Neurological disorders-associated anti-glycosphingolipid IgG-antibodies display differentially restricted IgG subclass distribution

Antibodies against several self-glycans on glycosphingolipids are frequently detected in different neurological disorders. Their pathogenic role is profusely documented, but the keys for their origin remain elusive. Additionally, antibodies recognizing non-self glycans appear in normal human serum during immune response to bacteria. Using HPTLC-immunostaining we aimed to characterize IgM and IgG subclass antibody responses against glycosphingolipids carrying self glycans (GM1/GM2/GM3/GD1a/GD1b/GD3/GT1b/GQ1b) and non-self glycans (Forssman/GA1/“A” blood group/Nt7) in sera from 27 randomly selected neurological disorder patients presenting IgG reactivity towards any of these antigens. Presence of IgG2 (p = 0.0001) and IgG1 (p = 0.0078) was more frequent for IgG antibodies against non-self glycans, along with less restricted antibody response (two or more simultaneous IgG subclasses). Contrariwise, IgG subclass distribution against self glycans showed clear dominance for IgG3 presence (p = 0.0017) and more restricted IgG-subclass distributions (i.e. a single IgG subclass, p = 0.0133). Interestingly, anti-self glycan IgG antibodies with simultaneous IgM presence had higher proportion of IgG2 (p = 0.0295). IgG subclass frequencies were skewed towards IgG1 (p = 0.0266) for “anti-self glycan A” subgroup (GM2/GM1/GD1b) and to IgG3 (p = 0.0007) for “anti-self glycan B” subgroup (GM3/GD1a/GD3/GT1b/GQ1b). Variations in players and/or antigenic presentation pathways supporting isotype (M-G) and IgG-subclass pattern differences in the humoral immune response against glycosphingolipids carrying non-self versus self-glycans are discussed.

www.nature.com/scientificreports/ Human sera commonly display anti-glycan antibodies (i.e. antibodies recognizing saccharide sequences on one or more types of glycoconjugates, regardless of the inducer immunogen) 1 . Certain membrane lipids (known as glycosphingolipids) exhibit oligosaccharides as their hydrophilic head groups, granting access for binding by viruses, toxins and antibodies 2 . Normal subjects routinely display naturally-occurring antibodies recognizing non-self glycosphingolipids: archetypal examples are the ABO blood group agglutinins, arisen when blood group "0" individuals develop antibodies able to agglutinate blood group "A"/"B" red blood cells 3 . These antibodies appear within the context of a normal immune response against bacteria colonizing intestinal or respiratory tract 4 . Our laboratory described a similar origin for IgM antibodies recognizing a few self glycosphingolipids such as gangliosides GM1 and GD1b 5 ; however, these low affinity, cross-reactive IgM are non-pathogenic (normal anti-GM1 IgM antibodies 6 ). On the contrary, autoimmune diseases frequently exhibit immune reactivity towards self-glycosphingolipids 7 . In particular, gangliosides (glycosphingolipids abundantly found in nervous system) are often targeted by antibodies present in a variety of neurological diseases 8 . Multiple triggering mechanisms for nervous system dysfunction elicited by anti-ganglioside antibodies have been well documented: formation of a membrane attack complex (MAC) at motor nerve terminals by complement activation on the nerve cell membrane, impairment of axonal membrane properties at the nodes of Ranvier causing disfunction of voltagegated sodium channels (Nav) and conduction block; induction of apoptotic cascade activation in dorsal root ganglion cells; blockade of neurotransmitter release at motor nerve terminals by presynaptic inhibitory effect on voltage-gated Ca channel currents; complement-independent function alteration of certain receptors at lipid rafts acting as signaling platforms; and so forth 9 . Remarkably, the origin of anti-ganglioside antibodies is much less clear. Molecular mimicry between lipopolysaccharides from specific Campylobacter jejuni serotypes and ganglioside structures can either cause Guillain Barré syndrome (by inducing anti-GM1 and anti-GD1a antibodies) or Miller-Fisher syndrome (by inducing anti-GQ1b antibodies) [10][11][12] . Intriguingly, only a small fraction of individuals develops further neuropathy after infection with proper C. jejuni serotypes, implying additional bacterial or host constraints 13,14 . For anti-GM1 antibodies, we have proposed this additional requirement to be randomly elicited mutations affecting the binding site of normal anti-GM1 IgM antibodies ("binding site drift" hypothesis) 15 . Altogether, "molecular mimicry" and "binding site drift" hypotheses complement each other to depict how neuropathy-associated IgM and IgG anti-GM1 antibodies originate 16 .
