We review how polyreactive natural IgM autoantibodies (IgM-NAA) protect the host from invading micro-organisms and host neo-antigens that are constantly being produced by oxidation mechanisms and cell apoptosis. Second, we discuss how IgM-NAA and IgM anti-leukocyte antibodies (IgM-ALA) inhibits autoimmune inflammation by antiidiotypic mechanisms, enhancing removal of apoptotic cells, masking neo-antigens, and regulating the function of dendritic cells (DC) and effector cells. Third, we review how natural IgM prevents autoimmune disorders arising from pathogenic IgG autoantibodies, triggered by genetic mechanisms (e.g., SLE) or micro-organisms, as well as by autoreactive B and T cells that have escaped tolerance mechanisms. Studies in IgM knockout mice have clearly demonstrated that regulatory B and T cells require IgM to effectively regulate inflammation mediated by innate, adaptive, and autoimmune mechanisms. It is, therefore, not surprising why the host positively selects such autoreactive B1 cells that generate IgM-NAA, which are also evolutionarily conserved. Fourth, we show that IgM-ALA levels and their repertoire can vary in normal humans and disease states and this variation may partly explain the observed differences in the inflammatory response after infection, ischemic injury, or after a transplant. We also show how protective IgM-NAA can be rendered pathogenic under non-physiological conditions. We also review IgG-NAA that are more abundant than IgM-NAA in plasma. However, we need to understand if the (Fab)2 region of IgG-NAA has physiological relevance in non-disease states, as in plasma, their functional activity is blocked by IgM-NAA having anti-idiotypic activity. Some IgG-NAA are produced by B2 cells that have escaped tolerance mechanisms and we show how such pathogenic IgG-NAA are regulated to prevent autoimmune disease. The Fc region of IgG-NAA can influence inflammation and B cell function *in vivo* by binding to activating and inhibitory FcγR. IgM-NAA has therapeutic potential. Polyclonal IgM infusions can be used to abrogate on-going inflammation. Additionally, inflammation arising after ischemic kidney injury, e.g., during high-risk elective cardiac surgery or after allograft transplantation, can be prevented by pre-emptively infusing polyclonal IgM or DC pretreated *ex vivo* with IgM or by increasing *in vivo* IgM with a vaccine approach. Cell therapy is appealing as less IgM will be required.

Keywords: natural IgG autoantibodies, natural IgM autoantibodies, natural autoantibodies, natural IgM antileukocyte antibodies, allograft rejection, TH-17, renal IRI, autoimmune insulitis, regulation of co-stimulatory receptors, regulatory dendritic cells

### *Edited by:*

*Nichol E. Holodick, The Feinstein Institute for Medical Research, USA*

### *Reviewed by:*

*Ziaur S. M. Rahman, Pennsylvania State University, USA Christoph J. Binder, Medical University of Vienna, Austria*

> *\*Correspondence: Peter Isaac Lobo<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to B Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 16 December 2015 Accepted: 06 May 2016 Published: 06 June 2016*

### *Citation:*

*Lobo PI (2016) Role of Natural Autoantibodies and Natural IgM Anti-Leucocyte Autoantibodies in Health and Disease. Front. Immunol. 7:198. doi: 10.3389/fimmu.2016.00198*

### INTRODUCTION

The study of natural IgM autoantibodies (IgM-NAA) has given us a good insight on how nature, by creating polyreactive pentavalent antibodies, has accomplished a difficult task of trying to protect the host from both diverse foreign pathogens and from diverse self neo-antigens that are constantly produced within the host. Poly-reactivity with low binding affinity, but with high avidity, has enabled these IgM-NAA-producing B cell clones to be rapidly activated by a foreign or an auto neo-antigen for deploying protective mechanisms to the host. Such a response provides time for the adaptive immune system to mount a highly specific immune response to foreign antigens and, in addition, lessens the burden on the host to maintain diverse B cell clones producing highly specific IgG auto-antibodies, which has the potential of causing autoimmune disease owing to their high-affinity binding. Second, since both foreign and self neo-antigens can induce an inflammatory response, these IgM-NAA have taken over another task of subduing an excessive inflammatory response that can injure the host. This regulation of inflammatory cells without damage is made possible by the low binding affinity of IgM-ALA to live leukocytes, combined with its inability to effectively activate the lytic component of complement at body temperature (37°C). Hence, it is not surprising why these IgM-NAA antibodies, which first arose in cartilaginous fish, have been conserved during evolution [reviewed in Ref. (1)] and why IgM-NAA makes up about 70–80% of circulating IgM (2, 3). Additionally, some studies have shown that the binding of natural IgM to autologous receptors is also evolutionarily conserved among mammalian species as human IgM has the same functional effect as murine IgM on murine cells *in vitro* or when used *in vivo* in mice (4–6).

Natural autoantibodies of different isotypes have been intensively studied during the last 40 years (7–17). These autoantibodies have been termed "natural antibodies" as they are produced at birth in the absence of exposure to foreign antigens. The full repertoire of NAA develops by early childhood. In mice, NAA are predominantly produced by the CD5+ B1 cells, while marginalzone splenic B (MZB) cells contribute the remainder. These B1 cells produce predominantly IgM, IgA, and IgG3 autoantibodies (18, 19), independently of T cell help, and exhibit an enhanced response to innate immune signals, such as TLR agonist. Hence, B1 and MZB cells differ from B2 cells in that the response of these cells *in vivo* is rapid and can be driven by TLR agonists independently of antigen binding to their BCR (20–23). Additionally, there are data to indicate that autoantibody-producing B1 cells, unlike self-reactive T cells, are positively selected for their selfreactivity, thus implying that NAA are conserved by design (24–27). Further support for their importance comes from studies in chimeric mice demonstrating that IgM-NAA comprise the majority of circulating IgM (2, 3). Most cross-sectional studies in humans and rodents would indicate that IgM-NAA decrease with age (28–31) or lose their effectiveness with age (32) except for one report where follow-up of five healthy individuals for 25 years revealed no change in IgM-NAA levels (33). However, IgG-NAA can increase (34) but do not decrease with age (35, 36).

Innately produced natural IgM-NAA should not be confused with immune IgM and IgG that are produced several days later after exposure to foreign antigens or pathogens. Such immune IgM and IgG are not natural autoantibodies and in general are antigen specific and are produced by B2 cells that require antigen binding to BcR and additional T cell help to generate anti-protein antibodies. However, production of immune IgM is limited as these IgM secreting B2 cells migrate to B cell follicles, where with the help of T cells, these B2 cells undergo isotype switching and somatic hyper-mutation, thus generating long-lived memory B cells and differentiating into plasma cells that produce IgG antibodies with high-affinity binding.

The human equivalent of the murine CD5+ B1 subset has been recently identified and characterized. This CD20+ CD43+ CD27+ human B1 subset that can spontaneously secrete antibody represents about 50% of umbilical cord B cells and 15–20% of circulating adult B cells, and is the predominant source of human IgM-NAA (29). In humans, CD5 is not a specific marker of B1 as this marker is expressed by both B1 and B2 cells. Similarly, CD43 and CD27 are not specific markers for human B1 as about 20% of CD43+ CD 27+ B cells have characteristics of pre-plasmablasts that are derived from T-dependent B cells present in germinal centers (37, 38). Human IgM-NAA are also polyreactive and bind similar autoantigens as in mice, including oxidized neodeterminants and leukocyte receptors (4, 39–41).

### IgM-NAA, IgG-NAA, and Pathogenic IgG Autoantibodies

One physiological role of NAA is to protect the host from pathogenic IgG autoantibodies. We will, therefore, briefly describe the biology of natural IgM and IgG-NAA and pathogenic IgG autoantibodies in health and disease and then discuss the different mechanisms used by NAA to counter pathogenic IgG. B1 cells have been shown to secrete IgM, IgA, and IgG3-NAA that are encoded by minimally or non-mutated germ line genes that are enriched for heavy chain variable region rearrangements and with H-L pairings that allow for poly-reactivity (18, 19). NAA generated by B1 cells rarely undergo isotype switching or somatic hyper-mutation to acquire antigen specificity. IgA in the gut is predominantly produced by B1 cells present in the gut lymphoid tissue but there is increasing evidence to show that B2 cells can also participate in gut mucosal immunity and this could account for the observed somatic hyper-mutation and antigen specificity of gut mucosal IgA (42–46).

### Natural IgM Autoantibodies

In mice, the majority of IgM-NAA are produced by splenic B1 and MZB cells (47–49). B1 and MZB cells, such as memory B cells, express CD27 and spontaneously produce IgM, but IgM production can also be increased by TLR activation [e.g., lipopolysaccharide (LPS)] or via the BCR in response to pathogens. Normal levels of IgM-NAA are present at birth even under germ-free conditions and in nude mice, indicating that IgM-NAA production is not dependent on exposure to foreign antigens, TLR or T cell activation (50). IgM-NAA repertoire is shaped by T-independent antigen activation, especially of MZB cells (51, 52). An important characteristic of these antibodies is their low binding affinity (53). It is possible that the membrane expressed IgM or BCR of B1 and MZB cells producing NAA also exhibit low binding affinity and perhaps this latter characteristic may be involved in preventing autoreactive B1 cells from being deleted or undergoing negative selection. There are data to show that autoreactive B1 cells are positively selected and this process requires both the autoantigen and the relevant BCR (24–27). The need to positively select B1 cells secreting IgM-NAA would indicate that these antibodies have an important physiological role that will be reviewed later.

Natural IgM autoantibodies have been shown to be polyclonal with clones having specificity for some, but not all self-antigens, i.e., certain common epitopes present on phylogenetically conserved self-antigens. Some of these IgM clones with reactivity to selfantigens have been identified, e.g., IgM clones with specificity for leukocyte receptors [IgM anti-leukocyte antibodies (IgM-ALA)] (4, 39), Fc domain of IgG (rheumatoid factor) (14, 15), complement components (17), collagen, thyroglobulin, intracellular constituents, such as cytoskeletal proteins, cytosolic enzymes, dsDNA, or nucleosomes, neutrophil cytoplasmic enzymes (ANCA) (50, 54) and oxidized neo-determinants [e.g., phosphorylcholine (PC)] that are exposed when lipids are oxidized or cells undergo apoptosis (55, 56). While some IgM-ALA have mono-reactivity, e.g., to some cytokines, most are polyreactive with each polyreactive IgM-NAA clone having a selective binding profile (50). For example, IgM anti-PC NAA will bind to ABO blood type antigens, endotoxins, and oxidized neo-determinants on apoptotic cells but this autoantibody has no binding reactivity to nuclear antigens or to IgG (57). Additionally, these IgM-NAA, by virtue of being polyreactive, also cross-react with pathogen-expressed molecules, for example, PC on *Streptococcus pneumoniae* and other antigens expressed by various viruses and parasites (55–57) Hence, it has been suggested that these natural IgM antibodies are protective, serving as a first line of defense against infections and protecting the host from pathogen-mediated apoptotic cells and oxidized neo-determinants that can induce pathogenic IgG autoantibodies (55, 56). Additionally, polyreactive IgM-NAA have been shown to bind to idiotypic determinants on self-reactive IgG, thus providing another mechanism to protect the host from high-affinity binding IgG autoantibodies that are potentially pathogenic (31, 50).

In mice, B1 cells are rare in the bone-marrow, lymph nodes, and splenic B cell follicles (white pulp) while significant numbers of B1 cells are located in the splenic marginal zone as well as in the peritoneal and pleural cavities. However, under normal conditions, these peritoneal B1 cells do not contribute significantly to circulating IgM-NAA, but during sepsis, peritoneal B1 cells rapidly migrate to the splenic marginal zone (23, 58) where most of the circulating IgM-NAA are produced (59). B1 cells are distinct from B2 cells in many respects and they are derived from different progenitors (18, 19, 60). Importantly B1cells express CD27, a memory B cell marker, and such as memory B cells, on encountering antigen, spontaneously secrete IgM without requiring signaling via co-stimulatory molecules (60). B1 cells do not require to traffic or reside in the splenic B2/T cell follicles as they are T independent and secrete IgM without isotype switching or somatic mutation (25, 59, 61). In fact, isotype switching and somatic hyper-mutation of immunoglobulins in B1 cells is kept in check during an immune response to prevent the development of high affinity, anti-self IgG antibodies (61, 62). In this regard, SPA-1 in B-1 cells inhibits Rap-1 GTP, which enhances somatic hyper-mutation and hence SPA-1 deficient mice generate high-affinity IgG anti-dsDNA and anti-red blood cell autoantibodies and develop lupus nephritis as they age (62). Recent data would indicate that levels of peritoneal B1 and splenic B1 and MZB cells is regulated by serum levels of polyclonal (but not monoclonal) IgM (63–65) that interacts with FcμR expressed by these cells (66) as well as by IgG binding to FcγRIIB (67).

### IgG-NAA

One can demonstrate the existence of IgG-NAA in normal adult murine or human serum under conditions where serum IgM is either removed or diluted out (31, 68–70), indicating therefore that in serum the functional activity of IgG-NAA is blocked by polyreactive IgM with anti-idiotypic activity (31, 50, 54). There is, however, a large amount of IgG-NAA as significant levels of IgG-NAA can be detected even after diluting serum at 1:500 (34). Both IgG-NAA and IgM-NAA bind to the same phylogenetically conserved selfantigens and 15–20% of normal mouse IgG has been found to be polyreactive (50, 68). IgG produced by B1 cells are characteristically of the IgG3 isotype that have been shown to be polyreactive (18, 19). Clones of B cells secreting IgG4κ with high-affinity binding to dsDNA has been shown to exist in human umbilical cord blood and in the same umbilical cord there were other B cell clones producing IgM antibodies with binding reactivity to idiotypic determinants on IgG4 anti-dsDNA but not to idiotypic determinants on other IgG4 antibodies (34). The above observations would suggest that B cells generating IgG-NAA of different isotypes and affinities are present at birth and the IgG-NAA are rendered functionally inactive by polyreactive IgM with anti-idiotypic activity.

