Anti-LGI4 Antibody Is a Novel Juxtaparanodal Autoantibody for Chronic Inflammatory Demyelinating Polyneuropathy.

BACKGROUND AND OBJECTIVES: The objective of this study was to discover novel nodal autoantibodies in chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: We screened for autoantibodies that bind to mouse sciatic nerves and dorsal root ganglia (DRG) using indirect immunofluorescence (IFA) assays with sera from 113 patients with CIDP seronegative for anti-neurofascin 155 and anticontactin-1 antibodies and 127 controls. Western blotting, IFA assays using HEK293T cells transfected with relevant antigen expression plasmids, and cell-based RNA interference assays were used to identify target antigens. Krox20 and Periaxin expression, both of which independently control peripheral nerve myelination, was assessed by quantitative real-time PCR after application of patient and control sera to Schwann cells. RESULTS: Sera from 4 patients with CIDP, but not control sera, selectively bound to the nodal regions of sciatic nerves and DRG satellite glia (p = 0.048). The main immunoglobulin G (IgG) subtype was IgG4. IgG from these 4 patients stained a 60-kDa band on Western blots of mouse DRG and sciatic nerve lysates. These features indicated leucine-rich repeat LGI family member 4 (LGI4) as a candidate antigen. A commercial anti-LGI4 antibody and IgG from all 4 seropositive patients with CIDP showed the same immunostaining patterns of DRG and cultured rat Schwann cells and bound to the 60-kDa protein in Western blots of LGI4 overexpression lysates. IgG from 3 seropositive patients, but none from controls, bound to cells cotransfected with plasmids containing LGI4 and a disintegrin and metalloprotease domain-containing protein 22 (ADAM22), an LGI4 receptor. In cultured rat Schwann and human melanoma cells constitutively expressing LGI4, LGI4 siRNA effectively downregulated LGI4 and reduced patients' IgG binding compared with scrambled siRNA. Application of serum from a positive patient to Schwann cells expressing ADAM22 significantly reduced the expression of Krox20, but not Periaxin. Anti-LGI4 antibody-positive patients had a relatively old age at onset (mean age 58 years), motor weakness, deep and superficial sensory impairment with Romberg sign, and extremely high levels of CSF protein. Three patients showed subacute CIDP onset resembling Guillain-Barré syndrome. DISCUSSION: IgG4 anti-LGI4 antibodies are found in some elderly patients with CIDP who present subacute sensory impairment and motor weakness and are worth measuring, particularly in patients with symptoms resembling Guillain-Barré syndrome.

A new clustering method identifies multiple sclerosis-specific T-cell receptors.

OBJECTIVE: To characterize T-cell receptors (TCRs) and identify target epitopes in multiple sclerosis (MS). METHODS: Peripheral blood mononuclear cells were obtained from 39 MS patients and 19 healthy controls (HCs). TCR repertoires for α/ß/δ/γ chains, TCR diversity, and V/J usage were determined by next-generation sequencing. TCR ß chain repertoires were compared with affectation status using a novel clustering method, Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH). Cytomegalovirus (CMV)-IgG was measured in an additional 113 MS patients and 93 HCs. Regulatory T cells (Tregs) were measured by flow cytometry. RESULTS: TCR diversity for all four chains decreased with age. TCRα and TCRß diversity was higher in MS patients (P = 0.0015 and 0.024, respectively), even after age correction. TRAJ56 and TRBV4-3 were more prevalent in MS patients than in HCs (pcorr  = 0.027 and 0.040, respectively). GLIPH consolidated 208,674 TCR clones from MS patients into 1,294 clusters, among which two candidate clusters were identified. The TRBV4-3 cluster was shared by HLA-DRB1*04:05-positive patients (87.5%) and predicted to recognize CMV peptides (CMV-TCR). MS Severity Score (MSSS) was lower in patients with CMV-TCR than in those without (P = 0.037). CMV-IgG-positivity was associated with lower MSSS in HLA-DRB1*04:05 carriers (P = 0.0053). HLA-DRB1*04:05-positive individuals demonstrated higher CMV-IgG titers than HLA-DRB1*04:05-negative individuals (P = 0.017). CMV-IgG-positive patients had more Tregs than CMV-IgG-negative patients (P = 0.054). INTERPRETATION: High TCRα/TCRß diversity, regardless of age, is characteristic of MS. Association of a CMV-recognizing TCR with mild disability indicates CMV's protective role in HLA-DRB1*04:05-positive MS.