Although recent work on IgM and IgG isotypes has extended this view to explain the origin of other anti-self glycosphingolipid antibodies associated with neurological disorders 17 , some questions persist. These types of IgG antibodies are absent in healthy humans 6,17 . Polysaccharides, some other nonprotein antigens (e.g. glycosphingolipids), and few proteins (e.g. flagellin) are regarded as T-cell independent (TI) antigens: i.e. they are able to activate B-1b and splenic marginal zone (MZ) B cells without intracellular processing and lacking assistance from CD4 + T helper (Th) cells 18 . B-1b or splenic MZ B cells exposed to cytokines such as B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL)-generated mostly by dendritic cells-can undergo antibody class switching 19 . In contrast, most proteins are internalized by antigen-presenting cells (B-2 cells, macrophages, and dendritic cells), digested into peptide fragments and combined with MHC-class molecules to form MHC-peptide complexes that are displayed on the surface of the antigen-presenting cells to be recognized by Th-cell receptors (TCR). The specific recognition activates the B-2 cells (linked recognition), inducing antibody production and class switching. Human IgG isotype response is in turn divided into four subclasses (1 to 4) with different heavy chains influencing their own properties (e.g. Fc receptor affinity) and biological functions (e.g. complement system activation ability) 20 . Total IgG subclass levels in autoimmune disease patients do not differ substantially from those measured in healthy individuals; however, certain specific antibodies can exhibit variable subclass restrictions [21][22][23] . Generally speaking, IgG1 and IgG3 subclasses are mainly elicited against protein antigens, whereas certain glycan antigens preferentially induce IgG2 responses 24 . While antigen nature can have an impact on the type of IgG subclass elicited 25 , IgG subclass can also depend on the type of T helper cell (Th) response 26 . Th1 cells produce interferon-gamma (IFN-γ) and interleukin (IL) 2,Th2 cells produce IL-4 and IL-5 27 , and Th17 cells produce IL-17, IL-21, and IL-22 28 . Nevertheless, distinction between Th1, -2 and -17 cells is less pronounced in humans than in experimental mouse models 29 . Prior studies on neuropathy-associated anti-GM1, anti-GD1a and anti-GQ1b IgG antibodies indicate predominance for IgG1, IgG3 or both [30][31][32][33] ; however, these studies lacked simultaneous evaluation of subclass distribution against non-self glycans for comparison purposes. In the present work, we assessed IgG-subclass humoral immune response against various self and non-self glycan-carrying glycosphingolipids in patients with diverse neurological disorders. Reactivity pattern differences were thoroughly analyzed in the context of potential origin diversity for these antibodies.

Results
IgG antibody subclass distribution is different between responses against self glycan-and non-self glycan-carrying glycosphingolipids. We comparatively analyzed the percentage distribution for anti-non-self glycan and anti-self glycan IgG-subclass antibodies in 27 randomly selected patient serum samples (positive for diverse anti-self glycan IgG antibodies) by HPTLC-I. Figure 1A shows patient # 11 serum analysis as an example: anti-non-self glycan reactivity comprised anti-Forssman antibodies (IgM), anti-"A" glycosphingolipid antibodies (IgM), anti-Nt7 antibodies (IgM, IgG1 and IgG2), and anti-GA1 antibodies (IgM). Anti-self glycan reactivity was formed by anti-GM2 (IgM), anti-GM1 (IgM and IgG1) and anti-GD1b (IgM and IgG1) antibodies. These analyses were repeated for all patient serum samples evaluated (see Supplementary Figure S1). Figure 1B summarizes the different distributions of anti-self and anti-non self-glycan IgG-subclass antibodies for each neurological disorder patient. When necessary, inhibition experiments using soluble self glycan-carrying glycosphingolipids were performed to confirm or discard differences in IgM and IgG fine specificities (results not shown). Initial evaluation of HPTLC-I results indicated the observed patterns of anti-self glycan IgG antibodies correlated with the specific diseases for some of the neurological disorders most repre-Scientific RepoRtS | (2020) 10:13074 | https://doi.org/10.1038/s41598-020-70063-5 www.nature.com/scientificreports/ sented in our samples: IgG anti-GM1 in Guillain Barré syndrome (7 of 8; 88%) and IgG anti-GQ1b in Miller Fisher syndrome (3 of 4; 75%) 8 . For IgG subclass distribution analyses of each given antigen group, we plotted the percentage of serum samples positive for each IgG subclass (considering the number of samples positive for any IgG subclass towards that antigen as the 100%; see Materials and Methods). Anti-non self glycan IgG antibodies had a significantly higher frequency of IgG1 (p = 0.0078) and IgG2 (p = 0.0001). In contrast, sera positive for anti-self glycan IgG antibodies showed a more frequent presence of IgG3 (p = 0.0017; Fig. 2).
