Manipulation of the glycan-specific natural antibody repertoire for immunotherapy

Abstract

Natural immunoglobulin derived from innate-like B lymphocytes plays important roles in the suppression of inflammatory responses and represents a promising therapeutic target in a growing number of allergic and autoimmune diseases. These antibodies are commonly autoreactive and incorporate evolutionarily conserved specificities, including certain glycan-specific antibodies. Despite this conservation, exposure to bacterial polysaccharides during innate-like B lymphocyte development, through either natural exposure or immunization, induces significant changes in clonal representation within the glycan-reactive B cell pool. Glycan-reactive natural antibodies (NAbs) have been reported to play protective and pathogenic roles in autoimmune and inflammatory diseases. An understanding of the composition and functions of a healthy glycan-reactive NAb repertoire is therefore paramount. A more thorough understanding of NAb repertoire development holds promise for the design of both biological diagnostics and therapies. In this article, we review the development and functions of NAbs and examine three glycan specificities, represented in the innate-like B cell pool, to illustrate the complex roles environmental antigens play in NAb repertoire development. We also discuss the implications of increased clonal plasticity of the innate-like B cell repertoire during neonatal and perinatal periods, and the prospect of targeting B cell development with interventional therapies and correct defects in this important arm of the adaptive immune system.

Keywords: B-1 B cell; clonotype; glycan neodeterminant; innate-like B lymphocyte; natural antibody; repertoire development.

Figure 1. Natural antibodies fulfill multifunctional roles in host immunity

Pentameric IgM natural antibodies perform several important functions in immunological homeostasis. (A) Natural IgM is an important first-line-of-defense against invading pathogens, implicated in protection against infection by bacterial, viral, fungal, and multicellular pathogens including helminths. (B) The specificity of these antibodies also includes reactivity toward certain autologous antigens. Natural antibodies facilitate the efficacious clearance of apoptotic cells through recognition of danger-associated molecular patterns generated during apoptosis, such as phospholipid and glycan neoantigens. (C) Natural Ab-reactive neoantigens are generated on the vascular endothelium following ischemia reperfusion injury and nAbs suppress inflammatory lesions associated with deposition of atheroscerlotic plaques. (D) Natural Abs additionally suppress sensitization to allergens through reactivity with conserved T lymphocyte-independent epitopes present on allergenic particulates. Following opsonization of target antigens, nAbs can alter phagocyte activation and reduce the resulting T lymphocyte-dependent responses necessary for production of pathogenic IgG autoantibodies or mast-cell sensitizing IgE.

Figure 2. GlcNAc-specific natural antibodies recognize apoptosis-associated neoepitopes generated on irradiated thymocytes

(A) C57BL/6 thymocytes were irradiated to induce apoptosis, cultured overnight, then stained with Annexin-V, the membrane impermeable DNA dye 7-AAD, and for glycan neodeterminants using various polysaccharide-reactive monoclonal mouse antibodies for flow cytometric analysis. (B) Annexin-V and 7-AAD staining (left) were used to discriminate between live (R1, black, Annexin-V⁻,7AAD⁻) and apoptotic thymocytes (R2, red, Annexin-V⁺,7AAD⁺). Binding analysis of a GlcNAc-specific monoclonal mouse antibody induced by immunization with Group A Streptococcus (GAC78, right) reveals strong reactivity with apoptotic but not live thymocytes. A mouse monoclonal antibody specific for dextran (A16, middle) shows no reactivity. These observations strongly suggest that immunoreactive cryptic GlcNAc epitopes are accessible to antibodies during apoptotic processes.

Figure 3. Mouse antibodies elicited by immunization with Group A Streptococcus, and polyclonal human serum IgM recognize aberrantly truncated mammalian glycans

(A) Analysis of the fine-specificity exhibited by the mouse monoclonal antibody GAC78 for mammalian glycans by glycan microarray reveals specific reactivity with beta-GlcNAc-terminating glycans. Highest affinity was observed for β-1,4-, β-1,6- and monomeric β-GlcNAc structures. (B) Open source mammalian glycan array data examining polysaccharide autoreactivity represented in polyclonal human serum IgM derived from healthy donors (n=9) demonstrates wide-spread recognition of epitopes contained within the mammalian glycome. IgM reactivity for GlcNAc-terminating glycans was observed in all human serum samples analyzed. (C) Glycan microarray results were formatted to emphasize reactivity for the constituent glycan structures of a mature human glycan identified in pancreatic tissue by mass spectrometry. (top) Illustrations depicting the glycan structures for which respective binding, measured by relative fluorescence units, was obtained following microarray analysis are shown (bottom). Mouse mAb GAC78 (red) exhibits high reactivity for the chitobiose (GlcNAcβ-1,4-GlcNAcβ) core elements, as well as reactivity for terminal branching structures of GlcNAcβ-1,6-(GlcNAcβ-1,2-)Mannoseα-1,6- and GlcNAcβ-1,4-(GlcNAcβ-1,2-)Mannoseα1–3 (not shown). β-1,2-GlcNAc epitopes are not recognized by GAC78, nor are GlcNAc epitopes contained within the context of a mature glycan chain; reactivity is associated only with glycans terminating in particular configurations of GlcNAc. Polyclonal serum IgM from healthy human donors (grey) also exhibits reactivity with chitobiose core elements; however, reactivity for GlcNAcβ-1,6-(GlcNAcβ-1,2-)Mannoseα-1,6-structures was not detected in these samples. Collectively, these results indicate that natural IgM reactivity for cryptic glycan epitopes in mammalian N-linked glycan chains are conserved between mice and humans.

