional immune system. In summary, FL is a malignant disease where the control by the immune system ultimately decides about progression and transformation rate. The advent of monoclonal antibodies has changed the way we treat FL and new approaches restoring the individual immune control will hopefully improve results further.

Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity.

Binding of the complement component C1q to the CH2 domain of antigen-bound immunoglobulin gamma (IgG) activates the classical complement pathway and depends on its close proximity to Fc fragments of neighboring antibodies. IgG subclasses contain a highly conserved asparagine 297 (N)-linked biantennary glycan within their CH2 domains, the core structure of which can be extended with terminal galactose and sialic acid residues. To investigate whether Fc-glycosylation regulates effector functions of human IgG subclasses, we cloned the antigen-binding region of the CD20-specific monoclonal antibody rituximab into IgG isotype expression vectors. We found that Fc-galactosylation enhances the efficacy of CD20-targeting complement-fixing antibodies for C1q binding and complement-mediated tumor cell lysis. Increased efficacies were restricted to IgG1 and IgG3 subclasses indicating that Fc-galactosylation alone is not sufficient for IgG2 and IgG4 to acquire complement-fixing properties. Addition of terminal galactose to the N-glycan specifically improved binding of C1q without changing antigen- and FcγRIIIa-binding affinities of IgG isotypes. These data indicate that Fc galactosylation can be harnessed to enhance the complement-activating properties of IgG1 and IgG3 antibodies.

Mucosal administration of CD3-specific monoclonal antibody inhibits diabetes in NOD mice and in a preclinical mouse model transgenic for the CD3 epsilon chain

CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-β and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.

OR2 Variable expression of HLA-C impacts T versus B cell crossmatch outcomes

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibodies (DSA) and determine eligibility for transplantation and desensitization regimens. We measured HLA class I and HLA-C surface expression on T and B cells from various sources and examined the impact of HLA-C expression on FCXM reactivity in the presence of HLA-C DSA. HLA class I and HLA-C expression was examined on cells isolated from 18 donors using monoclonal antibodies (clone G46.2 and mAb DT9). T and B cells were differentiated using CD3 and CD19 specific monoclonal antibodies. Cells were acquired on a BD FACSCanto II; HLA expression was determined using median channel fluorescence (MCF) values and FCXM reactivity was assessed as a ratio of test serum to negative control serum. The mean HLA-C expression was significantly higher on T (11693 MCF) than B cells (5480 MCF, p < 0.05, Figure 1) while class I expression was lower on T (10167 MCF) than B cells (23602 MCF, p < 0.001) when comparing cells isolated from the same donors. This difference in HLA-C expression was mirrored in FCXM tests using sera containing HLA-C specific DSA. Mean reactivity with T cells as targets yielded consistently higher ratios than tests using B cells as targets (p < 0.001). Seven of 10 FCXM tests yielded positive T and negative B results. HLA-C expression is known to vary among HLA-C alleles (Apps et al., Science 2013); however, this study shows variability also exists between lymphocyte lineages. HLA-C is expressed higher on T than B cells and this difference impacts crossmatch reactivity. These findings should be considered when interpreting FCXM results.Download : Download high-res image (108KB)Download : Download full-size image

A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway.

CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

Subcutaneous versus Intravenous Administration of Rituximab: Pharmacokinetics, CD20 Target Coverage and B-Cell Depletion in Cynomolgus Monkeys

The CD20-specific monoclonal antibody rituximab (MabThera®, Rituxan®) is widely used as the backbone of treatment for patients with hematologic disorders. Intravenous administration of rituximab is associated with infusion times of 4–6 hours, and can be associated with infusion-related reactions. Subcutaneous administration of rituximab may reduce this and facilitate administration without infusion-related reactions. We sought to determine the feasibility of achieving equivalent efficacy (measured by endogenous B-cell depletion) and long-term durability of CD20 target coverage for subcutaneously administered rituximab compared with intravenous dosing. In these preclinical studies, male cynomolgus monkeys were treated with either intravenous rituximab or novel subcutaneous formulation of rituximab containing human recombinant DNA-derived hyaluronidase enzyme. Peripheral blood samples were analyzed for serum rituximab concentrations, peripheral B-cell depletion, and CD20 target coverage, including subset analysis according to CD21+ status. Distal lymph node B-cell depletion and CD20 target coverage were also measured. Initial peak serum concentrations of rituximab were significantly higher following intravenous administration than subcutaneous. However, the mean serum rituximab trough concentrations were comparable at 2 and 7 days post-first dose and 9 and 14 days post-second dose. Efficacy of B-cell depletion in both peripheral blood and distal lymph nodes was comparable for both methods. In lymph nodes, 9 days after the second dose with subcutaneous and intravenous rituximab, B-cell levels were decreased by 57% and 42% respectively. Similarly, levels of peripheral blood B cells were depleted by >94% for both subcutaneous and intravenous dosing at all time points. Long-term recovery of free unbound surface CD20 levels was similar, and the duration of B-cell depletion was equally sustained over 2 months for both methods. These results demonstrate that, despite initial peak serum drug level differences, subcutaneous rituximab has similar durability, pharmacodynamics, and efficacy compared with intravenous rituximab.