Examined in close detail across the different serum samples, some IgG antibody responses comprised two or more different IgG subclasses, whereas others were formed by a single IgG subclass. The reactivity against self glycans was significantly associated to the presence of a single IgG subclass (p = 0.0133), thus representing a more restricted antibody response compared to that towards non-self glycans (Fig. 3). www.nature.com/scientificreports/

Changes in IgG antibody subclass distribution within responses against different self gly-
can-carrying glycosphingolipids. IgM populations recognizing certain self glycan-carrying glycosphingolipids (GM2/GM1/GD1b) have been reported in normal human sera 6 , leaving room for potential antibody response differences within the various self glycan glycosphingolipids tested. Based on this, reactivities against anti-self glycan glycosphingolipids were subgrouped in "anti-self glycan A" (GM2/GM1/GD1b) and "anti-self glycan B" (GD3/GQ1b/GD1a/GM3/GT1b, see "Methods"). The proportion of IgG1 subclass antibody response was significantly higher for anti-self glycan A than for anti-self glycan B subgroup (p = 0.0266). On the contrary, anti-self glycan B subgroup reached a significantly higher IgG3 proportion compared to anti-self glycan A subgroup (p = 0.0007; Fig. 4), supporting the notion for a distinct type of IgG response within the antibodies against self glycans.

The presence of anti-self glycan IgM antibody counterpart is associated with differences in
anti-self glycan igG antibody subclasses. Irrespective of their specificity, some populations of antiself glycan IgG antibodies were present along with their IgM counterpart, while others had their IgM counterpart absent (Fig. 1B). We therefore grouped anti-self glycan IgG antibody subclass results in different antibody subpopulations based on the simultaneous presence or absence of IgM counterpart. Anti-self glycan IgG responses present along with their IgM counterpart displayed a higher proportion of IgG2 compared to those IgG responses without IgM counterpart (p = 0.0295; Fig. 5).  www.nature.com/scientificreports/

Discussion
Even though invertebrates can distinguish self-from non-self components, it is not until the level of terrestrial vertebrates-amphibians, reptiles, birds, and mammals-that a complete immune system with thymus, spleen, bone marrow, and lymph nodes is present and IgM and IgG antibodies are made 34 . Besides their crucial role in host defense and homeostasis, improper control of antibody production can generate antibodies towards certain self-antigens (such as glycan antigens) and cause autoimmune diseases 8 . The structural similarities between non-self and self glycans could indicate connections between both responses 16 . In the present work, we characterized the antibody immune response (IgM, total IgG and IgG subclasses) against diverse non-self and self glycosphingolipids in a cohort of patients with neurological disorders. Parallel evaluation of the humoral immune response against glycosphingolipids carrying either non-self glycans or self glycans allowed comparisons between both types of responses, where anti-non-self glycan IgM antibodies are considered anti-bacterial antibodies 4,5,35 .