Figure 4. The population of the B-1 B cell repertoire is highly temporal, and is shaped by genetic restrictions on B cell receptor recombination and by positive-selection mechanisms

B-1 B cell committed progenitors have been identified in embryonic mice as early as day 9. There is a clear hierarchy regarding the emergence of various B cell clonal-specificities shaped by both the absence of terminal deoxynucleotidyl transferase activity in fetal B cells (A) and preferential IGHV gene usage (B). B-1 B cell lymphopoiesis continues through neonatal and perinatal development, during which period TdT is active at low levels. Together, these mechanisms result in a dynamic appearance of characteristic clonal BCR rearrangements (indicated by distinct colors). (C) Following successful rearrangement of clonal B cell receptors, B-1 B cells undergo positive selection in order to become mature B-1 B cells. These selection events occur in the context of autologous antigens, giving rise to autoreactive, CD5⁺ B-1a B cells, or through exogenous antigen, giving rise to CD5- B-1b B cells. Once mature, these cells populate the peritoneal and pleural cavities, where they are maintained through self-renewal. (D) Clonal specificities represented in the B-1 B cell pool are highly stable throughout adult life. Thus, the neonatal and perinatal periods represent a critical time of B-1 cell repertoire plasticity, wherein environmental antigens significantly influence the clonal composition of this population. The outcomes of these early developmental and environmental influences on the B-1 B cell repertoire have significant impacts on immunity and the propensity to develop aberrant allergic or autoimmune phenomenon during adult life.

Figure 5. The interplay of endogenous and exogenous antigen signaling on innate-like B cell repertoire formation

Exogenous antigen contributes significantly to clonal representation in the mature B-1 B cell pool, and the complexity of these events is demonstrated through analysis of α-1,6-glucan, β-GlcNAc, and α-1,3-Gal B cell specificities. (A) α-1,3-glucan structures are absent from the mammalian glycome and Dex-specific B cells develop in the absence of stimulation by endogenous glucan epitopes. There are two canonical clonotypes within the Dex-reactive B cell pool, M104E (orange) and J558 (red), the latter of which is more highly represented in the adult repertoire. Glucan-specific B cells possess mainly a B-1b and MZ B cell phenotype, suggesting that exogenous antigen promotes selection of this clonotype into the B cell pool. In support of this interpretation, the numbers of Dex-specific B cell precursors are significantly reduced in mice raised in germ-free conditions, suggesting exogenous antigen is solely responsible for selection or maintenance of cells bearing this specificity. (B) Contrary to Dex-specific B cells, GlcNAc-specific B cells develop in the context of several forms of GlcNAcylated autoantigen, although these cryptic epitopes may provide a limiting developmental signaling to GlcNAc-reactive B cell precursors. In conventionally housed mice, GlcNAc specific cells are predominantly represented in the B-1b (lightgreen) compartment, but are also observed within the B-1a (darkgreen) population. In germ-free conditions, there is a stark decrease in the numbers of GlcNAc-specific cells in both B-1 B cell compartments (blue). Given that the B-1a phenotype is generally associated with autoreactivity, these observations suggest GlcNAc-specific B-1b B cells depend solely on exogenous antigen for their development, whereas exogenous antigen may rescue GlcNAc specific B-1a clonotypes from negative selection following engagement of autologous glycans. (C) Lastly, α-1,3-galactose epitopes, generated by α-Galactosyl Transferase (GalT) are common terminating moieties of N-linked and O-linked glycan chains in mice and drive negative selection of Gal-reactive B cells. Thus, Gal-reactivity is absent from the adult repertoire. In GalT-deficient mice, however, α-1,3-galactose-specific B cells develop, adopt a B-1b B cell phenotype, and are clonally expanded through interactions with commensal microbiota-derived antigens. These examples of three independent B-1b B cell glycan specificities illustrate how integration of signals derived from both endogenous and exogenous forms of homologous carbohydrate epitopes contribute to selection of the anti-glycan repertoire. As the B-1 B cell pool represents precursors of both natural IgM and secretory IgA producing plasma cells, the representation of certain clonal specificities within these expressed natural antibody and mucosal secretory IgA repertoires relies on exogenous antigen.