Importantly, IgG-NAA-producing B cells, unlike IgM-NAAproducing B cells, are in an inactive state at birth and in mice these B cells start producing IgG-NAA after exposure to bowel bacteria or foreign antigens (71–73). In humans, it may take more than 2 years before significant levels of IgG-NAA can be detected in the serum (74). It is also possible that autoreactive T cells, which have been shown to exist in normal individuals (75) or infectious agents could activate B cells to produce IgG-NAA later on in life. For example, levels of polyreactive IgG anti-dsDNA have been shown to increase after various infections and these polyreactive IgG anti-dsDNA have been found to cross-react with antigens on micro-organisms, including bacteria (76). It is, therefore, not surprising that low levels of IgG-NAA at birth increase after exposure to infections (71–73).

Studies on IgG-NAA isotypes in normal sera or on the B cell subsets that produce these antibodies are lacking. There are, however, extensive data on an IgG-NAA, i.e., IgG anti-DNA, that is both mono-reactive and polyreactive and present in both normal sera and SLE sera and some of the findings with IgG anti-DNA may apply to other IgG-NAA. Several lines of evidence would favor that B2 cells, with the help of T cells, can generate IgG-NAA. Wardemann et al. clearly demonstrated that 55–75% of early B cell precursors, present in human adult bone-marrow, display auto-reactivity to conserved intracellular cytoplasmic and nuclear constituents. About 80% of these autoreactive B cells are removed at two check points, i.e., centrally in the bone-marrow and in the periphery (77). Second, in human umbilical cord, B cells secreting high-affinity binding IgG4 anti-dsDNA have been isolated and IgG1 antimyeloperoxidase (ANCA) antibodies have been found in normal human sera, indicating that these high-affinity isotypeswitched antibodies are produced by the T cell-dependent B2 cells (34, 78). Furthermore, in the MLR/*lpr* murine model of SLE, IgG anti-dsDNA has been shown to be produced by B2 cells under the influence of excess T helper cells as a result of Fas deficiency (79). Hence, removing T cells ameliorates SLE and reduces production of IgG anti-DNA in this model of SLE (80–82).

Under normal conditions, it is unclear whether the (Fab)2 region of IgG-NAA is physiologically active especially since their activity is blocked by poly reactive IgM with anti-idiotypic activity (31, 50). Hence, most of the physiological functions of IgG-NAA have been derived from purified IgG or pooled purified IgG (IVIG). *In vivo* infusion of IVIG has been shown to ameliorate cell-mediated inflammatory processes, e.g., in Kawasaki's disease or autoantibody-mediated disorders, e.g., in idiopathic thrombocytopenic purpura, anti-factor VIII autoimmune disease, or myasthenia gravis [reviewed in Ref. (83)]. One can show, in *in vitro* studies, that the beneficial effects of purified IgG-NAA can be mediated by both the (Fab)2 region and the Fc region of IgG-NAA. In these *in vitro* studies, the (Fab)2 region has been shown to (i) block function of activating FcγR receptors expressed by monocytes/macrophages. Such activating FcγR are involved in phagocytosis of IgG autoantibody complexed to platelets, (ii) block idiotypic determinants on IgG autoantibodies, thus neutralizing these antibodies, e.g., their binding to Factor VIII or platelets, (iii) inhibit function of certain proinflammatory cytokines by binding to these cytokines or their receptors, and (iv) provide protection against bacterial infection by binding of IgG-NAA to lectins on bacterial surfaces thus enhancing opsonization (84). Similarly, in *in vitro* studies, the Fc region of IgG-NAA has been shown to inhibit the function of B cells, plasma cells, dendritic cells (DC), macrophages, and neutrophils by binding to the inhibitory FcγRIIB receptor expressed by these cells. Activation of FcγRIIB by Fc region of IgG-NAA inhibits production of autoantibodies by B cells and plasma cells. Additionally IVIG ameliorates cell-mediated inflammation, e.g., in Kawasaki's disease, by activating FcγRIIB and inhibiting the function of dendritic cells, macrophages, and neutrophils.

IgG with anti-idiotypic activity have been found in pathological conditions especially when there is minimal or no IgM with antiidiotypic activity (85) and increased levels of *in vivo* anti-idiotypic IgG has been associated with amelioration of disease activity mediated by pathogenic IgG autoantibodies, thus indicating that under pathological conditions, *in vivo* IgG with anti-idiotypic activity is functionally active (85, 86). There are data to show that normal levels of IgG autoantibodies, e.g., to myeloperoxidase (MPO) increase >10-fold during disease states (87) and it is possible that under pathological conditions when IgM-NAA with anti-idiotypic activity is low, there is initially a compensatory increase in IgG antibodies with anti-idiotypic activity and the disease does not manifest clinically (85). However, with time, the data would also indicate that there is a decrease in the protective IgM/IgG anti-idiotypic antibodies and the disease manifests clinically (85). Hence, administering IVIG, which is known to have anti-idiotypic activity, could be beneficial in this situation especially if levels of both IgG and IgM-NAA are relatively low.

### Pathogenic IgG Autoantibodies

Pathogenic autoantibodies that cause disease differ from IgM and IgG3-NAA in that they are predominantly of the IgG isotype, undergo somatic mutation with addition of N regions and these IgG autoantibodies can be either polyreactive or exhibit mono-reactivity. IgG autoantibodies, unlike IgM-NAA, have high-binding affinity (53). However, not all IgG autoantibodies with high-binding affinity are pathogenic *in vivo* and this is best exemplified by IgG anti-dsDNA autoantibodies (88–91). There are conflicting data on the B cell precursors that generate these pathogenic IgG autoantibodies. Data would indicate that both B1 and B2 cells can generate pathogenic IgG autoantibodies depending on the animal model used. For example, studies in the Fas-deficient MRL-*lpr* murine SLE model would indicate that pathogenic self-reactive IgG antibodies are generated by B2 cells (79). However, in the NZB/W disease model of SLE and in a murine transgenic model of hemolytic anemia, the pathogenic murine IgG anti-dsDNA and anti-erythrocyte transgenic autoantibodies were shown to be produced by B1 lymphocytes (21, 92–94). Hence, removing T cells did not affect production of IgG anti-dsDNA or disease activity in the NZB/W model of SLE (80).

There are several mechanisms that increase pathogenic IgG antibody production and IgG anti-dsDNA has been most studied in this regard. In adolescent females with SLE and rheumatoid arthritis (RA) patients, there are data to show that production of IgG polyreactive autoantibodies results from breakdown of B cell tolerance mechanisms where during B cell development, B cells producing high-affinity IgG autoantibodies have not been removed or silenced either centrally in the bone-marrow or in the periphery before they mature into naïve immuno-competent lymphocytes (95, 96). Studies in healthy individuals indicate that 55–70% of newly generated bone-marrow B cells express self-reactive antibodies and most of these B cells are removed or silenced such that in healthy humans there remains 5–20% of circulating naïve B cells that continue to generate self-reactive IgG autoantibodies (77). However, in SLE and RA patients, owing to breakdown of B cell tolerance mechanisms, 25–50% of mature naïve B cells were found to generate self-reactive IgG autoantibodies with diverse specificities (95, 96). Second, excess BAFF could also contribute to the breakdown of tolerance in the periphery as BAFF has been shown to inhibit deletion or apoptosis of selfreactive B cells (92, 97). Elevated BAFF levels have been shown to be present in serum of RA and Sjogren's syndrome (98)**.** Third, there are murine studies to show that IgM-NAA-producing B1 cells can be induced, especially with repeated immunization, to generate polyreactive IgG anti-self that can also react to the immunizing antigen. For example, B1 cells can be induced to generate pathogenic polyreactive IgG anti-DNA and anti-myosin antibodies by immunization with a hapten or an antigen derived from a micro-organism (99–101). Other mechanisms may, however, operate in certain other autoimmune disorders where highly specific IgG autoantibodies are produced, e.g., in autoimmune hemolytic anemia and in Factor VIII deficiency. It is possible that such autoimmune disorders could arise from a breakdown in peripheral mechanisms such as specific deficiency of antiidiotypic IgM-NAA that blocks pathogenic autoantibody or specific deficiency of IgM-NAA that binds and masks autologous neo-determinants such as dsDNA.

Based on observations in the preceding paragraph, it appears that different mechanisms could operate in generating pathogenic IgG autoantibodies depending on the murine disease model. Additionally, the mechanisms may differ in humans even though the disease has similar disease manifestations. A summary of the existing evidence would indicate that IgG autoantibodies can be induced by the following mechanisms:

and anti-GBM nephritis, frequently occur in elderly patients who, with aging, can develop lower levels of IgM-NAA (28–32) but not IgG-NAA (33, 35, 36), which can increase with aging (34).

**Figure 1** summarizes the role of natural and immune antibodies in normal and pathological states.

### Role of IgM-NAA in Protecting Against Pathogenic IgG Autoantibodies

SLE is the most studied autoimmune disorder where high levels of IgM and IgG autoantibodies, directed against nuclear antigens, especially dsDNA, are produced in the majority of patients. Before we discuss the role of IgM-NAA in protecting against pathogenic IgG anti-dsDNA, it would be important to indicate that a subset of lupus nephritis (approximately 25%) can occur in the absence of detectable circulating IgG anti-DNA and this is commonly referred to as C1q nephropathy (113, 114). The latter observations have led some investigators to question whether IgG anti-dsDNA is indeed pathogenic (115). In some patients with C1q nephropathy, circulating IgG anti-dsDNA can be detected several years later but this subset of patients with C1q nephropathy have in general a better prognosis even though histologically there is similar glomerular immunoglobulin staining and immune deposits as in classical lupus nephritis (114). It is tempting to speculate that patients with C1q nephropathy may have high IgM-NAA and IgM anti-dsDNA that may be preventing detection of circulating IgG anti-dsDNA. A murine model with lupus nephritis but with no IgG anti-dsDNA in both the circulation and in kidney eluates has also been described (116). It is possible that in this murine model, the immune deposits detected in the kidney may be caused by

IgG complexes containing another antigen, other than dsDNA, and a similar mechanism could also operate in some patients with C1q nephropathy without circulating IgG anti-dsDNA.

However, there is evidence indicating that IgG anti-dsDNA, together with C1q, is an active participant in inducing or initiating glomerular damage in classical lupus nephritis with circulating IgG anti-dsDNA. First, SLE in the MRL*/lpr* lupus-prone mice is ameliorated when rendered IgG deficient by crossing these mice with either activation-induced deaminase (AID) or CD40Ldeficient mice (117, 118). Second, one can induce lupus nephritis in normal mice by either infusing purified IgG anti-dsDNA or by increasing *in vivo* IgG anti-dsDNA with use of transgenic cells or hybridoma cells (88–91). Importantly, not all IgG anti-dsDNA are pathogenic as defined by induction of lupus nephritis *in vivo* and this also may explain why certain patients with high levels of circulating IgG anti-DNA are spared from lupus nephritis (88–91). In SLE, a potential protective mechanism involves binding of IgM anti-dsDNA to DNA with masking of the antigenic determinants that bind to IgG anti-dsDNA. Hence, lupus nephritis can occur if there is excess IgG anti-dsDNA or if there is a relative deficiency of IgM anti-dsDNA. Such a concept will explain the accelerated development of lupus nephritis in MRL*/lpr* mice lacking IgM and will also explain the lack of lupus nephritis in patients having high IgG anti-dsDNA and high IgM anti-dsDNA or IgM-NAA (119, 120). This latter possibility may also explain why FcγRIIBdeficient Balb/c mice are more resistant to developing lupus nephritis when compared to FcγRIIB-deficient C57BL/6 mice (121). In both mice, there is excess IgG anti-dsDNA production due to lack of the inhibitory FcγRIIB receptor but Balb/c mice have significantly higher levels of plasma IgM when compared to C57BL/6 mice (122–124).

Several observations would suggest a role of IgM-NAA in protecting normal individuals and patients with autoimmune disorders from such self-reactive IgG antibodies. The protective role of IgM-NAA is best exemplified in murine models of SLE, where enhancing IgM anti-dsDNA levels or infusing IgM anti-dsDNA, ameliorates disease activity (107, 125, 126). Well studied mechanisms by which IgM-NAA can induce protection and prevent inflammation include (i) removing neo-antigens by binding of IgM-NAA to oxidized neo-determinants and removal of IgM/C1q bound antigens by phagocytosis, e.g., IgM anti-PC (127), (ii) inhibition of the pathogenic IgG autoantibody induced inflammatory response by IgM-NAA, e.g., IgM-ALA (128), (iii) neutralization of pathogenic IgG autoantibodies by IgM-NAA having anti-idiotypic activity (129), (iv) preventing binding of IgG to antigenic determinants by competitive inhibition even though these IgM-NAA have lower binding affinity (129), (v) inhibiting complement activity by binding of IgM to complement components (17), and (vi) inhibiting generation of IgG autoantibodies possibly by binding of IgM-NAA to endogenous autoantigens/ neo-antigens or to the B cell FcμR (64, 65, 119, 130–132).

The above mechanisms may explain why mice without secretory IgM have increased IgG autoantibodies, including anti-dsDNA, with aging (119, 120). This would also explain why B1-deficient SJL mice are more susceptible to induction of experimental autoimmune thyroiditis or allergic encephalitis cells (133). Conversely, such a concept would also explain why disease activity in SLE patients is less severe with high levels of IgM anti-dsDNA and other IgM-NAA (125, 134, 135). The above mechanisms may also explain why several autoimmune disorders are helped by either increasing production of *in vivo* IgM-NAA (126) or administering either pooled polyclonal IgM or a specific IgM-NAA (110, 129, 136). The latter is best exemplified by IgM anti-dsDNA where infusion of a specific IgM-NAA ameliorated SLE-induced nephritis in NZB mice (107).