After TGN1412: Recent developments in cytokine release assays

The failure of regulatory science to keep pace with and support the development of new biological medicines was very publically highlighted in March 2006 when the first-in-man Phase I clinical trial of the immunomodulatory CD28-specific monoclonal antibody (mAb) TGN1412 ended in disaster when all six volunteers suffered a life-threatening adverse reaction termed a ‘Cytokine Storm’. The poor predictive value of standard pre-clinical safety tests and animal models applied to TGN1412 demonstrated the need for a new generation of immunotoxicity assays and animal models that are both sensitive and predictive of clinical outcome in man. The non-predictive result obtained from pre-clinical safety testing in cynomolgus macaques has now been attributed to a lack of CD28 expression on CD4+ effector memory T-cells that therefore cannot be stimulated by TGN1412. In contrast, high levels of CD28 are expressed on human CD4+ effector memory T-cells, the source of most TGN1412-stimulated pro-inflammatory cytokines. Standard in vitro safety tests with human cells were also non-predictive as they did not replicate in vivo presentation of TGN1412. It was subsequently shown that, if an immobilized therapeutic mAb-based assay or endothelial cell co-culture assay was used to evaluate TGN1412, then these would have predicted a pro-inflammatory response in man. New in vitro assays based on these approaches are now being applied to emerging therapeutics to hopefully prevent a repeat of the TGN1412 incident. It has emerged that the mechanism of pro-inflammatory cytokine release stimulated by TGN1412 is different to that of other therapeutic mAbs, such that standard pro-inflammatory markers such as TNFα and IL-8 are not discriminatory. Rather, IL-2 release and lymphoproliferation are optimal readouts of a TGN1412-like pro-inflammatory response.

CD28 Superagonist Antibody Treatment Attenuated Obliterative Bronchiolitis in Rat Allo-Orthotopic Tracheal Transplantation by Preferentially Expanding Foxp3-Expressing Regulatory T Cells

Obliterative airway disease (OAD) due to chronic alloantigen rejection remains a major challenge for long-term graft survival in lung transplantation. It is known that superagonistic CD28-specific monoclonal antibody JJ316 (supCD28 MAb) has the ability to induce regulatory T cells (Tregs) efficiently. Here we used a rat orthotopic tracheal transplantation model to investigate the effects of supCD28 MAb on expanding Tregs in vivo and its application in suppression of acute and chronic airway allograft rejection. SupCD28 MAb administration revealed a significant increase in the CD4+CD25+ T cells, CD4+FoxP3+ T cells, and CD4+CD25+ FoxP3+ T cells population among CD4+ T cells in spleen, peripheral blood, as well as cervical lymph nodes. The allografts from animals treated with supCD28 MAb showed significantly less airway obliteration and rejection of the respiratory epithelium compared with allografts of the mouse immunoglobulin G-treated group on the 5th day and the 60th day after transplantation. Overall, our data demonstrated that an intraperitoneally administrated low dose of supCD28 MAb was sufficient to induce Treg cell expansion in vivo and was effective in protecting the airway graft from early rejection and chronic OAD development. These findings provide the basis for new therapies to prevent OAD and perhaps rejection of allografts in other human transplantations.

