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

Abstract

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.

Figure 1

Distribution of anti-non-self glycan and anti-self glycan IgM and IgG subclass antibodies in neurological disorder patients. (A) A representative HPTLC-I result, corresponding to patient # 11. After serum incubation, proper specific secondary antibody (see M&M) for binding detection of each isotype (IgM or IgG) or each IgG subclass (IgG1, IgG2, IgG3 or IgG4) was added. On left plate the different glycosphingolipids were visualized with orcinol reagent. (B) Summary of anti-non-self glycan and anti-self glycan IgM and IgG subclasses found in 27 randomly chosen, anti-self glycan IgG Ab-positive, neurological disorder patients. Presence (yellow squares) or absence (blue squares) of IgM and IgG antibody subclasses reactive for each glycosphingolipid (by HPTLC-I) is shown. Patient number (Patient #) and neurological disorder diagnosis (Dx) are detailed in far-left columns. ALS amyotrophic lateral sclerosis, AMN asymmetric motor neuropathy, CIDP chronic inflammatory demyelinating polyneuropathy, DN diabetic neuropathy, GBS Guillain-Barré syndrome, LMND lower motor neuron disease, MFS Miller Fisher syndrome, MMN multifocal motor neuropathy, PNS paraneoplastic syndrome, SMN sensory-motor neuropathy.

Figure 2

Anti-self glycan antibodies have a different IgG subclass distribution compared to those against non-self glycans. Percentage of samples having antibody reactivity against antigens grouped in non-self glycan (blue bars) and self glycan (red bars) are shown for each IgG subclass. The different anti-non-self glycan and anti-self glycan IgG subclass antibody reactivities were determined using HPTLC-I (**: p < 0.01; ***:p < 0.001; Fisher's exact test).

Figure 3

The reactivity against self glycans is associated to a more restricted IgG subclass antibody response. Stacked bars show proportion of IgG antibody populations composed by single (“one”) versus multiple different (“two”, “three” or “four”) IgG subclasses in anti-non-self glycan and anti-self glycan IgG antibody responses. The presence of single IgG subclass populations is considered indicative of a more restricted response (*: p < 0.05, Fisher’s exact test).

Figure 4

The IgG subclass frequencies are skewed towards different IgG subclasses between anti-self glycan A and anti-self glycan B responses. IgG subclass frequency distributions for antigens grouped in self glycan A (peach bars) and self glycan B (purple bars) are shown. Antibody reactivities from each subclass were determined using HPTLC-I. (**: p < 0.01; ***: p < 0.001; Fisher's exact test).

Figure 5

IgM counterpart presence for anti-self glycan IgG antibody responses is associated with an increased frequency of IgG2 subclass. Antibody reactivities of anti-self glycan antibodies detected along with IgM (striped bars) versus anti-self glycan antibodies without IgM counterpart (red-filled bars) were determined for each subclass using HPTLC-I. *: p < 0.05 (Fisher’s exact test).

Figure 6

IgG antibody subclass responses are qualitatively different between self glycans and non-self glycans. Trends are displayed proportionally to percentage distributions for each isotype and subclass. TI T-independent antibody response, TD T-dependent antibody response.