As already observed, anti-non-self glycan IgG antibodies always had their IgM counterpart 17 , and presented a less restricted IgG subclass response (i.e. two or more different IgG subclasses). Displaying IgG2 and IgG1 as prevailing IgG subclasses, they resembled antibody responses against bacterial glycans 36,37 , with IgG2 hinting at a relevant contribution of a TI immune response to these antibody populations 38 . On the contrary, IgG antibodies against self glycans frequently exhibited a more restricted IgG-subclass distribution (i.e. a single IgG subclass) and an increased proportion of IgG3 subclass (consistent with a T-cell dependent (TD) immune response 39    www.nature.com/scientificreports/ B cells (often referred to as B-2 cells) are present in secondary lymphoid organs. Although both B-2 populations can experience Ig class switching and differentiate into memory cells 40,42 , FO B cells are primarily responsible for generating long-lasting, high affinity IgG antibodies against T-dependent antigens 40 , while MZ B cells can also recognize TI carbohydrate and phospholipid antigens (like B-1 cells do) 42 . TI type II immune responses generate memory B cells 43 . IgM-expressing memory B cells with IgM + IgD + CD27 + phenotype can undergo secondary germinal center reactions upon reactivation with glycan antigens, differentiate to plasma cells and switch their isotype to IgG2 44,45 . We observed significantly more IgG2 contribution when IgM was present in anti-self glycan responses. IgG2 has lower ability to activate complement, to induce antibody-dependent cell-mediated cytotoxicity and to trigger antibody-dependent cellular phagocytosis than IgG1 and IgG3 20 : competition of IgG2 with other IgG subclasses for antigen binding in a context of IgG2 anti-self glycan relative enrichment could lead to lessen pathogenic events, and to an overall reduced severity of the neurological disorder. Glycan-antibody interactions occur via a number of attractive forces of hydrophilic and hydrophobic nature 46 : the acquisition, modification or loss of interaction points allows emergence of clones with specificity changes. Successive B-cell activation events introducing single nucleotide changes at high speed (~ 1 mutation every 10 3 base pairs/generation) can provide fast BCR diversification 47 and some variants with potential affinity maturation through somatic hypermutation 48 . In the context of a process we have coined as "binding site drift" hypothesis 16 , new specificities could now lead these anti-non self glycan antibodies to cross-react with sequences similar to those from self glycan. Bacterial-driven B-cell activations can expand these anti-self glycan IgG B-cell clones. Upon reaching a certain antibody affinity threshold value, these B-cell clones can now be activated by self glycan oligosaccharide structures, either on self molecules or on foreign components from certain microorganisms (e.g. Campylobacter jejuni LPS O-antigen with oligosaccharide motifs similar to GM1, GD1a, GD3, GT1a, GQ1b; Haemophilus influenzae mimicking GM1 and GT1a; Cytomegalovirus resembling GM2 8 ). Even though glycans are considered TI antigens, our findings regarding IgG subclasses suggest anti-self glycan responses can develop by two mechanisms: one TI (for anti-self glycan with IgM counterpart) and the other TD (for both types of anti-self glycan). T-cell involvement is indeed a likely event: MZ B cells can present lipoglycans and glycosphingolipids to CD1-restricted α/β and γ/δ T-cells. Activation via CD40L-CD40 and T cell multiplication provide the context for proliferation and antibody responses with eventual class switch 49 . That would not necessarily be detrimental for IgM: IgG antibody responses from MZ B-cells tend to maintain IgM production, even for long-term memory B-cell populations 50 .
How do anti-self glycan IgG antibodies occur if they have no IgM counterpart in normal human serum? One possibility would be a response against self glycan initiated by anti-self glycan (autoreactive) B-cells with surface IgM, leaving only anti-self glycan memory B-cells carrying surface IgG remaining after this first response. We hypothesize anti-self glycan IgM + B-cell clones populations that experienced a "binding site drift" process and are activated by self glycan would have the chance to expand. Some selected B-cell clones could then change their isotype to some of the different IgG subclasses, overall resulting in two or more IgG subclasses elicited in addition to IgM. On the contrary, clones originated from a "binding site drift" event at the IgG level are already committed to an IgG subclass. Therefore, expansion of one of those clones would generate only anti-self glycan antibodies from that specific IgG subclass, with neither anti-self glycan IgM antibodies nor the remaining IgG subclasses. This could account for the less restricted IgG subclass distribution observed for anti-non-self glycan IgG antibodies, compared to anti-self glycan IgG antibodies. Implying a potential association between IgM presence, a broader IgG subclass response and a substantial TI component, these and the aforementioned findings are represented in Fig. 6.
Within anti-self glycan IgG antibody reactivity (showing increased IgG3 subclass proportion, i.e. TD response), antibodies recognizing "self glycan B" subgroup had significantly higher proportion of IgG3 than those against "self glycan A" subgroup, suggesting origin differences. It is important to note that the "self glycan B" subgroup (GM3/GD3/GD1a/GT1b/GQ1b) comprises glycosphingolipids carrying a N-Acetyl Neuraminic Acid (NeuNAc) bound to a terminal galactose (NeuNAcα2,3Gal). This is a very important factor regarding anti-self glycan antibody specificity: for example, antibodies towards GM 1 (part of "self glycan A" subgroup) require a free terminal galactose and (in some patients) an internal NeuNAc for binding, rendering them unable to cross-react with self glycans carrying terminal NeuNAc ("self glycan B"). The inbalance in IgG1-/IgG3-subclass proportion could have consequences in pathology development, since IgG3-subclass has a better complement activator ability than IgG1 25 albeit its three-times shorter plasma half-life 20 . Considering antibodies against terminal NeuNAcα2,3Gal epitope have been preferentially associated with axonal damage 51 , one can speculate that the higher biological activity of IgG3 can have an influence on the greater severity and slower recovery observed in the axonal forms (compared to demyelinating variants) of Guillain-Barré syndrome 52 .