Abstract 81: B Lymphocytes Trigger MCP3-Dependent Mobilization of Monocytes and Promote Adverse Ventricular Remodeling After Myocardial Infarction

Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and persistent infiltration of innate immune cells, such as neutrophils and Ly6Chi monocytes, has been shown to promote adverse cardiac tissue remodeling. However, little is known regarding the role of mature B lymphocytes, which play a crucial role in the activation of the inflammatory response in several immune-mediated diseases. Here, we hypothesized that B lymphocytes might modulate the inflammatory response and affect the immune-dependent adverse cardiac remodeling. In a mouse model of myocardial infarction, cardiac B lymphocytes levels peaked at day 5 after the onset of infarction. Of interest, treatment with a CD20-specific monoclonal antibody decreased circulating and infiltrating B cell numbers (p=0.0008 and p=0.0002 vs control), reduced infarct size and post-ischemic immunoinflammatory response, and improved cardiac function (p=0.02 vs control) assessed by echocardiography. Intriguingly, B cell depletion was associated with an impairment of Ly6Chi monocytes mobilization from bone marrow (p=0.02 vs control), leading to reduced levels of circulating and infiltrating cardiac monocytes. The acute infarction led to transient increase of both MCP-1 and MCP-3 levels. Interestingly, B cell depletion was associated with a significant and selective reduction of MCP-3 (p=0.03 vs control) but did not alter MCP-1 levels (p=0.11). Cultured activated B cells released MCP-3 and treatment with a neutralizing MCP-3 antibody abrogated B lymphocytes-induced migration of cultured monocytes. Finally, transfer of B cell-depleted splenocytes into Rag1 -/- mice improved cardiac function after myocardial infarction compared to the transfer of non-depleted splenocytes (p=0.005). This effect was abrogated after re-supplementation with B lymphocytes isolated from wild-type mice (p=0.0007) but not from MCP-3-deficient animals (p=0.7008). In conclusion, we show that following acute myocardial infarction, B lymphocytes, trigger an MCP-3-dependent mobilization of Ly6Chi monocytes from the bone marrow to the blood, leading to their recruitment into the injured myocardium and to exacerbation of tissue inflammation, thereby promoting adverse cardiac remodeling.

Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.

Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412

AbstractHuman volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-γ, and other toxic cytokines in response to this CD28 “superagonist” (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on “tonic” TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs.

Membrane microdomain sphingolipids are required for anti-CD20-induced death of chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia remains incurable, despite the addition of rituximab to chemotherapy as an available means of treatment. The resistance of certain patients to this monoclonal antibody prompted us to set up in vitro studies of another CD20-specific monoclonal antibody, B1 (later termed tositumomab). We hypothesized that the membrane lipid organization of leukemic B cells might be instrumental in the cells' sensitivity to the B1 monoclonal antibody. B lymphocytes from 36 patients with chronic lymphocytic leukemia and 13 patients with non-Hodgkin's lymphoma were investigated for B1-triggered cell death. Membrane components, such as sphingomyelin and ganglioside M1, were investigated by flow cytometry, immunofluorescence and co-immunoprecipitation, together with the Csk-binding protein. Chronic lymphocytic leukemia patients segregated into two groups: B cells from one group were sensitive to B1, whereas those from the second group were not. Further results ascribed the resistance of these latter cases to a defective recruitment of Csk-binding protein, resulting in a lack of sphingomyelin and ganglioside M1 at the outer leaflet of the plasma membrane of their malignant B cells. Sphingolipids were indeed retained in the cytoplasm, because of lowered activity of P-glycoprotein. Supporting this mechanism, rifampicin, an inducer of P-glycoprotein, improved the activity of this transmembrane efflux pump, normalized the quantity of sphingomyelin within the membrane, and thereby restored the efficacy of the B1 monoclonal antibody in the formerly B1-resistant cases of chronic lymphocytic leukemia. The lipid organization of membranes of B cells from patients with chronic lymphocytic leukemia differs from one patient to another. In practice, given the relevance of the membrane lipid distribution to the efficacy of biotherapies, this observation is of potential importance.