Early decrease in intermediate monocytes in peripheral blood is characteristic of multiple system atrophy-cerebellar type.

To identify biomarkers for multiple system atrophy-cerebellar type (MSA-C), we used flow cytometry to measure surface marker expression of peripheral blood monocytes from patients with MSA-C or hereditary spinocerebellar degeneration (hSCD) and from healthy controls (HCs). The percentage of intermediate monocytes was significantly lower in MSA-C patients than in hSCD patients and HCs and showed significant positive correlations with disease duration and unified MSA rating scale scores. The percentage of CD62L+ intermediate monocytes was significantly lower in MSA-C patients than in hSCD patients and HCs. Early decrease of peripheral blood intermediate monocytes is characteristic of MSA-C and is a biomarker.

Central nervous system-specific antinuclear antibodies in patients with multiple sclerosis.

BACKGROUND: Nuclear antigen released from central nervous system (CNS) cells undergoing destruction may induce production of antinuclear antibodies (ANA). We characterized the CNS-specific production of ANA in multiple sclerosis (MS). METHODS: We assessed CNS-ANA binding to mouse cerebellar cell nuclei by immunofluorescence assay (IFA) with sera from 104 MS patients (91 relapsing-remitting; 13 secondary progressive), 30 patients with neuromyelitis optica spectrum disorders (NMOSD), and 30 healthy controls (HCs). Conventional ANA (cANA) was detected by IFA using human epithelial type-2 cells. CNS-ANA-positive cANA-negative patients were termed CNS-specific ANA-positive. Western blotting (WB) was performed using mouse cerebellar nuclear fractions. RESULTS: CNS-specific ANA were more frequent in MS than in NMOSD patients or HCs (13.5% vs 0% for both comparisons, both p < .05) and were associated with HLA-DRB1*15:01 (p = .0174). WB revealed a common 55 kDa band in seven MS patients. Compared with CNS-specific ANA-negative MS patients, those with 55 kDa band-immunoreactive CNS-specific ANA showed a higher frequency of secondary progressive MS (42.9% vs 10.0%, p = .0387) and greater Expanded Disability Status Scale scores (4.50 ± 2.02 vs 2.92 ± 2.27, p = .0506). CONCLUSIONS: The CNS-specific ANA was more frequently detected in MS patients than NMOSD patients or HCs. 55 kDa band-reactive CNS-specific ANA may reflect clinical disease progression in MS.

Long-term use of interferon-ß in multiple sclerosis increases Vδ1-Vδ2-Vγ9- γδ T cells that are associated with a better outcome.