Presence of pathogenic IgG autoantibodies leads to a compensatory increase in IgM–NAA, such as IgM anti-PC, IgM-ALA, and IgM/IgG anti-idiotypic antibodies, to counter these pathogenic IgG antibodies as well as inflammation initiated by the excess IgG self-reactive antibody. It is also possible, that over time, the production of IgM and IgG anti-idiotypic antibodies may not be able to keep up with the relatively high production of pathogenic IgG autoantibody and the disease becomes symptomatic. Hence, the initial increase in IgM-NAA and IgG anti-idiotypic antibody levels could explain the asymptomatic nature of the disease despite increased levels of IgG anti-dsDNA and other anti-nuclear antibodies several years before clinical onset of disease (85, 137). Additionally, there could be a heightened compensatory response of other immuno-regulatory mechanisms, e.g., increase in T-regs or IL-10 in B1 cells, which could inhibit the clinical onset of disease or ameliorate disease activity in these patients and other animal models. The latter is best exemplified in mice with secretory IgM deficiency, where despite total lack of circulating IgM and increased IgG3 anti-dsDNA (120), these cellular compensatory mechanisms inhibit development of autoimmune disease including lupus nephritis (63, 119). It is also possible that IgG3 anti-DNA is not pathogenic and their increased production by B1 cells, in secretory IgM deficiency (120), may be protective. However, in situations where inflammation has been experimentally induced or there is an excessive increase in production of pathogenic IgG autoantibodies or increased apoptosis, then these cellular regulatory mechanisms cannot effectively compensate for lack of IgM and this leads to a more severe and rapid worsening in disease activity. For example, SLE disease is more severe in the MRL-*lpr* mice when these mice are crossed with secretory IgM deficient mice (120). Similarly, secretory IgM-deficient mice are prone to develop a more rapid and severe inflammatory response after renal ischemia or allogeneic cardiac transplantation (138).

### Physiological Role of IgM-NAA

IgM–NAA, which are spontaneously secreted by B1 and MZB cells, are polyclonal and exhibit a diverse repertoire of antigen specificities, many of which are polyreactive and, hence, crossreact with foreign antigens or pathogens. Additionally, B1 and MZB cell clones can be activated either by their TLR or BCR by both endogenous and foreign antigens. These special characteristics of IgM-NAA, together with the low-binding affinity (but with high-binding avidity owing to its pentameric structure), have allowed these antibodies to acquire functions that are not characteristic of the highly specific immune IgM and IgG antibodies. Physiological functions that have been attributed to IgM-NAA have included the following:

(iii) *Inhibiting IgG autoantibody production and inflammatory responses by clearing apoptotic cells and binding to oxidized neo-determinants*. Apoptotic cells that are normally produced in large numbers, express oxidized neo-determinants. Several homeostatic mechanisms, including binding of C1q and mannose binding lectin (MBL) to apoptotic cells, are involved in inducing macrophages and DC to phagocytose apoptotic cells and nuclear antigens [reviewed in Ref. (145)]. IgM-NAA are also involved in enhancing this phagocytic process, especially after tissue injury of nerves (69), and in inducing an anti-inflammatory effect (56). Several IgM-NAA antibodies have been identified, each with binding specificity to certain oxidized neo-determinants, including PC,

representative examples of five separate experiments. Figure and legend reproduced with permission from Ref. (144). Copyright 2008. The American

Association of Immunologists, Inc.

malondialdehyde (MDA), cardiolipin, phosphatidylserine, and Annexin IV present on apoptotic cells and not live cells (146). The best-studied IgM-NAA reactive to apoptotic cells is the IgM anti-PC antibody (also referred to as T-15), which binds to a dominant oxidized neo-determinant on apoptotic cells. Both *in vitro* and *in vivo* studies, using monoclonal IgM anti-PC, have clearly shown that phagocytosis by DC and macrophage is enhanced when C1q or MBL binds to the IgM/apoptotic cell complex. In these *in vivo* studies, either endogenous production of IgM anti-PC was increased by administering large numbers of apoptotic thymocytes (2.5 × 107 ) or mice were given 1.5–2 mg of a monoclonal IgM anti-PC preparation that bound to endogenous apoptotic cells. Hence, natural IgM anti-PC inhibits production of pathogenic IgG autoantibodies by masking oxidized neo-determinants and enhancing removal of apoptotic cells. However, enhancing apoptotic cell phagocytosis by DC also inhibits DC maturation/activation and induces an anti-inflammatory effect with decreased production of pro-inflammatory cytokines and an increase in levels of splenic regulatory B cells (127, 147). Additionally, repeated weekly infusions of monoclonal IgM anti-PC or apoptotic thymocytes inhibited development of arthritis and induced regulatory B cells in mice (127, 147).

Natural IgM autoantibodies has also been shown to bind to various oxidation-specific neo-determinants, specifically PC and MDA present on oxidized LDL lipids, but not native LDL (146, 148, 149). High levels of IgM anti-PC and IgM anti-MDA were also found in ApoE-deficient mice that have high circulating cholesterol and severe atherosclerosis. It has been suggested that oxidized lipids, in this murine model, induces these high IgM anti-PC/MDA antibodies as a protective mechanism. Supporting such a concept are other observations showing an association of high *in vivo* levels of IgM anti-PC and reduced atherosclerotic plaques in both patients and different animal models [reviewed in Ref. (56)]. Similarly, there are studies showing that immunization of LDL receptor knockout mice with the *S. pneumoniae* vaccine induces IgM anti-PC and this is associated with less atherosclerotic lesions (150). Several mechanisms have been postulated to explain the amelioration of atherosclerotic lesions by IgM anti-PC in these murine models of atherosclerosis. First, IgM anti-PC masks the PC containing neo-determinants on oxidized lipids, thus inhibiting uptake of these lipids by macrophages and this leads to a decrease in foamy macrophages that are part of the atherosclerotic plaque (151). Second, IgM anti-PC, together with C1q, enhances phagocytosis of apoptotic foamy macrophages containing high levels of lipids. Enhanced phagocytosis of apoptotic macrophages, especially by DC, inhibits DC maturation/activation and induces an anti-inflammatory milieu (127, 147). Data demonstrating increased atherosclerosis in secretory IgM-deficient mice or in C1q ko mice would lend support to the role of IgM anti-PC and C1q in inhibiting atherosclerosis (102, 141, 152–154).

The finding that both IgM-deficient mice and C1q knockout mice have increased apoptotic cells as well as develop SLE would indicate that both IgM anti-PC and C1q are required to effectively remove or clear apoptotic cells (155, 156). The latter studies would also indicate that inefficient clearance of apoptotic cells, including nuclear antigens, induces development of pathogenic IgG autoantibodies and SLE. However, ineffectual clearance of apoptotic cells cannot entirely explain the increased propensity of IgM-deficient mice to develop autoimmunity and more severe atherosclerosis when compared to C1q ko mice as both mice have similar number of apoptotic cells in the atherosclerotic plaques and the C1q ko mice has in addition, circulating apoptotic cells that should have worsened atherosclerosis (102, 141, 155). Such observations would indicate that IgM-NAA regulates inflammation by other additional mechanisms (to be presented below) besides enhancing phagocytosis of apoptotic cells.

(iv) *Inhibiting inflammation by binding of IgM-NAA to receptors on live leukocytes, i.e., via IgM-ALA*. The existence of this IgM-NAA subset was known since 1970 (157). Their role in inflammation was recognized when several investigators showed that the level of these antibodies, like IgM anti-PC, increased with diverse infections and inflammatory states [reviewed in Ref. (39)]. However, it was unclear whether IgM-ALA were pathogenic or not. The idea that IgM-ALA may have anti-inflammatory function and may be protective came from observations in allograft recipients where patients with high levels of IgM-ALA were found to have significantly less rejections and developed less alloantibodies after alloimmunization (**Figure 3**) (39, 131). We hypothesized that these natural IgM-ALA increase during inflammatory states to regulate leukocyte function and prevent excess inflammation that may be detrimental to the host. Two characteristics inherent in IgM-ALA allowed us to make this hypothesis. First, we showed that these antibodies bind with low affinity to different leukocyte receptors, including co-stimulatory molecules and chemokine receptors (4, 39). Second, we and other investigators observed that these complement fixing antibodies did not lyse leukocytes at body temperature but readily lysed cells at room temperature or colder temperatures in the presence of complement (131, 157, 159). This non-lytic nature of these antibodies at body temperature led us to hypothesize that IgM-ALA, by binding to receptors on live leukocytes at body temperature, could alter or regulate their function. Initially, with *in vitro* studies using human IgM, we showed that the repertoire of IgM-ALA varies among individuals during health and in disease and, second, that IgM-ALA regulates the function of both human and murine T cells and dendritic cells (4, 39, 138). Additionally, in murine models, we showed that increasing *in vivo* levels of IgM-ALA with physiological doses of purified polyclonal IgM, i.e., 150 μg/mouse, every 3 days, protected mice from (a) renal ischemia reperfusion injury (IRI), (b) ameliorated rejection in a fully mis-matched cardiac allograft model, and (c) protected NOD mice from developing autoimmune insulitis and diabetes. In further experiments, we show that the protective effect in the model of innate inflammation (i.e., renal IRI) is mediated in part by IgM-ALA binding

differences in the level of IgM bound to donor T lymphocytes (IgM-ALA) after addition of pre-transplant serum from different ESRD patients. (B) Alloantibody titer in sera measured by cytotoxicity is correlated to presence of IgM autoantibody to leukocytes. (C) The difference in percentage of acute rejections and graft loss comparing high IgM-ALA vs. the no and low IgM-ALA groups. *MCF* indicates increase in mean channel fluorescence of anti-IgM staining after addition of serum. Figure and legend reproduced with permission from Ref. (131) copyright 1981 Wolters Kluwer Health Inc. (B) Figure and legend reproduced with permission from Ref. (158). Copyright 2010 Springer Science + Business Media LLC (A,C).

to activated/mature DC and regulating their function (4). In a later section, we will review in more detail the above data on IgM-ALA. We plan to show that the observed antiinflammatory effects of polyclonal IgM was predominantly from IgM-ALA and not from IgM anti-PC that bound to apoptotic cells.

(v) *Inhibiting expansion of B1 cells and enhancing antigen presentation to B2 and helper T cells in splenic lymphoid follicles.* This physiological function of IgM became obvious in mice with secretory IgM deficiency as well as in mice with FcμR deficiency. B1 cells in the splenic marginal zone increased several fold in mice deficient in IgM while levels of IgM increased in FcμR-deficient mice indicating that IgM maintains B1 cell homeostasis and levels of natural IgM by binding to FcμR (63, 64, 66, 160). Second, both these mice have impaired immune IgG response to protein antigens, especially with low dose antigen, indicating that IgM facilitates antigen trafficking from the splenic marginal zone to the B2 cell rich lymphoid follicles via FcμR bound IgM/ antigen complexes (63, 160).

The above observations indicate that IgM-NAA protect the host from invading organisms and more importantly maintain several homeostatic mechanisms primarily aimed at preventing autoimmunity and over exuberant inflammation, which can have detrimental effects on the host. **Table 1** summarizes some of the physiological and pathological concepts outlined above. Several observations indicate that infective and other inflammatory states increase all IgM-NAA subsets, especially IgM anti-PC to clear the increased production of apoptotic cells that could trigger autoimmunity and, second, increase IgM-ALA to subdue excess inflammation that can be detrimental to the host [reviewed in

### TABLE 1 | Physiologic function of non-pathogenic autoantibodies.

Ref. (56) and next section]. Based on the preceding observations, one could predict that a decrease in IgM-NAA, as can occur in aging (28–30), could predispose to increased autoimmunity and increased morbidity and mortality from an excess inflammatory response, for example with infections.

### Pathogenic Effects of IgM-NAA Under Non-Physiological Conditions

Certain IgM-NAA which under physiological conditions are protective and anti-inflammatory can under non-physiological conditions become pathogenic and induce inflammation. In this section, we will present some of the conditions that can induce certain protective IgM-NAA to become pathogenic.

Interestingly, one does not observe innate inflammation mediated by IgM anti-NMM after murine renal ischemia even though endothelial cells in glomeruli and peri-tubular capillaries express NMM (167, 168). One possibility is that NMM in the peri-tubular capillaries or in the tubules is not unmasked after ischemia. After renal IRI, most of the ischemia induced kidney injury occurs in the outer medullary renal tubules. However, after renal IRI, one can detect increased IgM binding to glomeruli but not to the extensive network of NMM containing capillaries that surround the tubules or the renal tubules (168). Additionally, depleting B1 cells, decreased binding of IgM to glomeruli, and decreased glomerular injury, but did not protect tubules from inflammation-mediated renal injury, thus indicating that the innate inflammation seen after renal IRI is not mediated by natural IgM and complement (168). Other studies would indicate that the inflammatory response after renal IRI is mediated by ischemia-induced renal tubular injury that activates innate immune cellular mechanisms involving NK and NKT cells (169). This latter study would also explain why Rag-1 ko mice and IgM ko mice without secretory IgM are not protected from renal IRI (4, 138, 170–172).

(iii) *Induction of pathogenesis by non-physiologic expansion of specific IgM*-*NAA clones*. This is best exemplified by expansion of certain B1 cell clones that specifically secrete rheumatoid factor (RhF), an IgM-NAA that binds to self IgG. Excess production of RhF predisposes to generation of large circulating IgM/IgG complexes (referred to as cryoglobulins as these complexes precipitate *ex vivo* in the cold) that cause thrombosis of small blood vessels especially in the kidney glomeruli and skin (173, 174). This serious clinical problem is treated by plasmapheresis (to remove cryoglobulins) and agents to deplete B cells. The most common etiology for monoclonal or polyclonal expansion of RhF secreting B1 cell clones is chronic hepatitis C infection. Currently, it is unclear why this viral infection is associated with clonal expansion of RhF secreting B1 clones. More importantly, even though RhF was the first IgM-NAA to be discovered, we do not understand their normal physiological role.

### Nature of IgM-ALA and Understanding Their Anti-Inflammatory Function

In this section, we review some of our pertinent observations on IgM-ALA that initially were discovered because of their lymphocytotoxic activity at room temperature in the presence of complement [reviewed in Ref. (39)]. Here, we show (a) that a substantial subset (8–10%) of IgM secreting human B cell clones, obtained from the umbilical cord, has IgM-ALA activity, (b) that polyclonal IgM-ALA clones have binding specificity to certain leukocyte subsets and that polyclonal IgM-ALA will bind to only some leukocyte receptors and not others. More importantly, we show that the repertoires of IgM-ALA vary in disease states among different individuals, and (c) that IgM-ALA exhibits anti-inflammatory effects, both *in vitro* and *in vivo.* We show that the anti-inflammatory effects are in part explained by IgM-ALA binding to co-stimulatory receptors and regulating the function of T effector cells and DC without decreasing Tregs. These *in vitro* studies involve human and murine cells, while *in vivo* studies were performed in mice.