Anti-self glycan antibodies are an important component in the multifactorial process defining clinical evolution for certain neurological disorders. Evidence from GBS patients with anti-GM1 IgG antibodies indicates they do not present too many different populations simultaneously, although different patients exhibit different anti-GM1 antibody fine specificities 53 . We could consider they together represent different possibilities of antibody specificity changes and expand this perspective to other anti-self glycan IgG-mediated neurological disorders, assuming that emergence of different anti-self glycan IgG antibody populations results from a random process. As we discussed earlier, in the context of the "binding site drift" hypothesis we could potentially foresee different types of antibody responses for the same self glycan target. One response, originated from an antiself glycan IgM + B-cell population that drifted and is now activated by self glycan can lead to some IgM clones changing their isotype to produce various different IgG subclasses in combinations of TI responses (mainly IgG2) and TD responses (mainly IgG1 and IgG3). In contrast, a response generated from anti-self glycan IgG + B-cell clones (already committed to a determined IgG subclass) that drifted at the IgG level (upon expansion of one of those clones in a TD response) would make only that specific IgG subclass. Based on biological activity disparities exhibited by each IgG subclass, the differences in IgG subclass responses generated could have clinical

Methods
Human sera. Disease serum samples were collected before any immune treatment from patients attending Neurology services from Hospital "Ramos Mejía" and Hospital Nacional de Clínicas "José de San Martín" (Buenos Aires, Argentina) with early symptoms of neurological disease. After clot separation, sera were submitted to our laboratory for routine determination of anti-glycosphingolipid antibodies (GM1, GM2, GM3, GD1a, GD1b, GD3, GT1b, GQ1b). From samples resulting positive for IgG antibodies against any of these self glycan-carrying glycosphingolipids, we randomly selected 27 patients for further analysis (amyotrophic lateral sclerosis, n = 2; asymmetric motor neuropathy, n = 2, chronic inflammatory demyelinating polyneuropathy, n = 1, diabetic neuropathy, n = 1; Guillain-Barré syndrome, n = 8; lower motor neuron disease, n = 2; Miller Fisher syndrome, n = 4; multifocal motor neuropathy, n = 1; paraneoplastic syndrome, n = 1; sensory-motor neuropathy, n = 5). With different extents, all these neurological disorders have been reported to present autoimmune components 8,54,55 . All procedures were performed in accordance with Ethical Guidelines on Research Involving Human Subjects 56 and with ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments, with prior approval by the Ethics Committee of CIQUIBIC-CONICET. Informed consent was obtained from the patients. www.nature.com/scientificreports/ Glycosphingolipids. Glycosphingolipids were obtained from the following biological materials: Sandhoff disease human brain for GM2; dog erythrocytes for GM3; chick brain for GD3; human brain for GM1, GD1a, GD1b, GT1b, and GQ1b; sheep erythrocytes for Forssman glycosphingolipid (Forssman); human blood group "A" meconium for blood group "A" glycosphingolipid; Calliphora vicina pupae for Nt7 glycosphingolipid 57 . Folch upper phase from partitioned lipid extract 58 was sequentially purified using DEAE -chromatography 59 and HPLC on Iatrobeads silica-gel column 60 . Acid hydrolysis of cow brain gangliosides was used to prepare asialo-GM1 (GA1) 61 . Lipid purity was checked by HPTLC with orcinol reagent.
Statistical analyses. Antibody reactivity reported as categorical data was combined into groups for statistical purposes. Immunostaining against non-self glycans (GA1, Forssman, Nt7 and blood group "A" glycosphingolipid for "0" and "B" blood group individuals) was grouped as "non-self glycan" reactivity. IgM populations against GM1, GD1b and GM2 are present in normal human sera 6 ; hence, IgG reactivity against GM1, GD1b and GM2 was at times considered a subgroup ("self glycan A"). Finally, IgG response against GM3, GD3, GD1a, GT1b and GQ1b was counted as another subgroup of self glycans ("self glycan B"). For each given antigen group, IgG subclass distribution analyses were done by comparing the number of samples positive for each IgG subclass to the number of samples positive for total IgG antibodies towards that antigen (considered as the total sample number). Data were examined by Fisher's exact test with Prism 6 (GraphPad software, La Jolla, CA). Differences with P value < 0.05 were considered significant. Degrees of statistical significance are presented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001, or ****, p < 0.0001.