The MB20-11 monoclonal antibody reacts with mouse CD20. CD20 is a B cell-specific 33-37 kDa transmembrane protein which is also known as B-lymphocyte antigen, B1, and Bp35. CD20 plays roles in intracellular calcium regulation and B cell activation and is critical for an optimal B cell immune response against T-independent antigens. CD20 is first expressed after the induction of CD19 together with IgM during the pre-B to immature B cell transition in the bone marrow. It’s expression then increases during maturation with almost all mature B cells expressing some level of CD20. However, CD20 is not expressed by plasma blasts or plasma cells. CD20 is expressed by most B cell neoplasms and is useful in diagnosing B cell lymphomas and leukemia. Many anti-CD20 monoclonal antibodies are currently being used to successfully treat leukemia, lymphomas, and various autoimmune diseases. A single injection of the MB20-11 antibody has been reported to deplete circulating B cells in mice within 1 hour of treatment, with a durable effect for 57 days before B cells begin to repopulate in the blood and spleen.
Haas, K. M., et al. (2010). "Protective and pathogenic roles for B cells during systemic autoimmunity in NZB/W F1 mice" J Immunol 184(9): 4789-4800. PubMed
Delineating the relative contributions of B lymphocytes during the course of autoimmune disease has been difficult. Therefore, the effects of depleting all mature B cells using a potent CD20 mAb, or of depleting circulating and marginal zone B cells using a ligand-blocking CD22 mAb, were compared in NZB/W F(1) mice, a model for human systemic lupus erythematosus. Single low-dose mAb treatments depleted B cells efficiently in both NZB/W F(1) and C57BL/6 mice. Prophylactic B cell depletion by repeated CD20 mAb treatments prolonged survival during pristane-accelerated lupus in NZB/W F(1) mice, whereas CD22 mAb had little effect. Despite effective B cell depletion, neither mAb treatment prevented autoantibody generation. In addition, CD20, CD22, and control mAb-treated NZB/W F(1) mice developed anti-mouse IgG autoantibodies in contrast to parental NZB and NZW strains, which may have reduced the effectiveness of B cell depletion. Despite this, low-dose CD20 mAb treatment initiated in 12-28-wk-old mice, and administered every 4 wk thereafter, significantly delayed spontaneous disease in NZB/W F(1) mice. By contrast, B cell depletion initiated in 4-wk-old mice hastened disease onset, which paralleled depletion of the IL-10-producing regulatory B cell subset called B10 cells. B10 cells were phenotypically similar in NZB/W F(1) and C57BL/6 mice, but were expanded significantly in young NZB/W F(1) mice. Thus, B cell depletion had significant effects on NZB/W F(1) mouse survival that were dependent on the timing of treatment initiation. Therefore, distinct B cell populations can have opposing protective and pathogenic roles during lupus progression.
in vivo B cell depletion
Hamaguchi, Y., et al. (2006). "Antibody isotype-specific engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy" J Exp Med 203(3): 743-753. PubMed
CD20 monoclonal antibody (mAb) immunotherapy is effective for lymphoma and autoimmune disease. In a mouse model of immunotherapy using mouse anti-mouse CD20 mAbs, the innate monocyte network depletes B cells through immunoglobulin (Ig)G Fc receptor (FcgammaR)-dependent pathways with a hierarchy of IgG2a/c>IgG1/IgG2b>IgG3. To understand the molecular basis for these CD20 mAb subclass differences, B cell depletion was assessed in mice deficient or blocked for stimulatory FcgammaRI, FcgammaRIII, FcgammaRIV, or FcR common gamma chain, or inhibitory FcgammaRIIB. IgG1 CD20 mAbs induced B cell depletion through preferential, if not exclusive, interactions with low-affinity FcgammaRIII. IgG2b CD20 mAbs interacted preferentially with intermediate affinity FcgammaRIV. The potency of IgG2a/c CD20 mAbs resulted from FcgammaRIV interactions, with potential contributions from high-affinity FcgammaRI. Regardless, FcgammaRIV could mediate IgG2a/b/c CD20 mAb-induced depletion in the absence of FcgammaRI and FcgammaRIII. In contrast, inhibitory FcgammaRIIB deficiency significantly increased CD20 mAb-induced B cell depletion by enhancing monocyte function. Although FcgammaR-dependent pathways regulated B cell depletion from lymphoid tissues, both FcgammaR-dependent and -independent pathways contributed to mature bone marrow and circulating B cell clearance by CD20 mAbs. Thus, isotype-specific mAb interactions with distinct FcgammaRs contribute significantly to the effectiveness of CD20 mAbs in vivo, which may have important clinical implications for CD20 and other mAb-based therapies.