### Human Umbilical Cord B Cell Clones Produce IgM-ALA that Exhibit Leukocyte Receptor Specificity – Binding of IgM to Leukocytes was Not Mediated by Fc**μ**R

We initially wanted to determine if IgM-ALA exhibited leukocyte receptor specificity as natural antibodies are polyreactive and, hence, each monoclonal IgM could non-specifically bind to carbohydrate or other moieties on several leukocyte receptors. We isolated B cell clones from human umbilical cord blood and observed that >90% of B cells were IgM secreting with about 10% of the IgM clones having IgM-ALA binding activity when examined by flowcytometry on a cell mixture comprising of B (Daudi), T (Jurkat, Sup T1), and macrophage (U937) human cell lines. The lack of demonstrable leukocyte reactivity by the majority of IgM clones would indicate that IgM did not bind to FcμR on human B cells and macrophages (175). Second, we

observed that these IgM-ALA clones exhibited leukocyte subset specificity in that some of the IgM-ALA monoclonal antibodies only bound to receptors expressed by all leukocytes or either T cells (SupT-1, Jurkat) or macrophages (U937) or B cells (Daudi) (**Figure 4**) (39). Further studies revealed that only human monoclonal IgM, having T cell reactivity, immunoprecipitated CD4 from cell lysates and bound to recombinant soluble CD4, thus indicating that IgM-ALA can exhibit both receptor and cell specificity.

### Polyclonal IgM from Different Human Sera Differ in Their Repertoire for Receptor Binding. IgM, in Addition, Regulates Human T Effector Cells and DC Without Affecting Tregs or Chemokine Production

We used polyclonal IgM, purified by size exclusion chromatography, from sera of normals, HIV patients and renal dialysis (ESRD) patients (39). Ammonium chloride precipitation was not used as this method affected IgM-ALA binding [see method details in Ref. (39)]. We showed that IgM could immunoprecipitate CD3,

control, agarose beads without IgM were added to lysate to determine whether the leukocyte receptor non-specifically bound to the bead (B plus Ly). Note that several fold more receptors were immunoprecipitated by ESRD and HIV IgM when compared with normal IgM. (C) Significantly increased binding of IgM from B cell clone 4G4 to CD4+ T cells when compared with CD4− T cells (MCF 71.6 vs. 16.6). Clone 4G4 secreted IgM with anti-CD4 reactivity. Note that no increased binding was observed on CD4+ T cells using a B cell clone (IE12) secreting IgM without anti-CD4 reactivity (MCF 22.5 vs. 22.3). Figure and legend reproduced with permission from Ref. (39). Copyright 2008. The American Association of Immunologists, Inc.

CD4, CCR5, and CXCR4 from lysates of cell lines (**Figure 5**). However, the repertoire of IgM-ALA was found to be different among individuals, especially patients as exemplified with HIV patients (see **Figure 5**). Such differences in the repertoires of IgM-ALA may explain why clinical manifestations of inflammation are different among different individuals. It is also possible that prior exposure to different infective agents or foreign antigens may explain the observed differences in the repertoire of IgM-ALA among different individuals (31).

In *in vitro* studies with human peripheral blood mononuclear cells (PBMC), addition of polyclonal human IgM, but not human IgG or Waldenstrom's IgM lacking IgM-ALA, differentially inhibited co-stimulatory receptor upregulation, cytokine production, and proliferation of T cells (39). Both normal and patient IgM downregulated expression of CD4, CD2, and CD86 but not CD8 and CD28 on blood PBMC activated with

TNF-α and IL-13 but not that of IL-6, IL-8, MIG, and MCP-1 produced in response to alloantigen activation of T cells. Supernatants were obtained from day 5 MLR cultures stimulated in the presence or absence of pooled IgM (15 μg/ml), added on day 0. (F) Pooled human IgM inhibits anti-CD3-mediated Zap-70 phosphorylation of human T cells. IgM was added 30 min before anti-CD3/28 and cells were cultured overnight before quantitation with flowcytometry. Figures and legend reproduced with permission from Ref. (138) (A–D) and (39) (E,F). Copyright 2008 and 2012. The American Association of Immunologists, Inc.

alloantigens (MLR) (39). Additionally, both normal and patient IgM inhibited production of the same set of cytokines, i.e., TNFα, IL-13, and IL-2 but not IL-6 and chemokines when human PBMC were activated by alloantigens [**Figure 6E**; Ref. (39)]. Other investigators working with a monoclonal IgM-ALA with reactivity to TcR have shown that IgM-ALA can inhibit IL-2 production and T cell proliferation by binding to the TcR (5, 176). Similarly, we showed that IgM inhibited T cell proliferation induced by alloantigens or CD3 ligation and also inhibited Zap-70 phosphorylation induced by anti-CD3 [**Figure 6F**; Ref. (39)]. However, IgM did not alter chemokine production by activated PBMC but inhibited chemokine-induced chemotaxis by binding to the receptor and blocking chemokine binding (39). Importantly, IgM did not alter Treg levels in an MLR, despite inhibiting T cell proliferation. In general, identical quantities of patient IgM had a more inhibitory effect in the above studies when compared to normal IgM (39). These functional differences between normal and patient IgM may be explained by differences in their quantity and IgM-NAA repertoire (**Figure 5**). Other investigators have also shown that polyclonal human IgM can inhibit proliferation of human T cells (5, 177).

In summary, the data indicate that polyclonal IgM, in physiological doses, inhibit human T effector cell activation and proliferation as well as regulates production of certain cytokines by binding to certain co-stimulatory molecules (CD4, CD3, TcR). IgM does not inhibit T regs. We also show that the quantity and repertoire of IgM-ALA varies in different individuals especially in disease. Interestingly, IgM-ALA does not appear to affect the production of chemokines by leukocytes, but interferes with their action by binding to chemokine receptors.

### Polyclonal Murine IgM Binds to Specific Co-Stimulatory Receptors and Regulates the Function of Murine T Effector Cells, DC, and NKT Cells but Not Tregs

In murine studies, we initially observed that IgM-ALA bound to pronased splenic leukocytes (**Figures 7B,C**) and that IgM-ALA had several fold increased binding to live splenic granulocytes, DC, and B cells when compared to T cells and IgM binding to all leukocytes was enhanced when cells were activated (see **Figure 7A**). Furthermore, we showed that IgM-ALA bound to splenic leukocytes independently of FcμR and, second, showed that IgM, after binding to leukocytes, could immunoprecipitate several different leukocyte receptors (**Figure 7D**) (138). These observations, together with the above findings with human leukocytes, led us to investigate if IgM had an inhibitory effect on the function of T cells, DC, and NKT cells by binding to receptors, e.g., antigen-presenting receptors and co-stimulatory receptors, that get upregulated during activation. Both T cells and DC have an important role in adaptive immunity, e.g., in transplantation

FIGURE 7 | Polyclonal murine WT IgM bind to membrane receptors on leukocytes. (A) Polyclonal IgM has several fold increased binding to activated murine splenic B cells and dendritic cells (DC). Splenic leukocytes, activated for 48 h were interacted with purified mouse IgM at 4°C and evaluated for IgM binding using IgG anti-IgM (clone 11/41). Isotype monoclonal IgM with reactivity to KLH did not bind to activated leukocytes (data not shown). IgM binding to B cells was evaluated by blocking intrinsically expressed IgM with unlabeled IgG anti-IgM (clone 11/41). (B) It depicts immunofluorescence microscopy images of IgM binding to cell membranes of splenic T lymphocytes. (C) It compares binding of IgM and isotype IgM on CD3+WT-B6 pronase pretreated splenic leukocytes. Spleen cells were pronase digested to remove FcμR and to show that IgM-ALA can bind to other receptors on cell membranes. (D) It depicts a representative example of a Western blot from two separate experiments demonstrating immunoprecipitation by WT-polyclonal IgM of biotinylated membrane proteins from the murine macrophage cell line J77. In this experiment, WT-polyclonal IgM is compared with an equal amount of isotype IgM that has no binding activity to leukocytes using flow cytometry. Figure and legend reproduced with permission from Ref. (4) (A) and Ref. (138) (B–D). Copyright 2012 and 2015. The American Association of Immunologists, Inc.

and NKT cells, together with DC, have an important role in innate immunity.

With murine leukocytes, we showed in *in vitro* studies, that physiological doses of murine polyclonal IgM inhibited naïve T cells from differentiating into TH-1 and Th-17 cells even when IgM was added 48 h after activation (**Figures 6B–D**) (138). This inhibitory effect of IgM-ALA on T cells did not depend on presence of DC as the same inhibitory effect was noted when T cells were activated with insoluble anti-CD3/28. IgM inhibition of anti-CD3-mediated Zap-70 phosphorylation would indicate that the inhibitory effect of IgM on T cell function is mediated by IgM binding to CD3 [**Figure 6F**; Ref. (39)]. IgM did not, however, inhibit differentiation of murine T cells into Foxp3+ cells and, furthermore, under Th-17 differentiating cytokine conditions, IgM inhibited sorted Foxp3+ cells from differentiating into TH-17 cells (**Figure 6D**) (138).

Since there are <1.5% of DC in murine splenic leukocytes, we used 7–8 day cultured murine bone-marrow DC (BMDC) to investigate the functional effects of IgM on DC (4). We showed that polyclonal murine IgM, but not IgM pre-adsorbed with activated splenic leukocytes, bound to recombinant soluble CD40 and PD1 but not PDL-1, CD40L, and CD80, indicating, therefore, that IgM-ALA has binding specificity to certain DC receptors, just as we observed with human T cell receptors where IgM bound to CD4, CD3, and CD2 but not to CD8 (39). The functional effect of IgM binding to certain specific co-stimulatory receptors was tested on LPS-activated BMDC. We show that IgM, even when added 2 h after LPS, inhibited LPS-induced CD40 upregulation, but not upregulation of CD86, PDL-1, and MHC-II of BMDC and downregulated basal expression of PD1 on BMDC. IgM, in addition, downregulated p65NF-κB activation induced by LPS (**Figure 8D**) but not by LPS + anti-CD40 (agonistic Ab), thus indicating that IgM can inhibit p65NF-κB upregulation mediated by TLR4 activation, but not when both TLR4 and CD40 are activated (4). Interestingly, IgM inhibited TLR4 activation by a mechanism that did not involve inhibition of LPS binding to cell receptors (4). However, despite downregulation by IgM of TLR4 induced p65NF-κB, there was no decrease in IL-12 production or increase in IL-10 production indicating that LPS activates other transcription factors, besides p65NF-κB, to upregulate these cytokines and certain other co-stimulatory receptors that were not downregulated with IgM (4). In *in vivo* studies (to be presented in the next section), we show that IgM pretreatment of LPS-activated BMDC switches these activated BMDC to a regulatory phenotype possibly by a mechanism involving downregulation of CD40 and NF-κB.

We next did *in vitro* studies to test the effect of polyclonal IgM on Type1 NKT cells, which together with DC, have an important role in inducing innate inflammation, e.g., after renal reperfusion injury (IRI). In these studies, we used α-gal-ceramide, a glycolipid that is taken up by DC and presented via the CD1d receptor to Type 1 NKT cells, which get activated and secrete IFN-γ. This assay is specific for determining Type 1 NKT function as only Type 1 NKT, but not T effector cells, will secrete IFN-γ after exposure to α-gal-ceramide, which is recognized, in context of CD1d presentation, by the invariant TcR on Type 1 NKT cells. We show that physiological doses of IgM inhibits α-gal-ceramide

induced IFN-γ production of splenic leukocytes even when IgM is introduced 1 h after α-gal-ceramide (**Figure 6A**) (138). We have not defined the mechanism for the inhibitory effect of IgM on Type 1 NKT function.

In summary (see **Table 2**), the *in vitro* data with both human and murine cells would indicate that IgM-ALA regulates leukocyte function by binding and downregulating certain leukocyte receptors (e.g., CD4 and CD2 on T cells, CD40, and CD86 on DC) and inducing regulatory function in DC. Importantly, IgM does not decrease Foxp3+ Tregs. IgM regulates leukocyte activation, proliferation, and chemotaxis to attenuate excess inflammation [**Figure 6**; Ref. (39)]. The marked individual variation in the repertoire of IgM-ALA, with specificity to the different leukocyte receptors, observed in both normal and disease states could potentially explain the differences in the vigor and character of inflammatory responses in different individuals exposed to the same inciting agent. Additionally, differences in inflammatory response may also be influenced by total levels of IgM-NAA or IgM-ALA as we observed in transplant recipients (**Figure 3**). Finally, IgM-ALA, by binding to leukocyte receptors and inhibiting cell activation, can provide another mechanism to limit viral entry into cells and replication as we have shown with the HIV-1 virus (**Figure 2**) (144).

### IgM-ALA Inhibits the Innate Immune Inflammatory Response in Renal Ischemia Reperfusion Injury

To test the *in vitro* inhibitory effects of IgM-ALA on DC and NKT cells, we used an *in vivo* murine model of renal IRI (169). In this model, renal vessels to both kidneys are completely occluded with clamps for 26 or 32 min to induce either mild or severe ischemic renal tubular injury. The kidneys are then allowed to re-perfuse by unclamping the blood vessels and the extent of renal injury or decrease in renal function is quantitated at 24 h of reperfusion by measuring for accumulation in the plasma of waste products (e.g., creatinine) that are normally only removed by the kidneys. In this model, the initial ischemic injury is not sufficient to impair renal function as quantitated by measuring plasma creatinine, but it is the innate inflammatory response to products released by ischemic renal cells (e.g., DAMPS and glycolipids) that significantly worsen kidney injury that leads to loss of function. In this model, DAMPS and glycolipids released by ischemic renal cells are taken up by DC and in the splenic marginal zone, DC present glycolipids in the context of CD1d to activate NKT cells, which rapidly release IFN-γ to activate innate effector cells especially granulocytes, macrophages, and NK cells (169). Activated innate effectors migrate to the kidney, where chemokines, released by ischemic cells, enhance extravasation of inflammatory cells into the kidney interstitium. The inflammatory effector cells in the kidney interstitium cause further renal tubular injury with loss of kidney function that leads to an increase in plasma creatinine. This acute loss in kidney function is referred to as acute kidney injury (AKI).