The effects of Foxp3-expressing regulatory T cells expanded with CD28 superagonist antibody in DSS-induced mice colitis

Regulatory T (Treg) cells play an important role in the pathogenesis of inflammatory bowel disease (IBD). In the present study, we found that a superagonistic CD28-specific monoclonal antibody (supCD28mAb, D665) could preferentially stimulate expansion of CD4+Foxp3+ Treg cells. Foxp3 EGFP mice were orally administrated with 3.5% DSS for 5 days, and intraperitoneally injected supCD28mAb 1 mg/mice in treated group. All of the mice were sacrificed on day 8, and both clinical and histological parameters showed that the severity of colitis was significantly reduced in treated group compared to controls. In treated group, the proportion of CD103, CD152 and CD62L expression on Foxp3+Treg cells in the spleen and mesenteric lymph node were higher than controls. Furthermore, qRT-PCR analysis showed that expression of anti-inflammatory cytokines such as IL-10, TGF-β was significantly increased in treated group. Taken together, our data demonstrated that supCD28mAb targets CD4+Foxp3+Treg cells expansion in vivo, maintains and enhances their regulatory functions, to reduce the damage of colon in dextran sulfate sodium (DSS)-induced mouse colitis by secreting a large amount of IL-10. It represents a major advance towards the therapeutic use of polyclonally activated Treg cells as cellular therapy for treatment of IBD.

The B Cell in Atherosclerosis: Teaming Up with the Bad Guys?

Atherosclerosis has long been recognized as a disease characterized by chronic arterial inflammation. More-recent studies have indicated that this inflammation is driven by adaptive immune responses against modified self antigens (such as oxidized LDL) that have been trapped in the artery wall. Does this mean that atherosclerosis should be seen as an autoimmune disease? In a traditional sense, that is probably not the case, but there is considerable support for the notion that autoimmune responses against oxidized LDL and other modified vascular self antigens play an important role in modulating disease activity and hence represent interesting targets for the development of novel interventions to prevent cardiovascular disease. The picture emerging from studies that have used hypercholesterolemic mouse strains deficient in various components of the adaptive immune system suggests that activation of immune responses of T helper 1 (Th1)3 cells toward vascular antigens are strongly proatherogenic, whereas activation of immune responses involving regulatory T cells and B lymphocytes are protective. The balance between these pathogenic and protective immune responses would then determine if lesions progress or regress (1). The concept of the atheroprotective B cell is now being challenged, however, by a recent study from Ait-Oufella and coworkers published in the Journal of Experimental Medicine (2). In this provocative and interesting study, the authors used injections of a CD20-specific monoclonal antibody (mAb) to deplete apolipoprotein E–deficient and LDL receptor–deficient mice of B cells. CD20 mAb treatment reduced the number of B cells in the blood, spleen, and bone marrow by >90%, did not affect plasma cholesterol concentrations, and reduced the growth of atherosclerotic lesions by between 20% and 50%, depending on diet, length of the treatment period, and mouse model. The most pronounced reductions were observed in mice with lower plasma cholesterol (chow diet) and a longer treatment period …

CD28 superagonist-induced regulatory T cell expansion ameliorates mesangioproliferative glomerulonephritis in rats

Naturally occurring regulatory T cells (Treg) are essential for the prevention of autoimmunity and overshooting immune responses to pathogens; however, the involvement of Treg in mesangioproliferative glomerulonephritis, a major cause of chronic kidney disease, remains unclear. Superagonistic CD28-specific monoclonal antibodies (CD28SA) are highly effective activators of Treg in rats. To confirm our hypothesis that CD28SA reduces the severity of experimental glomerulonephritis, anti-Thy1 nephritis model rats were treated with CD28SA or saline. CD28SA significantly suppressed the increase in proteinuria and serum creatinine levels. CD28SA-treated nephritic rats exhibited an increase in the infiltration of Treg in the glomeruli accompanied by infiltration of CD163-positive macrophages ("alternatively activated" macrophages). In addition, CD28SA significantly induced interleukin-10 mRNA expression in glomeruli, thereby ameliorating mesangial cell proliferation and extracellular matrix expansion. We established a new therapeutic approach to suppressing progressive glomerulonephritis. The therapeutic value of this approach warrants further attention and preclinical studies.

Primate testing of TGN1412: right target, wrong cell

The failure of toxicity studies in non-human primates to predict the cytokine release syndrome during a first-in-man study of the CD28-specific monoclonal antibody TGN1412 has remained unexplained so far. In this issue of the BJP, work from the NIBSC first identifies the effector memory subset of human T-lymphocytes as the most likely source of the pro-inflammatory cytokines released during the study, and goes on to show that in cynomolgus monkeys, this subset lacks CD28, the target molecule of TGN1412. We discuss the implications for the TGN1412 catastrophe and for preclinical evaluation of biologicals in animal models in general.

B cell depletion reduces the development of atherosclerosis in mice

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell–depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti–oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell–derived IFN-γ secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody–mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.