in vivo B cell depletion
Uchida, J., et al. (2004). "Mouse CD20 expression and function" Int Immunol 16(1): 119-129. PubMed
CD20 plays a role in human B cell proliferation and is an effective target for immunotherapy. In this study, mouse CD20 expression and biochemistry were assessed for the first time using a new panel of CD20-specific mAb, with CD20 function assessed using CD20-deficient (CD20(-/-)) mice. CD20 expression was B cell restricted and was initiated during late pre-B cell development. The frequency and density of CD20 expression increased during B cell maturation in the bone marrow, with a subpopulation of transitional IgM(hi) B cells expressing higher CD20 levels than the majority of mature recirculating B cells. Transitional T1 B cells in the spleen also expressed high CD20 levels, providing a useful new marker for this B cell subset. In CD20(-/-) mice, immature and mature B cell IgM expression was approximately 20-30% lower relative to B cells from wild-type littermates. In addition, CD19-induced intracellular calcium responses were significantly reduced in CD20(-/-) B cells, with a less dramatic effect on IgM-induced responses. These results reveal a role for CD20 in transmembrane Ca(2+) movement in mouse primary B cells that complements previous results obtained using human CD20 cDNA-transfected cell lines. Otherwise, B cell development, tissue localization, signal transduction, proliferation, T cell-dependent antibody responses and affinity maturation were normal in CD20(-/-) mice. Thus, mouse and human CD20 share similar patterns of expression and function. These studies thereby provide an animal model for studying CD20 function in vivo and the molecular mechanisms that influence anti-CD20 immunotherapy.
in vivo B cell depletion
Uchida, J., et al. (2004). "The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy" J Exp Med 199(12): 1659-1669. PubMed
Anti-CD20 antibody immunotherapy effectively treats non-Hodgkin’s lymphoma and autoimmune disease. However, the cellular and molecular pathways for B cell depletion remain undefined because human mechanistic studies are limited. Proposed mechanisms include antibody-, effector cell-, and complement-dependent cytotoxicity, the disruption of CD20 signaling pathways, and the induction of apoptosis. To identify the mechanisms for B cell depletion in vivo, a new mouse model for anti-CD20 immunotherapy was developed using a panel of twelve mouse anti-mouse CD20 monoclonal antibodies representing all four immunoglobulin G isotypes. Anti-CD20 antibodies rapidly depleted the vast majority of circulating and tissue B cells in an isotype-restricted manner that was completely dependent on effector cell Fc receptor expression. B cell depletion used both FcgammaRI- and FcgammaRIII-dependent pathways, whereas B cells were not eliminated in FcR common gamma chain-deficient mice. Monocytes were the dominant effector cells for B cell depletion, with no demonstrable role for T or natural killer cells. Although most anti-CD20 antibodies activated complement in vitro, B cell depletion was completely effective in mice with genetic deficiencies in C3, C4, or C1q complement components. That the innate monocyte network depletes B cells through FcgammaR-dependent pathways during anti-CD20 immunotherapy has important clinical implications for anti-CD20 and other antibody-based therapies.
Interaction of the endogenous antibody response with activating FcγRs enhance control of Mayaro virus through monocytes.
In PLoS Pathogens on 1 February 2025 by Dunagan, M. M., Dábilla, N., et al.
Mayaro virus (MAYV) is an emerging arbovirus. Previous studies have shown antibody Fc effector functions are critical for optimal monoclonal antibody-mediated protection against alphaviruses; however, the requirement of Fc gamma receptors (FcγRs) for protection during natural infection has not been evaluated. Here, we showed mice lacking activating FcγRs (FcRγ-/-) developed prolonged clinical disease with increased MAYV in joint-associated tissues. Viral reduction was associated with anti-MAYV cell surface binding antibodies rather than neutralizing antibodies. Lack of Fc-FcγR engagement increased the number of monocytes present in the joint-associated tissue through chronic timepoints. Single-cell RNA sequencing showed elevated levels of pro-inflammatory monocytes in joint-associated tissue with increased MAYV RNA present in FcRγ-/- monocytes and macrophages. Transfer of FcRγ-/- monocytes into wild type animals was sufficient to increase virus in joint-associated tissue. Overall, this study suggests that engagement of antibody Fc with activating FcγRs promotes protective responses during MAYV infection and prevents a pro-viral role for monocytes.
Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Mus musculus (House mouse)
,
Cancer Research
Blockade of TGF-β and PD-L1 by bintrafusp alfa promotes survival in preclinical ovarian cancer models by promoting T effector and NK cell responses.
In British Journal of Cancer on 1 June 2024 by Kment, J., Newsted, D., et al.