We used two approaches to test the protective role of IgM in the suppression of this ischemia-induced innate inflammatory response. First, we performed renal IRI in B6/S4-IgMko mice (referred to as IgM ko) that lack circulating IgM but have normal levels of other immunoglobulins. These mice have normal or increased levels of Tregs, B regs, and IL-10 and their normal functioning B cells express membrane IgM or BcR but are unable to secrete IgM. Unlike their WT counterpart, we demonstrated that these mice are very sensitive to renal ischemia, developing AKI with mild ischemia (26 min clamp time) that is insufficient to cause AKI in their WT counterparts (**Figures 9A,B**). Replenishing IgM in the IgM ko mice with a single 240 μg dose of polyclonal IgM, to achieve plasma levels similar to that in their WT counterparts, protected these IgM ko mice from developing AKI with mild ischemia, thus indicating that sensitivity to ischemia in the IgM ko mice resulted from a lack of circulating IgM (**Figures 9A,B**).

In the second approach, a single dose (150 μg) of normal purified polyclonal IgM was administered intravenously to wild-type C57BL6 (WT-B6) mice to increase baseline circulating IgM by about 30–50% and to determine if increasing IgM would protect these WT-B6 from severe renal ischemia (32 min clamp time). These studies clearly indicated that increasing circulating IgM levels protected mice from severe renal IRI (**Figure 9C**). We next determined that this protection was mediated by IgM-ALA as administering similar quantity of polyclonal IgM pre-adsorbed with activated splenic leukocytes to remove IgM-ALA failed to protect these WT-B6 mice from severe renal IRI (**Figure 9C**).

In both approaches, physiological doses of intravenous polyclonal IgM mediated protection by decreasing the innate ischemia-induced inflammatory response. Protected kidneys had a very minimal inflammatory response with no or minimal tubular injury that could be detected on histology. Based on our *in vitro* data, *in vivo* IgM-ALA could mediate protection through several mechanisms, including regulation of NKT and DC and maintaining or enhancing Tregs, which also mediates protection in this model of innate inflammation (178). Our prior observations showing that IgM-ALA had several fold increased binding to splenic DC, when compared to T cells, prompted us to investigate the role of IgM-ALA in regulating DC in this model. These studies are presented in the next section.

### Protection from Renal Ischemia is Mediated by IgM Induced Regulatory DC. Regulatory DC Require Tregs, B cells, Circulating IgM, and IL-10 to Mediate *In Vivo* Protection

To examine the *in vivo* role of IgM on DC, we used 7 to 8-dayold cultured BMDC. In these studies, BMDC were activated *ex vivo* for 48 h with LPS with or without polyclonal IgM. Activated BMDC were washed and then 0.5 × 106 BMDC were infused intravenously into mice 24 h before performing renal ischemia. In these studies, IgM + LPS-pretreated BMDC protected mice from ischemia induced AKI (**Figures 8A**) by inhibiting the increased generation of circulating granulocytes and inhibiting innate activated leukocytes from infiltrating the ischemic kidney [**Figure 10**, Ref. (4)]. Importantly, protection with LPS-activated BMDC was only observed when IgM was present during the 48-h culture and not when IgM was added at the end of the 48 h LPS activation (**Figure 8C**), indicating therefore that regulation of BMDC by IgM is an active process requiring both NF-κB and CD40 downregulation induced by IgM (**Figure 8D**). Preventing downregulation of NF-κB and CD40 by adding the agonistic anti-CD40 antibody to LPS + IgM during activation, negated the protective effect, thus supporting that NF-κB and CD40 downregulation are required to switch activated BMDC to a regulatory phenotype (4). It is possible that IgM by binding to CD40 induces this regulatory phenotype. Ex-vivo pretreatment of murine BMDC with human IgM was also protective in renal IRI (**Figure 8B**), indicating that the function of IgM-NAA is evolutionarily conserved among species.

However, since LPS activation of BMDC generates apoptotic cells, it is possible that protection from ischemia could be mediated by complexes of IgM anti-PC and apoptotic cells, which when injected, regulate endogenous DC (127). Such a possibility seemed unlikely, as in the *in vivo* murine studies to demonstrate the anti-inflammatory role of apoptotic cells, large quantities of apoptotic cells (2.5 × 107 thymocytes) were used (127), while in our studies, we only used 0.5 × 106 BMDC (4). However, to exclude the possibility of apoptotic cell-mediated protection, we used apoptotic BMDC by subjecting activated LPS + IgM pretreated BMDC (0.5 × 106 cells) to UV irradiation. Such apoptotic LPS + IgM pretreated BMDC failed to protect mice from ischemia induced AKI, thus excluding the role of apoptotic cell/IgM complexes in inducing protection (**Figure 8C**). The latter experiments indicated that IgM-ALA mediated protection by switching LPS-activated BMDC to a regulatory phenotype. BMDC required IL-10 but not IDO (indoleamine 2, 3-dioxygenase) to switch to a regulatory phenotype as IgM + LPS pretreatment of IL-10 ko BMDC, but not IDO ko BMDC, failed to protect mice from developing AKI after renal ischemia (4).

We hypothesize that intravenously injected regulatory BMDC inhibit the innate inflammatory response by entering the splenic marginal zone where they inhibit NKT function. However, in further studies, we show that injected regulatory

FIGURE 9 | B6/S4-IgMko mice are more sensitive to renal IRI when compared to their WT counterparts (WT-B6/S4). (A, B) Kidneys from B6/S4-IgMko mice and their WT counterparts (WT-B6/S4) were subjected to mild ischemia (26 min) and then reperfused. Data depict 24 h plasma creatinine comparing (WT-B6/ S4) WT mice with B6/S4-IgMko mice, and B6/S4-IgMko pretreated with 240 μg IgM, 24 h before ischemic injury. Histology depicts H&E staining of renal outer medulla after 24 h of reperfusion. (C) Polyclonal IgM, but not leukcocyte adsorbed IgM (Leu-Ads IgM), protects against renal IRI in WT-B6 mice. In these studies, WT-B6 mice were pretreated with equal quantities (150 μg in 0.75 ml) of IgM or Leu-Ads IgM or IgG, 24 h before subjecting the kidneys to severe ischemia (32 min). Kidneys were reperfused for 24 h prior to determining plasma creatinine (C). Control mice were pretreated with 0.75 ml RPMI containing 150 μg bovine albumin to exclude variables, such as volume/colloid, that can protect against ischemic injury. Figure and legend reproduced with permission from Ref. (138). Copyright 2012. The American Association of Immunologists, Inc.

BMDC require the presence of other *in vivo* suppressive mechanisms, such as circulating IgM, IL-10, Tregs, and B cells, to mediate protection (4).

In summary, IgM-ALA inhibits the ischemia-induced innate inflammatory response by several mechanisms, including switching activated DC to a regulatory phenotype, inhibiting

NKT cell IFN-γ production, and inhibiting chemotaxis of leukocytes by binding to chemokine receptors. However, IgM-ALA is in-effective on its own in inhibiting the ischemia-induced innate inflammatory response and requires the presence of other *in vivo* suppressive mechanisms, such as IL-10, Tregs and B cells. Conversely, these other *in vivo* suppressive mechanisms, such as Tregs and Bregs, cannot effectively protect against ischemia induced innate inflammation without IgM-NAA as evidenced in our renal ischemia experiments using IgM ko mice that lack secretory IgM (**Figures 9A,B**).

### Polyclonal IgM Inhibits Inflammation Mediated by Adaptive Immune Mechanisms in Allograft Transplantation

Because of our clinical observations (**Figure 3**) and the *in vitro* studies demonstrating that IgM (a) inhibited alloantigen-activated T cell proliferation and differentiation into Th-1 and Th-17 independently of DC (**Figure 6**) and (b) could induce regulatory function in DC, we performed experiments aimed at determining whether IgM could also inhibit allograft rejection, which is an *in vivo* model of inflammation mediated by alloantigen-activated DC and T cells (138). Two approaches were used to test the role of polyclonal IgM. First, cardiac transplants were performed intraabdominally in B6/S4-IgM ko mice (referred to as IgM ko) or their WT littermates using B6-bm12 donor hearts, which are minimally incompatible at the MHC class II locus (Ia) with the recipient. In this transplant model, there is a mild chronic form of cellular rejection and a vasculopathy that is initiated by a T cell-mediated inflammatory process and cardiac graft loss (defined as loss of intra-abdominal cardiac pulsation) occurs at >2 months in WT recipients. However, in IgM ko recipients, there is a more severe acute cellular rejection and graft loss occurs in 2–3 weeks, which is significantly earlier compared with their WT-B6/S4 counterparts, where cardiac graft loss occurs after >2 months (**Figure 11**). Histologically, there are considerably more TH-17 cells infiltrating the cardiac allograft in the IgM ko recipient despite no significant difference in infiltrating Tregs between the groups (138). The T cell findings on histology mirror the *in vitro* studies where IgM inhibited naïve T cells and Foxp3+ T cells from differentiating into TH-17 cells without affecting levels of Tregs (**Figure 6D**).

In the second approach, we wanted to determine whether increasing circulating levels of IgM in WT-B6 mice inhibited the severe and rapid rejection that occurs in the setting of fully MHC-incompatible donor hearts (i.e., from BALB/c donors). In this model, rejection in WT-B6 recipients is detectable by day 5 and graft loss occurs by 7–9 days (138). In these studies, 175 μg IgM was intravenously administered 24 h after ascertaining that cardiac surgery was successful, and the dose of IgM was repeated on days 3 and 5. Mice were euthanized on day 6. The data clearly show that IgM inhibited the severe inflammation in the cardiac allograft induced by rejection on day 6, as detected by H&E staining and immunofluorescence staining for neutrophils (7/4) and T cells (CD3). Importantly, with immunohistochemistry, this lack

legend reproduced with permission from Ref. (138). Copyright 2012. The

American Association of Immunologists, Inc.

of leukocyte infiltration in the cardiac parenchyma of IgM-treated recipients was also associated with no or minimal CXCL1+ leukocytes and with no or minimal fragmentation of capillaries, as identified by the endothelial cell marker CD31 (138).

In summary, we show that physiological doses (175 μg) of polyclonal IgM can subdue inflammatory responses mediated by an adaptive immune mechanism. Potential mechanisms include the following (a) a direct inhibitory effect of IgM-ALA on T effector cells, but not Tregs, possibly by binding and down-modulating CD3/TcR and certain specific co-stimulatory receptors, such as CD4 and CD2 but not CD8. As a result, T effector cells are inhibited from proliferating or producing certain specific cytokines (e.g., TNF, IFN-γ, IL-17 but not IL-6, and chemokines) or from differentiating into TH-1 and TH-17 pro-inflammatory cells. Importantly IgM-ALA does not affect levels of Foxp3+ Tregs, but prevents Foxp3 + cells from differentiating into TH-17 cells under pro-inflammatory conditions, (b) by binding to CD40 and switching activated DC to a regulatory phenotype with downregulation of CD40 and p65NF-κB, and (c) by inhibiting chemotaxis. It is highly unlikely that IgM anti-PC could have a significant role in inhibiting allograft rejection in our studies as we used small doses of polyclonal IgM (175 μg) while 1.5–2.0 mg of a monoclonal IgM anti-PC (T15 idiotype) was used to inhibit an arthritis model of inflammation mediated by adaptive immune mechanisms (127).

### Polyclonal IgM Antibodies Inhibit Autoimmune-Mediated Insulitis in NOD Mice

Insulitis in the NOD mouse is primarily mediated by autoimmune T cells but there are data to indicate that B cells are also involved. Depleting B cells or only B1 cells ameliorates insulitis indicating that some of the IgG autoantibodies detected in NOD mice may also be pathogenic (179–181). The role of B1 cells in generating pathogenic IgG autoantibodies has also been described in other autoimmune murine models (20, 94). Because our *in vitro* studies demonstrated that polyclonal IgM inhibited T cell proliferation and differentiation into Th-1 and Th-17 cells (**Figures 6B–D**) and can also counter pathogenic IgG autoantibodies (as previously discussed), we performed studies to determine whether IgM could inhibit autoimmune insulitis that results in islet cell destruction and diabetes mellitus (DM) in NOD mice (182). In these mice, the autoimmune inflammatory process begins spontaneously around 4–5 weeks after birth and the initial phase is characterized by a silent and non-destructive leukocyte infiltration of the perivascular and periductal regions in the pancreas as well as the peripheral islet regions by a heterogeneous mixture of CD4 and CD8 T cells, B cells, macrophages, and DC (peri-insulitis). In the invasive phase that begins at 8–12 weeks of age, the immune infiltrate enters the islet inducing beta cell destruction (insulitis). Significant destruction first becomes evident around 12–13 weeks of age with mice exhibiting overt diabetes (DM).

We wanted to determine the effect of increasing IgM levels on development of DM (182). NOD mice were administered biweekly intra-peritoneal polyclonal IgM (50 μg/dose) beginning either at 5 or 11 weeks of age and ending when mice were 18 weeks old. At 25 weeks of age, 80% of control mice (*n* = 30) became diabetic, while 0% of mice (*n* = 30) treated with IgM beginning at 5 weeks developed DM. Importantly, only 20% of pre-diabetic mice (*n*= 20) treated with IgM beginning at 11 weeks of age developed DM at 25 weeks of age. This latter observation is particularly notable as prior studies using co-stimulatory blockade failed to prevent DM in this murine model. At 18–25 weeks of age the pancreas revealed no or minimal insulitis in NOD mice treated with IgM beginning at 5 weeks of age. Other investigators using monoclonal polyreactive natural IgM in the neonatal period have also obtained similar results (183, 184).

In summary, these studies indicate that polyclonal IgM inhibits insulitis via several potential mechanisms, including inhibition of autoimmune T effectors and possibly countering IgG autoantibodies via anti-idiotypic mechanisms and by inhibiting the B cells that produce them. Additionally, IgM by switching activated DC to a regulatory phenotype and maintaining Tregs could enhance this protective effect.

