A Distinct Class of Antibodies May Be an Indicator of Gray Matter Autoimmunity in Early and Established Relapsing Remitting Multiple Sclerosis Patients

Abstract

*These authors contributed equally to the work in this manuscript.We have previously identified a distinct class of antibodies expressed by B cells in the cerebrospinal fluid (CSF) of early and established relapsing remitting multiple sclerosis (RRMS) patients that is not observed in healthy donors. These antibodies contain a unique pattern of mutations in six codons along VH4 antibody genes that we termed the antibody gene signature (AGS). In fact, patients who have such B cells in their CSF are identified as either having RRMS or developing RRMS in the future. As mutations in antibody genes increase antibody affinity for particular antigens, the goal for this study was to investigate whether AGS+ antibodies bind to brain tissue antigens. Single B cells were isolated from the CSF of 10 patients with early or established RRMS. We chose 32 of these B cells that expressed antibodies enriched for the AGS for further study. We generated monoclonal full-length recombinant human antibodies (rhAbs) and used both immunological assays and immunohistochemistry to investigate the capacity of these AGS+ rhAbs to bind brain tissue antigens. AGS+ rhAbs did not recognize myelin tracts in the corpus callosum. Instead, AGS+ rhAbs recognized neuronal nuclei and/or astrocytes, which are prevalent in the cortical gray matter. This pattern was unique to the AGS+ antibodies from early and established RRMS patients, as AGS+ antibodies from an early neuromyelitis optica patient did not display the same reactivity. Prevalence of CSF-derived B cells expressing AGS+ antibodies that bind to these cell types may be an indicator of gray matter-directed autoimmunity in early and established RRMS patients.

Peripheral VH4+ plasmablasts demonstrate autoreactive B cell expansion toward brain antigens in early multiple sclerosis patients

“…In addition to theCIS-PTM rhAbs, NMO rhAbs (n=3) and two antibodies from lupus patients (B1 as a negative control and G11 as a positive control) were utilized as controls in this part of the study [110]. Though the specificities of B1 and G11 have not been fully characterized, B1 does not bind to multiple types of brain tissue [57], NMDA receptors [19], DNA, LPS, or recombinant human insulin [110]. In contrast, G11 is a polyreactive antibody that binds to DNA [110] as well as NMDA receptors [19].…”

“…Additionally many antibodies recognize conformational epitopes, and the degree to which a technique may alter these epitopes can profoundly affect the outcome. For example, tissue staining done with MS CSF rhAbs on formalin fixed paraffin embedded brain tissue yielded almost no positive staining [74], but positive staining is detected with similar rhAbs when tested on paraformaldehyde fixed and gently unmasked tissue [12, 57]. However this unmasking may denature some conformational epitopes.…”

“…When rhAb reactivity is considered in aggregate (Supplemental Table 1), it is interesting to note that while the CSF-derived rhAbs from these patients were largely reactive to neurons and astrocytes in the gray matter of the brain [57], many of the rhAbs generated from the peripheral plasmablasts were directed towards both gray and white matter targets. These data suggest that peripheral plasmablasts have a wider array of autoreactive specificities compared to CSF-derived B cells, although their specificity and significance to MS pathology remains unknown.…”

“…Healthy and diseased (experimental autoimmune encephalomyelitis [EAE] or stroke) mouse brains were preserved, cryosectioned, and used for immunohistochemistry as described previously [57, 86]. Protocol details are provided in the supplemental methods.…”

“…Previous data from our laboratory demonstrated that B cells in the CSF of early and established MS patients express antibodies with a particular mutational pattern that bind gray matter targets in brain tissue, such as neurons and astrocytes [57]. Others have demonstrated that antibodies from clonally expanded MS CSF B cells that bind gray matter targets mediate complement deposition and damage to neurons in vitro , and pooled CSF antibodies from MS patients can mediate neurological dysfunction in vivo [12, 24].…”