Allergic airway disease (AAD) is an example of type 2 inflammation that leads to chronic airway eosinophilia controlled by CD4 Th2 cells. Inflammation is reinforced by mast cells and basophils armed with allergen-specific IgE made by allergen-specific B2 B cells of the adaptive immune system. Little is known about how AAD is affected by innate B1 cells, which produce natural antibodies (NAbs) that facilitate apoptotic cell clearance and detect damage- and pathogen-associated molecular patterns (DAMPS and PAMPS). We used transgenic mice lacking either B cells or NAbs in distinct mouse models of AAD that require either DAMPS or PAMPS as the initial trigger for type 2 immunity. In a DAMP-induced allergic model, driven by alum and uric acid, mouse strains lacking B cells (CD19DTA), NAbs (IgHEL MD4), or all secreted antibodies (sIgm-/-Aid-/-) displayed a significant reduction in both eosinophilia and Th2 priming compared with WT or Aid-/- mice lacking only germinal center-dependent high-affinity class-switched antibodies. Replenishing B cell-deficient mice with either unimmunized B1 B cells or NAbs during sensitization restored eosinophilia, suggesting that NAbs are required for licensing antigen-presenting cells to prime type 2 immunity. Conversely, PAMP-dependent type 2 priming to house dust mite or Aspergillus was not dependent on NAbs. This study reveals an underappreciated role of B1 B cell-generated NAbs in selectively driving DAMP-induced type 2 immunity.
Mus musculus (House mouse)
,
Cancer Research
,
Immunology and Microbiology
CMTM6 maintains B cell-intrinsic CD40 expression to regulate anti-tumor immunity
Preprint on BioRxiv : the Preprint Server for Biology on 10 March 2024 by Long, Y., Chen, R., et al.
SUMMARY Tumor cell CMTM6 is a novel tumor immunoregulator involved in maintaining membrane levels of several important molecules, such as PD-L1 and CD58. Host CMTM6 may also play a function in the tumor microenvironment. Here, we found that CMTM6 was highly expressed in splenic B cells and tumor-infiltrating B cells. CMTM6 deficiency resulted in impaired splenic development, germinal center B cell differentiation, memory B cell differentiation, and B cell anti-tumor immune responses. Through multi-omics data mining and B-cell agonist screening, we identified that CMTM6 interacted with CD40 and maintained CD40 membrane levels in B cells. CMTM6 regulated CD40 at the post-translational modification stage but not at the transcriptional stage. CMTM6 deficiency led to impaired CD40 signaling-mediated B cell activation, survival, proliferation, and differentiation. In vivo, CMTM6 deficiency leads to a significant decrease in the anti-tumor activity of B cell-dependent CD40 agonists. Collectively, B-cell intrinsic CMTM6 maintains B cell CD40 levels and signaling to promote B cell function and anti-tumor immunity. Highlights B cells participate in anti-tumor immunity by influencing the intratumoral infiltration and function of T cells; CMTM6 cis-interacts with CD40 to maintain CD40 cell membrane levels; Loss of B cell-intrinsic CMTM6 significantly reduces CD40 signaling-mediated B cell activation, survival, and differentiation; CMTM6 deficiency leads to a significant reduction in the anti-tumor activity of B cells and CD40 agonists. In Brief Long et al. demonstrate that B-cell intrinsic CMTM6 regulates CD40 signaling and function via maintaining the cell membrane level of B-cell CD40 through a post-translational modification pathway, thereby affecting anti-tumor B-cell immunity and the efficacy of CD40 agonist.
Mus musculus (House mouse)
Dnmt3amutations limit normal and autoreactive Tfh differentiation
Preprint on BioRxiv : the Preprint Server for Biology on 21 February 2024 by Shen, Y., Li, Z., et al.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation, strongly associated with the activity of autoreactive CD4+ T cells. DNMT3A mutations are the most common somatic mutations found in the hematopoietic system of patients with rheumatoid arthritis. However, the role of DNMT3A in CD4+ T cells and CD4+ T follicular helper (Tfh) cells is poorly understood. Since somatic mutations are not identified in standard genome-wide association studies, somatic mutations’ impact on the etiology of diseases could be underestimated. Here, we thoroughly characterized and used the KRN+ splenocyte transfer model of autoimmune joint inflammation and inactivated Dnmt3a using CRISPR-Cas9 and standard Cre/loxP approaches. Experiments with competitive bone marrow (BM) chimeras identified a positive role for Dnmt3a in Tfh differentiation, which was validated by comparing mice with Dnmt3a mutations in CD4+ cells to animals with WT Dnmt3a . In conclusion, We identify that Dnmt3a mutations limit normal and autoreactive Tfh differentiation. Key findings – Dnmt3a mutations limit Tfh differentiation, which could contribute to reduced immune responses in individuals with somatic DNMT3A mutations. – Deep characterization of the KRN+ splenocyte transfer model defines a dynamic process leading to reproducible autoimmune joint inflammation. – The immuno-CRISPR (iCR) methodology can be used to test the role of candidate genes in disease models.
Mus musculus (House mouse)
,
Immunology and Microbiology
Glycosylation-modified antigens as a tolerance-inducing vaccine platform prevent anaphylaxis in a pre-clinical model of food allergy.
In Cell Reports Medicine on 16 January 2024 by Cao, S., Maulloo, C. D., et al.