### CONCLUSION

**Figures 12** and **13** summarize our concepts of the inter-relationship between pathogens and natural antibodies. In both murine and human models, the evidence shows that these polyreactive and low-affinity binding IgM-NAA function under physiological conditions to (i) provide a first line of defense against invading organisms, (ii) protect the host from autoimmune inflammation mediated by autoimmune B and T cells that have escaped tolerance mechanisms, (iii) protect the host from endogenous oxidized neodeterminants and other neo-antigens that are unmasked during tissue damage, and (iv) regulate excess inflammation, mediated by both innate and adaptive immune mechanisms. Even though the full repertoire of IgM-NAA develop during the first few years of life, both their levels and repertoire differ in healthy individuals as well as in disease and could contribute to the varying inflammatory response, for example, after an infection or alloantigen exposure (see **Figures 2** and **5**). We hypothesize that high

protective levels of IgM-NAA are maintained by infections that have been shown to increase IgM-NAA, especially IgM-ALA and anti-PC [reviewed in Ref. (31, 39, 148)]. Such a hypothesis could explain the significantly low incidence of autoimmune disorders, such as SLE or sarcoidosis in rural parts of Africa where malaria and other infections are endemic (185–187). We have shown that IgM-ALA increases in active sarcoidosis (188). There are other suppressive mechanisms (e.g., Tregs, B regs, IL-10, TGF-β) that regulate inflammation and based on the different animal models of inflammation, it would appear that IgM-NAA have a more prominent role in regulating inflammation that involves pathogenic IgG autoantibodies, macrophages, and NKT cells. However, we show that IgM-NAA require Tregs, B cells, and IL-10 to be fully effective in controlling inflammation (4). Conversely, our studies and that of others, using mice deficient in IgM secretion, would also indicate that Tregs and Bregs also require IgM-NAA

### REFERENCES

to effectively control inflammation (39, 119, 120). Finally, it may be easier to develop a vaccine to increase IgM-NAA especially if we understand how diverse infectious agents increase IgM-ALA. Studies are also needed to determine if prolonged high IgM-NAA levels can induce excess immunosuppression. One could also use enriched IgM intravenous preparations to acutely treat patients with uncontrolled inflammation. Cell therapy, especially with IgM pretreated DC, could provide an alternative approach requiring minimal quantities of IgM to prevent ischemic acute renal failure (e.g., in high-risk patients undergoing cardiac surgery) or delayed graft function after renal transplantation (4).

### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

towards self antigens contrasts with age-dependent diversification of reactivities against bacterial antigens. *Eur J Immunol* (1995) **25**(9):2598–604. doi:10.1002/eji.1830250929

autoantibodies from patients with microscopic polyangiitis. *Rheumatology (Oxford)* (2011) **50**(7):1236–43. doi:10.1093/rheumatology/ker085

188. Lobo PI, Suratt PM. Studies on the autoantibody to lymphocytes in sarcoidosis. *J Clin Lab Immunol* (1979) **1**(4):283–8.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Lobo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Pneumococcal Polysaccharide Vaccination Elicits IgG Anti-A/B Blood Group Antibodies in Healthy Individuals and Patients with Type I Diabetes Mellitus

### *Edited by:*

*Ana María Hernández, Center of Molecular Immunology, Cuba*

### *Reviewed by:*

*Jean Van Den Elsen, University of Bath, UK Thomas Vorup-Jensen, Aarhus University, Denmark*

### *\*Correspondence:*

*Michael B. Fischer<EMAIL_ADDRESS>
*† Wendelin Wolfram and Kai M. T. Sauerwein have equally contributed as co-first authors, Hermann M. Wolf and Michael B. Fischer have equally contributed as co-last authors to this manuscript.*

### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 22 July 2016 Accepted: 25 October 2016 Published: 14 November 2016*

### *Citation:*

*Wolfram W, Sauerwein KMT, Binder CJ, Eibl-Musil N, Wolf HM and Fischer MB (2016) Pneumococcal Polysaccharide Vaccination Elicits IgG Anti-A/B Blood Group Antibodies in Healthy Individuals and Patients with Type I Diabetes Mellitus. Front. Immunol. 7:493. doi: 10.3389/fimmu.2016.00493*

*Wendelin Wolfram1†, Kai M. T. Sauerwein2†, Christoph J. Binder3 , Nicole Eibl-Musil4 , Hermann M. Wolf 2,5† and Michael B. Fischer1,6\*†*

*1Clinic for Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria, 2 Immunology Outpatient Clinic, Vienna, Austria, 3Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria, 4Rudolfstiftung Hospital of the City of Vienna, Vienna, Austria, 5Sigmund Freud Private University – Medical School, Vienna, Austria, 6Department for Health Science and Biomedicine, Danube University Krems, Krems, Austria*

Hypothesis: Blood group antibodies are natural antibodies that develop early in life in response to cross-reactive environmental antigens in the absence of antigen encounter. Even later in life structural similarities in saccharide composition between environmental antigens such as bacterial polysaccharides and blood group A/B antigens could lead to changes in serum levels, IgM/IgG isotype, and affinity maturation of blood group anti-A/B antibodies. We addressed the question whether immunization with pneumococcal polysaccharide (PnP) vaccine Pneumo 23 Vaccine "Pasteur Merieux" (Pn23) could have such an effect in patients with type I diabetes mellitus (DM I), an autoimmune disease where an aberrant immune response to microbial antigens likely plays a role.

Methods: Anti-PnP IgM and IgG responses were determined by ELISA, and the DiaMed-ID Micro Typing System was used to screen anti-A/B antibody titer before and after Pn23 immunization in 28 healthy individuals and 16 patients with DM I. In addition, surface plasmon resonance (SPR) technology using the Biacore® device and a synthetic blood group A/B trisaccharide as the antigen was applied to investigate IgM and IgG anti-A/B antibodies and to measure antibody binding dynamics.

Results: All healthy individuals and DM I patients responded with anti-PnP IgM and IgG antibody production 4–6 weeks after Pn23 immunization, while no increase in blood group anti-A/B antibody titer was observed when measured by the DiaMed-ID Micro Typing System. Interestingly, isotype-specific testing by SPR technology revealed an increase in blood group anti-A/B IgG, but not IgM, following Pn23 immunization in both patients and controls. No change in binding characteristics of blood group anti-A/B antibodies could be detected following Pn23 vaccination, supporting the assumption of an increase in IgG antibody titer with no or very little affinity maturation.

Conclusion: The study provides evidence for epitope sharing between pneumococcal polysaccharides and blood group ABO antigens, which leads to a booster of blood group anti-A/B antibodies of the IgG isotype after Pn23 immunization in healthy individuals. Manifest autoimmunity such as present in DM I patients has no additional effect on the cross-reactive antibody response against pneumococcal polysaccharides and blood group antigens.

Keywords: pneumococcal polysaccharide vaccine, 23-valent pneumococcal polysaccharide vaccine, anti-blood group A/B antibodies, surface plasmon resonance, isoagglutinins, natural antibodies

### INTRODUCTION

Natural antibodies are produced in the absence of overt external antigenic stimulation early in life in all healthy individuals with a functional immune system, presumably as a product of germline gene segment assembly (1). Therefore, they show low affinity to many microbial pathogens and certain crossreactivity, even to some self-antigens (1–4). Antibodies to carbohydrate epitopes share certain characteristics with natural antibodies, e.g., the absence of extensive class switching and affinity maturation due to absent T-cell help (5). Isoagglutinins, i.e., antibodies against carbohydrate epitopes that form the AB0 antigens on red blood cells (RBCs), are considered prototypic natural antibodies that were shown to be compromised in patients with immunodeficiency (1, 4, 6). Their appearance can be explained by inapparent stimulation particularly from intestinal bacteria because antigens cross-reactive with blood group polysaccharides are widely distributed in the environment (1, 7, 8). Natural anti-ABO antibodies have long been considered to be predominantly of the IgM isotype, but antibodies of the IgG isotype are also produced, especially later in life, e.g., after alloimmunization in pregnancy or ABO-incompatible blood transfusions (1). In the case of ABO blood groups, individuals with blood group A develop an immune response to B-like antigens of microorganisms and environmental antigens like plants containing these epitopes (1, 9). B blood group individuals develop anti-A antibodies, O blood group individuals produce both anti-A and anti-B antibodies, while AB blood group subjects have neither anti-A nor anti-B because they express both antigens on their RBCs.

 Blood group O individuals have carbohydrate structures named H-antigen terminated in the sequence alpha-Fuc(1,2)Gal. The blood group A antigen is then formed from the H-antigen by an *N*-acetylgalactosaminyltransferase that uses a UDP-*N*-acetylgalactosamine (GalNAc) donor, and the blood group B antigen is formed by a galactosyltransferase that uses a UDP-galactose (Gal) donor to convert the H-antigen (4). The human A and B blood group antigens differ from each other only in the substitution of an acetamino for a hydroxyl group on the terminal saccharide residue, and this substitution can be differentially recognized by specific antibodies (1, 4, 11, 12). Interindividual variations in the titer of anti-blood group ABO antibodies exist, representing modulation of the production of these natural antibodies in response to cross-reactive environmental antigens, e.g., through immunization (1). B cells could thus recognize shared carbohydrate epitopes due to structural similarities in saccharide composition, e.g., between microbial polysaccharides (PnPs) and blood group A/B antigens, which could also have an effect on blood group anti-A/B specific antibody production after PnP-immunization. Previous findings indicate shared epitopes between blood group oligosaccharides and pneumococcal capsular polysaccharides, as antibodies crossreacting with human erythrocytes and pneumococcal antigens have been described (7). Furthermore, it was shown that crossreactive carbohydrate determinants (CCDs) contained in blood group antigens and microbial polysaccharides can be targeted by immunoglobulins of different isotypes, in particular by IgE, but also by IgG antibodies (13).

In addition to their role as a first line of defense against infection, natural antibodies have been shown to play an important role in the initiation of autoimmunity (2, 8, 14–16). Natural antibodies reacting with self-antigens were initially considered to represent breakdown of tolerance, thus functioning as a template for the generation of pathogenic autoantibodies produced through a process of clonal selection, somatic hypermutation, and class switching driven by antigen (14). More recently, these antibodies have been attributed an additional role in the prevention of autoimmune disease, e.g., by inhibiting activation of the adaptive immune system by molecules released from apoptotic cells that could facilitate autoimmune events (15, 16). Formation of natural antibodies with high cross-reactivity could be regulated differently in healthy individuals as compared to individuals with an autoimmune disease such as type 1 diabetes mellitus (DM I) known to present with abnormalities in antibody formation (17). In addition to high-risk HLA genes, environmental factors are thought to play an important role in the pathogenesis of DM I, such as pancreatic virus infection, T cell-mediated and autoantibody-mediated molecular mimicry, and bystander activation of autoreactive lymphocytes by proinflammatory cytokines derived from dendritic cells activated by infectionrelated stimuli through innate pattern recognition receptors such as TLR (18–21). A single causative infectious agent for DM I autoimmunity has not been found, pointing toward the possibility that different infectious agents are capable of non-specifically enhancing the likelihood of autoimmunity. In addition to virus infection (19, 20), bacterial infection might play a role in the development of DM I, as antibodies against mycobacterial and proinsulin epitopes (22) as well as beta cell antigens (23) show cross-reactivity in children with new onset DM I. While human IDDM autoantigens are typically T-dependent protein antigens, microbial polysaccharides have been shown to modulate the autoimmune response leading to DM1 (24). Innate signaling *via* TLR and MyD88, pathways critical for T cell-independent (TI) immune defense (25), can reverse anergy in autoreactive B cells, suggesting that environmental factors associated with bacterial infection and inflammation may alter tolerance (26). Concerns that vaccination might lead to the development of autoimmune disease such as DM I have been raised (27), which could also relate to polysaccharide vaccines, as molecular mimicry in bacterial polysaccharide components is capable of inducing autoreactive antibodies, e.g., against some blood group antigens or neuronal gangliosides (28). In the present study, we investigated whether vaccination with Pneumo 23 Vaccine "Pasteur Merieux" (Pn23) has an effect on blood group ABO antibodies in healthy individuals and in patients with DM I. To screen for blood group anti-A/B antibodies, we used the commercially available DiaMed-ID Micro Typing System based on erythrocyte agglutination (6). To measure blood group-specific anti-A/B antibodies in healthy individuals and in patients with DM I in an isotype-specific manner, we used surface plasmon resonance (SPR) technology and synthetic A/B trisaccharides bound *via* amine-coupling to the CM5 chip (6, 11, 12). SPR using blood group-specific A/B synthetic trisaccharides is the ideal technology to investigate a potential IgM to IgG isotype shift and to investigate changes in binding characteristics of the relevant blood group anti-A/B antibodies under near to physiological conditions in real time.

### MATERIALS AND METHODS

### Vaccination of Healthy Individuals and Patients with Type I DM and Measurement of Anti-PnPs Antibodies

During a clinical study published previously (17), healthy individuals (*n* = 39) and patients with type I DM (*n* = 20) were immunized with the PnPs vaccine Pn23 (a 23-valent vaccine containing pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, Pasteur Merieux Connaught, Lyon, France) after informed consent was obtained (17, 29). Type I DM patients were, on average, 16 ± 7.4 years (SD) at the onset of disease, and at that time they tested positive for GAD autoantibodies. Age at initiation of the vaccination study was 31.7 ± 8.8 years (SD). For a detailed description of patients and controls, see Ref. (17). Blood samples were drawn 4–6 weeks after vaccination, the serum was immediately separated from the cellular blood component, dispensed into 500 μl aliquots, and stored until analysis in a freezer at −20°C. For this follow-up study, serum aliquots stored at −20°C were still available from 28 healthy controls and 16 DM I patients; selection of this subset of the previous study was only because no more serum was available from the other controls and DM I patients of the original study. Anti-PnPs antibodies were determined by a home-made ELISA detecting all serotypes as previously described (17, 30).

