VH2+ Antigen-Experienced B Cells in the Cerebrospinal Fluid Are Expanded and Enriched in Pediatric Anti-NMDA Receptor Encephalitis.

Pediatric and adult autoimmune encephalitis (AE) are often associated with Abs to the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Very little is known regarding the cerebrospinal fluid humoral immune profile and Ab genetics associated with pediatric anti-NMDAR-AE. Using a combination of cellular, molecular, and immunogenetics tools, we collected cerebrospinal fluid from pediatric subjects and generated 1) flow cytometry data to calculate the frequency of B cell subtypes in the cerebrospinal fluid of pediatric subjects with anti-NMDAR-AE and controls, 2) a panel of recombinant human Abs from a pediatric case of anti-NMDAR-AE that was refractory to treatment, and 3) a detailed analysis of the Ab genes that bound the NR1 subunit of the NMDAR. Ag-experienced B cells including memory cells, plasmablasts, and Ab-secreting cells were expanded in the pediatric anti-NMDAR-AE cohort, but not in the controls. These Ag-experienced B cells in the cerebrospinal fluid of a pediatric case of NMDAR-AE that was refractory to treatment had expanded use of variable H chain family 2 (VH2) genes with high somatic hypermutation that all bound to the NR1 subunit of the NMDAR. A CDR3 motif was identified in this refractory case that likely drove early stage activation and expansion of naive B cells to Ab-secreting cells, facilitating autoimmunity associated with pediatric anti-NMDAR-AE through the production of Abs that bind NR1. These features of humoral immune responses in the cerebrospinal fluid of pediatric anti-NMDAR-AE patients may be relevant for clinical diagnosis and treatment.

MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents.

MicroRNAs (miRNA, miR-) play important roles in disease development. In this study, we identified an anti-proliferative miRNA, miR-212-5p, that is induced in pulmonary artery smooth muscle cells (PASMCs) and lungs of pulmonary hypertension (PH) patients and rodents with experimental PH. We found that smooth muscle cell (SMC)-specific knockout of miR-212-5p exacerbated hypoxia-induced pulmonary vascular remodeling and PH in mice, suggesting that miR-212-5p may be upregulated in PASMCs to act as an endogenous inhibitor of PH, possibly by suppressing PASMC proliferation. Extracellular vesicles (EVs) have been shown recently to be promising drug delivery tools for disease treatment. We generated endothelium-derived EVs with an enriched miR-212-5p load, 212-eEVs, and found that they significantly attenuated hypoxia-induced PH in mice and Sugen/hypoxia-induced severe PH in rats, providing proof of concept that engineered endothelium-derived EVs can be used to deliver miRNA into lungs for treatment of severe PH.

Extracellular vesicles derived from endothelial cells in hypoxia contribute to pulmonary artery smooth muscle cell proliferation in-vitro and pulmonary hypertension in mice.

In the lung, communication between pulmonary vascular endothelial cells (PVEC) and pulmonary artery smooth muscle cells (PASMC) is essential for the maintenance of vascular homeostasis. In pulmonary hypertension (PH), the derangement in their cell-cell communication plays a major role in the pathogenesis of pulmonary vascular remodeling. In this study, we focused on the role of PVEC-derived extracellular vesicles (EV), specifically their microRNA (miRNA, miR-) cargo, in the regulation of PASMC proliferation and vascular remodeling in PH. We found that the amount of pro-proliferative miR-210-3p was increased in PVEC-derived EV in hypoxia (H-EV), which contributes to the H-EV-induced proliferation of PASMC and the development of PH.

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

Plasmablasts are a highly differentiated, antibody secreting B cell subset whose prevalence correlates with disease activity in Multiple Sclerosis (MS). For most patients experiencing partial transverse myelitis (PTM), plasmablasts are elevated in the blood at the first clinical presentation of disease (known as a clinically isolated syndrome or CIS). In this study we found that many of these peripheral plasmablasts are autoreactive and recognize primarily gray matter targets in brain tissue. These plasmablasts express antibodies that over-utilize immunoglobulin heavy chain V-region subgroup 4 (VH4) genes, and the highly mutated VH4+ plasmablast antibodies recognize intracellular antigens of neurons and astrocytes. Most of the autoreactive, highly mutated VH4+ plasmablast antibodies recognize only a portion of cortical neurons, indicating that the response may be specific to neuronal subgroups or layers. Furthermore, CIS-PTM patients with this plasmablast response also exhibit modest reactivity toward neuroantigens in the plasma IgG antibody pool. Taken together, these data indicate that expanded VH4+ peripheral plasmablasts in early MS patients recognize brain gray matter antigens. Peripheral plasmablasts may be participating in the autoimmune response associated with MS, and provide an interesting avenue for investigating the expansion of autoreactive B cells at the time of the first documented clinical event.

B cells from relapsing remitting multiple sclerosis patients support neuro-antigen-specific Th17 responses.

B cells are highly potent antigen presenting cells of their cognate antigens. However, it remains unknown whether B cells can orchestrate Th17-mediated responses against neuro-antigens. We report that MS patients and healthy donors had a similar frequency of antigen-specific Th1 and Th17 cells, and distribution of T effector and T central memory cells. Notwithstanding these similarities, the application of an in vitro assay demonstrated that the B cells derived from a subset of MS patients exhibited the capability of coordinating Th17 responses directed toward neuro-antigens. These observations underscore the B cell's contribution to the putative underpinnings of multiple sclerosis.

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

*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.

The effect of glatiramer acetate therapy on functional properties of B cells from patients with relapsing-remitting multiple sclerosis.

IMPORTANCE: This study describes what is, to our knowledge, the previously unknown effect of glatiramer acetate therapy on B cells in patients with relapsing-remitting multiple sclerosis (MS). OBJECTIVE: To determine whether glatiramer acetate therapy normalizes dysregulated B-cell proliferation and cytokine production in patients with MS. DESIGN, SETTING, AND PARTICIPANTS: Twenty-two patients with MS who were receiving glatiramer acetate therapy and 22 treatment-naive patients with MS were recruited at The University of Texas Southwestern Medical Center MS clinic. Cell samples from healthy donors were obtained from HemaCare (Van Nuys, California) or Carter Blood Bank (Dallas, Texas). Treatment-naive patients with MS had not received any disease-modifying therapies for at least 3 months before the study. EXPOSURES: Glatiramer acetate therapy for at least 3 months at the time of the study. MAIN OUTCOMES AND MEASURES: B-cell phenotype and proliferation and immunoglobulin and cytokine secretion. RESULTS: A restoration of interleukin 10 production by peripheral B cells was observed in patients undergoing glatiramer acetate therapy as well as a significant reduction of interleukin 6 production in a subset of patients who received therapy for less than 32 months. Furthermore, proliferation in response to high-dose CD40L was altered and immunoglobulin production was elevated in in vitro-activated B cells obtained from patients who received glatiramer acetate. CONCLUSIONS AND RELEVANCE: Glatiramer acetate therapy remodels the composition of the B-cell compartment and influences cytokine secretion and immunoglobulin production. These data suggest that glatiramer acetate therapy affects several aspects of dysregulated B-cell function in MS that may contribute to the therapeutic mechanisms of glatiramer acetate.