BACKGROUND: We previously reported that Vδ2+Vγ9+ γδ T cells were significantly decreased in multiple sclerosis (MS) patients without disease-modifying therapies (untreated MS) and were negatively correlated with Expanded Disability Status Scale (EDSS) scores, suggesting protective roles of Vδ2+Vγ9+ γδ T cells. Interferon-ß (IFN-ß) is one of the first-line disease-modifying drugs for MS. However, no previous studies have reported changes in γδ T cell subsets under IFN-ß treatment. Therefore, we aimed to clarify the effects of the long-term usage of IFN-ß on γδ T cell subsets in MS patients. METHODS: Comprehensive flow cytometric immunophenotyping was performed in 35 untreated MS and 21 MS patients on IFN-ß for more than 2 years (IFN-ß-treated MS) including eight super-responders fulfilling no evidence of disease activity criteria, and 44 healthy controls (HCs). RESULTS: The percentages of Vδ2+Vγ9+ cells in γδ T cells were significantly lower in untreated and IFN-ß-treated MS patients than in HCs. By contrast, the percentages of Vδ1-Vδ2-Vγ9- cells in γδ T cells were markedly higher in IFN-ß-treated MS patients than in HCs and untreated MS patients (both p < 0.001). A significant negative correlation between the percentages of Vδ2+Vγ9+ cells in γδ T cells and EDSS scores was confirmed in untreated MS but not evident in IFN-ß-treated MS. Moreover, class-switched memory B cells were decreased in IFN-ß-treated MS compared with HCs (p < 0.001) and untreated MS patients (p = 0.006). Interestingly, the percentages of Vδ1-Vδ2-Vγ9- cells in γδ T cells were negatively correlated with class-switched memory B cell percentages in all MS patients (r = - 0.369, p = 0.005), and the percentages of Vδ1-Vδ2-Vγ9- cells in Vδ1-Vδ2- γδ T cells were negatively correlated with EDSS scores only in IFN-ß super-responders (r = - 0.976, p < 0.001). CONCLUSIONS: The present study suggests that long-term usage of IFN-ß increases Vδ1-Vδ2-Vγ9- γδ T cells, which are associated with a better outcome, especially in IFN-ß super-responders. Thus, increased Vδ1-Vδ2-Vγ9- cells together with decreased class-switched memory B cells may contribute to the suppression of disease activity in MS patients under IFN-ß treatment.

Association of Decreased Percentage of Vδ2+Vγ9+ γδ T Cells With Disease Severity in Multiple Sclerosis.

We recently reported that deletion-type copy number variations of the T cell receptor (TCR) γ, α, and δ genes greatly enhanced susceptibility to multiple sclerosis (MS). However, the effect of abnormal TCR γδ gene rearrangement on MS pathogenesis remains unknown. In the present study, we aimed to clarify γδ TCR repertoire alterations and their relationship to clinical and immunological parameters in MS patients by comprehensive flow cytometric immunophenotyping. Peripheral blood mononuclear cells obtained from 30 untreated MS patients in remission and 23 age- and sex-matched healthy controls (HCs) were stained for surface markers and intracellular cytokines after stimulation with phorbol 12-myristate 13-acetate and ionomycin, and analyzed by flow cytometry. MS patients showed significantly decreased percentages of Vδ2+ and Vδ2+Vγ9+ cells in γδ T cells (pcorr = 0.0297 and pcorr = 0.0288, respectively) and elevated Vδ1/Vδ2 ratios compared with HCs (p = 0.0033). The percentages of interferon (IFN)-γ+Vδ2+ and interleukin (IL)-17A+IFN-γ+Vδ2+ cells in γδ T cells, as well as IFN-γ+ cells in Vδ2+ γδ T cells, were significantly lower in MS patients than in HCs (pcorr < 0.0009, pcorr = 0.0135, and pcorr = 0.0054, respectively). The percentages of Vδ2+ and Vδ2+Vγ9+ cells in γδ T cells were negatively correlated with both the Expanded Disability Status Scale score (r = -0.5006, p = 0.0048; and r = -0.5040, p = 0.0045, respectively) and Multiple Sclerosis Severity Score (r = -0.4682, p = 0.0091; and r = -0.4706, p = 0.0087, respectively), but not with age at disease onset, disease duration, or annualized relapse rate. In HCs, the percentages of Vδ2+ and Vδ2+Vγ9+ cells of total CD3+ T cells had strong positive correlations with the percentage of CD25+CD127low/- cells in CD4+ T cells (r = 0.7826, p < 0.0001; and r = 0.7848, p < 0.0001, respectively), whereas such correlations were totally absent in MS patients. These findings suggest that decreased Vδ2+Vγ9+ γδ T cells are associated with disability in MS. Therefore, the Vδ1/Vδ2 ratio might be a candidate biomarker for predicting disease severity in MS.