### Determination of Anti-A/B Antibody Titers by the DiaMed-ID Micro Typing System

Titers of blood group anti-A and anti-B antibodies were determined by the DiaMed-ID Micro Typing System (Bio-Rad Lab. Inc., Hercules, CA, USA) used according to the manufacturer's protocol. The ID-Card 50520 (NaCl) was used to measure IgM, while the ID-Card 50531 [LISS/Coombs containing polyspecific anti-human gamma globulin (AHG) serum] was used to determine IgG in addition to IgM. The serum samples were serially twofold diluted with 0.9% saline solution starting with 500 μl serum and 500 μl saline solutions. Also, 25 μl of each dilution were pipetted into a single column of the microtube gel card, and 50 μl of DiaMed-ID-DiaCell ABO/I-II test cell suspensions A1 or B (Bio-Rad Lab.) was added. ID-Card 50520 (NaCl) cards were incubated at room temperature for 15 min, the ID-Cards (LISS/Coombs) at 37°C, and thereafter centrifuged in a DiaMed-ID-Centrifuge 24 S for 10 min at 910 rpm. Results were read immediately after centrifugation and were subdivided into positive and negative results. The titer that led to visible agglutination of erythrocytes dispersed in the gel was selected positive. In an individual with blood group O, both anti-A and anti-B antibody titers were used for statistical analysis, while in an individual with blood group A, only anti-B antibody titers, and in an individual with blood group B, only anti-A antibody titers were used for statistical analysis; none of the study individuals had blood group AB.

### Determination of Anti-A/B Antibodies by Surface Plasmon Resonance

The erythrocyte aggregation assays based on the DiaMed-ID Micro Typing System are semiquantitative assays that cannot determine in an isotype-specific way a subtle increase of anti-A/B IgG and/or IgM antibodies against blood group A/B antigens. Therefore, we used SPR to measure anti-A/B antibodies as previously described (6, 11, 12). Amine-labeled blood group A and B trisaccharides (Dextra Laboratories Ltd., Reading, UK) were immobilized on two-channel sensor chips CM5 (General Electric Health Care, Freiburg, Germany) using standard protocols (31, 32). In brief, the CM5 sensor chip surface was activated with 100 μl 0.05 mol/l *N*-hydroxy-succinimide and 0.2 mol/l *N*-ethyl-*N*′-dimethylamino-propylcarbodiimide injected into the buffer stream of HBS-EP (0.01 mol/l HEPES buffer, pH 7.4, containing 0.15 mol/l NaCl, 3 mmol/l EDTA, and 0.005% surfactant P20) as described previously (6). The buffer stream was passed through FC-1 and FC-2 of the two-channel Biacore® X instrument (GE Health Care) at a flow rate of 5 μl/min for 20 min. Subsequently, 15 μl of a blood group A/B trisaccharide amine derivative solution at a concentration of 1 mg/ml, prepared with borate (pH 8.5), was injected into the buffer stream and passed through FC-1 at a flow rate of 5 μl/min and was halted after injection of 7 μl for 2 h; FC-2 served as a blank control. Residual active ester groups on the sensor surface were then deactivated by injecting 100 μl of 1 mol/l ethanolamine–HCl (pH 8.5) into the buffer stream and passing it through FC-1 and FC-2, and by passing the buffer stream through the cell until a stable baseline was achieved. To measure anti-blood group A/B antibodies, serum was diluted by half with the HBS-EP, and 100 μl of the diluted plasma samples were passed through FC-1 and FC-2 at a flow rate of 20 μl/min for 5-min duration. When the association phase was finished, binding of antibody was recorded and is expressed as resonance units (RU at "stability" in **Figure 1**, corresponding to RUt0 in **Figure 2**, panel 3). After each measurement, the antibodies bound to the blood group trisaccharides were removed with 50 μl of 50 mmol/l NaOH buffer at a flow rate of 60 μl/min for 50 s and the chip regenerated to reach the same baseline as prior to the measurement. The amounts of anti-A/B antibody that associated with the blood group A/B trisaccharide antigen immobilized on the sensor chip surface were obtained by subtracting the FC-2 value from the FC-1 value.

Alternatively, a four channel Biacore® T200 device (kindly provided by Florian Koelle, GE Health Care) was used, which enabled us to measure both anti-A and anti-B antibodies on one chip surface (CM5-chip) at the same time. The amount of trisaccharides and the coupling procedure remained similar to the two-channel system used previously, except the contact time was adapted to 600 s and the flow rate to 10 μl/min. Flow cell one (FC-1) and three (FC-3) were immobilized without trisaccharides and served as blank during binding analysis. FC-2 was immobilized with 1 mg/ml blood group A trisaccharide and FC-4 with 1 mg/ml blood group B trisaccharide. For binding analysis, serum was diluted by half with HBS-EP and injected in FC-2 and FC-4 for 180 s at a flow rate of 10 μl/min. The flow-path was 2–1 and 4–3. To determine IgM and IgG levels of A- and B-bound antibodies, anti-human IgG Abs [polyclonal aHIgG (γ-chain), Sigma-Aldrich, St. Louis, MO, USA] and anti-human IgM [polyclonal aHIgM (μ-chain), Sigma-Aldrich, St. Louis, MO, USA] were injected for 180 s at a flow rate of 10 μl/min directly after the serum sample. For measurement of anti-A/B antibodies RU at report point "stability" (see **Figure 1**, corresponding to RUt0 in **Figure 2**, panel 3) and for levels of IgM/IgG anti-A/B antibodies levels at report point "enhance\_level" (see **Figure 1**) relative to baseline were recorded. After determination of levels of IgM and IgG anti-A/B antibodies, the chip was regenerated twice with 50 mmol NaOH for 30 s at a flow rate of 10 μl/min with a stabilization period of 5 s after the second regeneration to reach the same baseline as prior to the measurement. For kinetic measurement, an association time of 200 s and dissociation time of 800 s was chosen. Dissociation of bound anti-A/B antibodies over time as a semiquantitative correlate for the strength of antibody binding was calculated according to the following formula: % dissociation = 100 − [RU at t1 (800 s)/ RU at t0 (200 s)] × 100 (see **Figure 2**, panel 3 as an example for measurement of RU at t0 and at t1). To compute an affinity constant *K*D the concentration of IgG antibodies in microgram per

FIGURE 1 | Exemplary binding curve of quantification of anti-A IgG antibodies in human serum, as generated by BIAevaluation**®** software (GE Healthcare). Injection of human serum was followed by a second injection of anti-IgG; response units (RU) at "stability" were recorded to assess the concentration of anti-A blood group antibodies, while response units at "enhance\_level" were representative for anti-A IgG concentration. The *x*-axis gives time in seconds, and the *y*-axis represents response units (RU).

### FIGURE 2 | Continued

Panel (1) different concentrations of monoclonal anti-A IgG (internal control) were injected to correlate response units to micrograms per milliliter, (2) anti-A IgG levels in human serum were determined utilizing a second injection of anti-IgG (Sigma), (3) different dilutions of human serum were measured without addition of second step reagent, and binding curves were recorded for a total of 1,200 s, (4) an estimate of *K*D was calculated using the BIAevaluation Software (GE Healthcare) with input of the concentrations of anti-A IgG determined in steps (2) and (3) as described in Section "Materials and Methods."

milliliter must be known, thus RUs for different concentrations of monoclonal anti-A/B IgG (clone 9A, Abcam, Cambridge, MA, USA; clone NaM87-1F6, BD Pharmingen, San Jose, CA, USA) were measured (**Figure 2**, panel 1). This enabled us to correlate an increase in response units derived from anti-A/B IgG measurements in patient serum with microgram per milliliter of anti-A/B IgG antibody by regression analysis in order to calculate the respective concentration of IgG anti-A/B (**Figure 2**, panel 2). Subsequently two dilutions of patient serum – one of them diluted 1:2 – and a blank were measured (**Figure 2**, panel 3), and the resulting binding curves were used to calculate *K*<sup>D</sup> (computed as *K*<sup>D</sup> = *K*off/*K*on) with the BIAevaluation® software from GE Healthcare. To fit the association/dissociation curve, a 1:1 binding model was chosen. The molecular weight of the analyte was assumed to be 150,000 Da (IgG). To assess the quality of the calculations, controls within the software were used. No external calculations were performed nor parameters added. Furthermore, as an internal control to test before each measurement was started, anti-blood group A or B IgM mAbs (anti-A murine monoclonal Abs clone MH04 and A3D3, anti-B murine monoclonal Abs clone NB1.19, NB10.5A5, and NB10.3B4, Ortho-Clinical Diagnostics, Neckargemünd, Germany), and anti-blood group A IgG mAb (clone 9A, Abcam, Cambridge, MA, USA; clone NaM87-1F6, BD Pharmingen, San Jose, CA, USA) were used to guarantee optimal chip performance and full recovery. For statistical analysis, both anti-A and anti-B antibody titers (RU) were used in an individual with blood group O, while in an individual with blood group A, only anti-B antibody titers, and in an individual with blood group B, only anti-A antibody titers were used for statistical analysis; none of the individuals in this study had blood group AB.

### Statistical Analysis

Statistically significant differences between study groups were calculated using the non-parametric two-tailed Mann–Whitney *U*-test. Results are depicted using box plot diagrams, with the median represented by a cross, the interquartile range (IQR) represented by the box, and 5- and 95-percentile values represented by the whiskers.

### RESULTS

### Anti-PnPs Response in Healthy Individuals and in Patients with Type I DM after Pneumovax**®**23 Immunization

The anti-PnPs antibody response prior and 4–6 weeks after immunization with the unconjugated PnPs vaccine Pn23, a 23-valent vaccine, was determined by ELISA in an isotype-specific manner in 28 healthy individuals and 16 patients with DM I. All healthy individuals included in this study showed a significant IgM (**Figure 3A**) and IgG (**Figure 3B**) anti-PnPs antibody response following vaccination. The results confirm previous findings in patients with type I DM (17) showing a normal antibody response to unconjugated pneumococcal polysaccharide, which is likely TI, while primary antibody responses to T-cell-dependent antigens are impaired (17).

### Increase in Blood Group IgG Anti-A/B Antibodies in Healthy Individuals and in Patients with Type I DM after Pneumovax**®**23 Immunization as Determined with the Biacore**®** Device

To address whether immunization with Pn23 has an effect on blood group anti-A/B antibodies in patients with DM-type I as compared to healthy individuals, we first determined antibody titers in the DiaMed-ID Micro Typing System, using the ID-Card 50520 (NaCl) as well as the ID-Card 50531 (LISS/ Coombs containing poly-specific AHG serum) to determine IgG in addition to IgM anti-A/B antibodies. We could observe no difference in blood group anti-A/B antibody titer prior and post immunization with Pn23 in both, healthy individuals and patients with DM-type I when the assay used was based upon erythrocyte aggregation in physiological NaCl (direct agglutination test, **Figure 4A**) or in the presence of anti-IgG Coombs serum (indirect agglutination test, **Figure 4B**). Interestingly, in a considerable percentage of study individuals of both groups (up to approximately 25%) the A/B isoagglutinin titers were lower than 1:4 in both agglutination systems, thus confirming previous findings of a relatively low sensitivity of the agglutination system to detect low isoagglutinin titers (6). To increase sensitivity, SPR technology was applied to further analyze anti-A/B antibodies, thus allowing real-time analysis of molecular interactions between antibodies and the isolated blood group trisaccharide antigen without molecular labeling leaving the structure of both molecules intact (6, 32). With this technology, biologically relevant antibody–antigen interactions can be assessed both quantitatively and qualitatively, as the carbohydrate antigens are presented in a physiological manner (11, 12, 31). Kinetics and affinity were investigated by analyzing the time curve and level of binding and concentration of anti-A/B antibodies in the sample by measuring mass binding, which further allowed the determination of the binding forces. The comparison of the response on the active and control surface allowed for the subtraction of bulk effects associated with buffer changes and for the high serum concentration with its mass binding, thus enabling the calculation of specific binding. Using SPR analysis, we found specific anti-A/B antibody binding to the respective blood group A/B trisaccharides in all individuals tested

between study groups were calculated using the non-parametric two-tailed Mann–Whitney *U*-test.

(**Figure 4C**), as has also been described previously for individuals with intact antibody responsiveness (6, 12). For all probes, a high association rate constant for the interaction and the use of a high ligand density on the sensor chip surface was found that promoted the mass transport limitation. As reference analyte with a known concentration, commercially available blood group anti-A or -B antibodies of IgM and blood group anti-A IgG were used (**Figure 2**, panel 1 shows monoclonal anti-A IgG as an example). For the example shown in **Figure 2**, panel 1, a sensor response of 1,000 RU corresponded to a shift of 0.1° in the SPR angle, which in turn correlates to approximately 16 μg/ml of anti-A IgG injected. Since the individuals selected for determination of *K*D [one healthy control (Ind1) and one DM I patient (Ind2) as well as a control individual not vaccinated] contained

(*Continued*)

resonance (SPR) technology.

### FIGURE 4 | Continued

Serum samples of healthy individuals (*n* = 28) and patients with type I DM (*n* = 16) were serially twofold diluted with 0.9% saline solution and blood group anti-A and/or anti-B IgM (A) were determined by ID-Card 50520 (NaCl), while the ID-Card 50531 (LISS/Coombs) was used to determine IgG and IgM isoagglutinins (B). The titer that led to visible agglutination of erythrocytes dispersed in the gel was selected positive. Alternatively (C), serum samples diluted in HES-EP buffer (1:2) were injected over either the blood group A or blood group B trisaccharide-coupled CM5 sensor chip. Binding kinetics, i.e., association and dissociation were recorded as sensorgrams in resonance units (RU) against time in FC-I and FC-II, and the amount of anti-A/B antibody that associated with the blood group A/B trisaccharide antigen immobilized on the sensor chip surface was obtained by subtracting the FC-II value from the FC-I value and is given as resonance units (RU). Results are depicted using box plot diagrams, with the median represented by a cross, the interquartile range (IQR) represented by the box, and 5- and 95-percentile values represented by the whiskers; no statistically significant differences were found between study groups as calculated using the non-parametric two-tailed Mann–Whitney *U*-test.

sufficient amounts of anti-A IgG for affinity analysis with very low to undetectable anti-A IgM, titration was only performed for IgG anti-A/B antibody. Of note, SPR technology is capable to detect anti-A/B antibodies with values of 50–180 RU in serum samples where the microtube column assay showed no erythrocyte aggregation. Even with this sensitive detection system, no change in isoagglutinin titers was found following vaccination with 23-valent PnPs, neither in healthy individuals nor in DM1 patients (**Figure 4C**).

Using the DiaMed-ID Micro Typing System in the direct and indirect agglutination assay as well as SPR technology under close to physiological conditions, both IgM- and IgG-antibodies were measured simultaneously, without discrimination between IgM and IgG response. To examine a possible booster effect of Pn23 vaccination on IgG anti-A/B antibodies only we applied isotypespecific second step antibodies to discriminate between blood group anti-A/B IgM and IgG antibodies. This system enabled us to detect an induction of blood group anti-A/B IgG (**Figure 5B**) but not IgM (**Figure 5A**) antibodies after immunization of healthy individuals with Pn23, an antibody response resembling a secondary/booster response. Interestingly, patients with type I DM showed a comparable induction of IgG isoagglutinins after immunization with Pn23 (**Figure 5B**), in agreement with their intact antibody responsiveness to pneumococcal polysaccharide, a T-independent antigen, while a significant impairment in primary antibody response to vaccination with T-cell-dependent antigens such as hepatitis A virus and diphtheria toxoid was described previously (17).

### Determination of Binding Characteristics of Anti-A/B Antibodies after Pneumovax**®**23 Immunization

To measure whether the induction of IgG-A/B antibodies observed after Pn23 immunization was accompanied by changes in binding characteristics of the blood group anti-A antibodies, as would be the case for a T-dependent IgG-booster response, we analyzed the binding dynamics of the anti-A/B antibodies as described above (**Figure 2**). We found no substantial change in the percentage of dissociation (**Figure 6A**) or the estimate of the affinity constant (*K*D, **Figure 6B**) of blood group-specific anti-A antibodies following pneumococcal vaccination, as calculated by BIAevaluation® software from GE Healthcare. For estimation of the affinity constant *K*D, healthy individuals and patients with type I DM were selected that had initially high anti-A-antibody titers (>1:256 or >700 RU), associated with high anti-A IgG and low to undetectable anti-A IgM antibody levels. Only three healthy individuals and two patients with type I DM fulfilled this criterion. As shown in **Figure 6** (depicting results from one healthy individual, Ind1 and one DM I patient, Ind2), our study gives evidence that immunization with Pn23 induces an increase in IgG anti-A/B antibody titers but has no effect on binding characteristics of blood group anti-A/B IgG antibodies, neither in the healthy individuals nor in the patients tested.

### DISCUSSION

Based on previous findings (7, 13) it was our hypothesis that structural similarities in saccharide composition between pneumococcal polysaccharides and blood group A/B saccharide antigens could lead to production of cross-reactive blood group anti-A/B antibodies after immunization with capsular polysaccharide vaccines, because B cells could recognize shared carbohydrate epitopes (2, 8, 10, 33, 34). In the current study, we indeed show that immunization with Pn23 vaccine is followed by induction of blood group anti-A/B antibodies of the IgG isotype in healthy individuals. Anti-A/B IgG antibodies could only be demonstrated when SPR technology using the Biacore® device was applied, while conventional technology employing erythrocyte aggregation assays using the DiaMed-ID Micro Typing System were unable to detect this side effect of Pn23 vaccination. Apparently, the increased sensitivity of SPR technology as compared to conventional erythrocyte agglutination assays as well as the near to physiological conditions of the molecular interaction between antibodies and the specific blood group trisaccharide antigen enabled us to detect the stimulation of small amounts of cross-reactive IgG antibodies (11, 12, 31, 32). A previous study described an alternative approach to measure isoagglutinins in an isotype-specific and sensitive manner by using synthetic blood group saccharides in ELISA and showed cross-reactivity between alpha-Gal and blood group B oligosaccharide (13). The increase in blood group anti-A/B IgG isotype response observed in our study provides additional evidence for a limited cross-reactivity between PnPs antigens and the carbohydrate moieties of blood group A/B trisaccharides. Whether the observed induction of IgG antibodies cross-reacting with A/B blood group antigens is of clinical relevance in healthy individuals remains to be determined but is unlikely due to the relatively small increase (approximately twofold) observed. Patients with type I DM

reagents as described in Section "Materials and Methods." Results are depicted using box plot diagrams, with the median represented by a cross, the interquartile range (IQR) represented by the box, and 5- and 95-percentile values represented by the whiskers; statistically significant differences between study groups were calculated using the non-parametric two-tailed Mann–Whitney *U*-test. For the IgM antibody response, no statistically significant differences were found between study groups.

displayed a comparable cross-reactive IgG antibody response against blood group antigens following Pn23 vaccination, although these patients present an autoimmune disease with profound immunological dysregulation (17, 18). Interestingly, cross-reactive IgM antibodies were not inducible, neither in DM1 patients nor in controls. This is characteristic of a booster response effecting IgG responses only and contrary to the effect of immunization with a conventional polysaccharide antigen such as PnPs inducing both anti-PnPs IgM and IgG antibody responses following booster vaccination (**Figure 3**).

An additional benefit of the SPR-based technology employed in this study is the capability for real-time measurements of affinity of antibody–antigen interactions without the use of molecular labeling, leaving the structure of both interacting molecules intact (11, 12, 32). Calculations of *K*D as performed in our study using human serum as a sample can only be an estimate for the true affinity of a given antibody since we are dealing with a variety of polyclonal antibodies of different isotypes contributing to the overall affinity of the blood group antibodies detected in the SPR analysis used. Isotype

FIGURE 6 | Binding characteristics of anti-A/B antibodies after immunization with Pn23 vaccine. The percentage of dissociation (A) of blood group anti-A/B antibodies before and after immunization with Pneumo 23 Vaccine "Pasteur Merieux" (Pn23) in healthy individuals (*n* = 28) and in patients with type I DM (*n* = 16) was investigated using the Biacore device, the CM5 chip, and the synthetic blood group specific A/B trisaccharides as described in Section "Materials and Methods." Furthermore, the estimated affinity constant *K*<sup>D</sup> (B) of blood group specific anti-A antibodies before and after pneumococcal vaccination were analyzed in serum samples of three healthy individuals and two patients with type I DM selected for their initially high anti-A antibody titer (>than 1:256 or >than 700 RU). Kinetic measurements of anti-A antibodies binding to surface bound trisaccharide before and after vaccination in two representative individuals (a healthy individual, Ind1, and a DM I patient, Ind2) who fulfilled these criteria are given; one healthy individual that was not included in the vaccination protocol but showed high titer for blood group anti-A antibodies was measured in parallel (Control). Green curves represent serum sample and red curves serum sample diluted by half; the black curves depict the calculated fit. n.d., not detectable.

bias has been limited since individuals with high blood group A IgG but low to undetectable blood group A IgM antibody were selected to estimate the binding affinity of anti-A IgG antibodies. To determine the affinity (*K*D) of serum antibodies of a single specificity, isolation and immobilization of the respective immunoglobulin clone would be required, which is only possible in individuals with monoclonal gammopathy of the desired specificity. Interestingly, we did not find a marked change in binding characteristics of the cross-reactive blood group anti-A/B antibodies, indicating that a relatively small increase in IgG antibody titer occurred without additional affinity maturation. An immune response to carbohydrate antigens such as to pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F is thought to be generally TI, which can limit the B cell response to the production of IgM antibodies mainly and limited amounts of IgG antibodies, and the absence of affinity maturation of the rearranged antibody genes might lead to the expression of germline sequences of antibodies with limited affinity (5, 34, 35). In previous studies, the affinity of anti-carbohydrate antibodies for their antigens was shown to be 3–5 orders lower in magnitude than affinities of anti-protein or anti-peptide antibodies for their antigens (2, 3, 8, 35). In this study, however, we found no evidence for low affinity binding of blood group A/B antibodies obtained either from healthy individuals or from patients with type I DM. When a mixture of commercially available monoclonal blood group anti-A IgM and IgG antibodies was formulated, simulating the amount and composition of naturally occurring blood group anti-A antibodies, the dissociation rate was comparable to the serum samples analyzed in **Figure 6** (data not shown). Germline antibodies recognizing carbohydrate epitopes might display significant cross-reactivity because the number of potential antigens the immune system must encounter appears to extensively outweigh the recombinational potential of the germline genes (2, 3, 34, 35). The recombinational potential of the germline genes for anti-Gal antibodies specific for blood group B substance in unimmunized individuals was shown to be limited, because anti-Gal antibodies are encoded by a group of 6–8 well-defined and structurally related germline progenitors in humans (36). Anti-Gal was shown to be the most abundant antibody in humans, constituting approximately 1% of immunoglobulins and found to be of IgG, IgM, and IgA isotype (37, 38). Cryptic antigens capable to bind anti-Gal are exposed on senescent human RBCs as well as on RBCs of patients with β-thalassemia and sickle cell anemia, but the amount of cryptic antigens expressed on RBCs was shown to be low (38). The preferential anti-Gal IgG subclass found by ELISA was IgG2 using Gal-specific synthetic saccharides, and these IgG2 antibodies showed cross-reactivity with blood group B antigen (13). ABO blood group antigens were shown to be the ideal targets to explore the concomitant effect of mutation on binding affinity and specificity of anti-carbohydrate antibodies (33). Mutant anti-carbohydrate antibodies with higher affinity achieved by single point mutations of specific residues lining the pocket on binding to the A and B blood group oligosaccharide antigens showed altered polarity, surface complementarity, and side chain aliphatic character, leading to improved binding affinity without loss of specificity (33). Furthermore, the poly-specificity in germline encoded antibodies to carbohydrate epitopes was shown to be due to greater conformational ability in their combining sites, and this flexibility helped to cope with new and changing pathogen surface structures by recognizing distinct highly conserved epitopes on bacterial polysaccharides (4, 33). Structural analysis of the affinity-matured antibody 48G7 and its germline precursor antibody showed that the germline precursor could undergo an induced fit upon binding to its cognate hapten, while the affinity-matured antibody 48G7 displayed lock and key binding. An immunological relationship between specific substances of ABO and type XIV pneumococcus gave further evidence for shared carbohydrate epitopes by showing that adsorption of the hemagglutinins in anti-type XIV horse serum with erythrocytes of each of the A/B blood groups removed the agglutinins not only of that group but also of the other groups (7). In addition to pneumococcus, other germs have been shown to cross-react with carbohydrate structures on the surface of human cells (39–43). The core oligosaccharides of low-molecular-weight LPS of pathogenic *Neisseria* spp. can mimic the carbohydrate moieties of glycosphingolipids (40). The core oligosaccharides of LPS of *Campylobacter jejuni* serotypes, which are associated with the development of Guillian–Barre' syndrome, can exhibit mimicry of gangliosides (41). Finally, the O-chain of a number of *Helicobacter pylori* strains exhibit mimicry of Lewis(x) and Lewis(y) blood group antigens, giving evidence that molecular mimicry can serve to camouflage the bacterial surface from the host (42).

In conclusion, our study provides evidence for a limited carbohydrate epitope sharing between PnPs and blood group sugar epitopes in healthy individuals as well as in patients with type I DM that could be observed after Pn23 immunization. Cross-reactive anti-polysaccharide antibody responses were comparable between patients and controls with respect to the titer and the affinity. Blood group anti-A/B antibodies are considered to be naturally occurring antibodies that occur basically in any individual with an intact immune response also in the absence of antigenic encounter. The clinical relevance of the slight increase in blood group anti-A/B IgG isotype response in healthy individuals after Pn23 immunization is certainly limited; our findings however indicate modulation of anti-A/B IgG antibodies, e.g., a booster response during life not only by encounter with the genuine antigen such as incompatible blood transfusions or alloimmunization during pregnancy but also through molecular mimicry of particular carbohydrate epitopes shared by blood group AB substances and bacterial polysaccharides such as PnPs.

### ETHICS STATEMENT

The study was approved by the ethics committee of the Rudolfstiftung Hospital of the City of Vienna. Informed consent was obtained from every study participant.

### AUTHOR CONTRIBUTIONS

MF and HW were the principal investigators, and they critically assessed data, performed statistical analysis, wrote the manuscript, and took primary responsibilities for the paper; WW

### REFERENCES

and KS performed the experiments and analyzed the data; CB critically assessed data and was critically involved in the initial draft; NE-M conducted the clinical study and provided clinical data. All the authors critically participated in all revisions of the manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Wolfram, Sauerwein, Binder, Eibl-Musil, Wolf and Fischer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# C

*Huaqun Zhu1,2,3†, Fanlei Hu1,2,3†, Xiaolin Sun1,2,3, Xiaoying Zhang1,2,3, Lei Zhu1,2,3, Xu Liu1,2,3, Xue Li1,2,3, Liling Xu1,2,3, Lianjie Shi4 , Yuzhou Gan1,2,3 and Yin Su1,2,3\**

*1Department of Rheumatology and Immunology, Peking University People's Hospital, Beijing, China, 2Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135), Beijing, China, 3Peking-Tsinghua Center for Life Sciences, Beijing, China, 4Peking University International Hospital, Beijing, China*

### *Edited by:*

*Ana María Hernández, Center of Molecular Inmunology, Cuba*

### *Reviewed by:*

*Taruna Madan, National Institute for Research in Reproductive Health, India Anil Chauhan, Saint Louis University, USA*

### *\*Correspondence:*

*Yin Su<EMAIL_ADDRESS>
*† Huaqun Zhu and Fanlei Hu contributed equally to this work.*

### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 24 August 2016 Accepted: 02 November 2016 Published: 21 November 2016*

### *Citation:*

*Zhu H, Hu F, Sun X, Zhang X, Zhu L, Liu X, Li X, Xu L, Shi L, Gan Y and Su Y (2016) CD16+ Monocyte Subset Was Enriched and Functionally Exacerbated in Driving T-Cell Activation and B-Cell Response in Systemic Lupus Erythematosus. Front. Immunol. 7:512. doi: 10.3389/fimmu.2016.00512*

response have not been well studied in systemic lupus erythematosus (SLE). Objective: The present study aimed to investigate the distribution of CD16+ monocyte

Background: The roles that CD16+ monocyte subset plays in T-cell activation and B-cell

subsets in SLE and explore their possible roles in T-cell activation and B-cell differentiation. Methods: The frequencies of monocyte subsets in the peripheral blood of healthy controls (HCs) and patients with SLE were determined by flow cytometry. Monocyte subsets

were sorted and cocultured with CD4+ T cells and CD19+ B cells. Then, T and B cells were collected for different subset detection, while the supernatants were collected for immunoglobulin G, IgA, and IgM or interferon-γ and interleukin-17A detection by enzyme-linked immunosorbent assay.