Laser-capture microdissection of plasma cells from subacute sclerosing panencephalitis brain reveals intrathecal disease-relevant antibodies

Abstract

Increased IgG and oligoclonal bands (OGBs) are found in the cerebrospinal fluid (CSF) of humans with chronic infectious CNS diseases such as neurosyphilis, cryptococcal and tuberculous meningitis, Lyme disease, some viral meningitides, varicella zoster virus vasculopathy, and subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus (MV). Studies in which the specificity of CSF OGBs was analyzed showed that the antibodies were directed against the agent that causes disease (1-8). For example, the OGBs in SSPE CSF and brain are antibodies directed against MV (1). These studies have led to the hypothesis that the oligoclonal IgG in the brain and CSF of patients with a chronic inflammatory CNS disease of unknown etiology, such as multiple sclerosis (MS), sarcoidosis, and Behcet's disease, is antibody directed against the agent that causes disease. Although OGBs are found in 88-100% of CSF from MS patients (9), the corresponding antigens remain unknown (10, 11). Such unanswered questions point to the need for improved techniques to identify disease-relevant antibodies and their cognate antigens and suggest the promise of such techniques in revealing the causes of inflammatory diseases with unknown etiologies.Here, we used laser-capture microdissection (LCM) to isolate individual CD38ϩ plasma cells from the brain of a patient with SSPE. Single-cell RT-PCR was used to amplify individual IgG heavy (H) and light (L) chain sequences expressed by each cell. Based on overrepresented Ig sequences, we constructed functional recombinant antibodies and identified their target antigens.Materials and Methods SSPE Brain. Brain removed from a 14-year-old male SSPE patient 5 h after death was flash-frozen and stored at Ϫ70°C. Sections (7 m) of frozen SSPE brain were prepared on nonplus glass slides (Fisher Scientific) at Ϫ30°C, fixed in acetone for 5 min at Ϫ20°C, and immunostained at 0°C for CD38ϩ cells. Briefly, sections were treated with 0.1% H 2 O 2 for 30 sec, incubated for 2 min with PBS containing 10% goat serum and then with a 1:50 dilution of mouse anti-human CD38 antibody (DakoCytomation, DAKO) for 10 min, rinsed in PBS, and incubated for 5 min with a 1:100 dilution of horseradish peroxidase-conjugated horse anti-mouse antibody (Vector Laboratories) and 5% goat serum in PBS. After rinsing in PBS, sections were incubated for 5 min with DAB substrate (Vector Laboratories), counterstained with hematoxylin, dehydrated in nuclease-free graded alcohols to 100% ethanol, and cleared with two rinses of H 2 0-free xylene. To preserve RNA, all aqueous solutions contained 200 units͞ml RNase inhibitor (Fisher Scientific), and the total time of tissue exposure to aqueous solutions was Ͻ25 min at 0°C. Staining was visualized by light microscopy.LCM and RT-PCR. LCM was performed on a PixCell IIe microscope (Arcturus Engineering, Mountain View, CA) with CapSure HS caps by using a pulse power of 70 mW, a 7.5-m laser spot diameter, pulse duration of 5 ms, and target voltage of 170 mV. Individual CD38ϩ cells ...

Clonally expanded plasma cells in the cerebrospinal fluid of MS patients produce myelin‐specific antibodies

“…PCR-amplified Ig H and L chain genes from each plasma cell clone were used to recombinantly reconstruct the antigen specificity of antibodies expressed by cePC preserving the original Ig H and L chain pairings of individual plasma cells. This approach has been demonstrated to faithfully resurrect pathogen-specific antibodies from individual plasma cells from patients with subacute sclerosing panencephalitis [25] and borreliosis, and to maintain the original antibody affinity [26].…”

Screening Random Peptide Libraries with Subacute Sclerosing PanencephalitisBrain-Derived Recombinant Antibodies Identifies Multiple Epitopes in the C-Terminal Region of the Measles Virus Nucleocapsid Protein

“…This approach is particularly useful in recapitulating in vitro the intrathecal antibody response in human CNS inflammatory diseases. Our previous sequence analysis of H-and L-chain V regions of plasma cells microdissected from SSPE brain revealed features of a targeted antigen-driven response, including clonal expansion, somatic mutation, intraclonal variation, and receptor editing (3). Furthermore, we demonstrated that the antibody response in SSPE brain is directed largely against MV, particularly the N protein, as evidenced by the specificity of five of eight rAbs generated from SSPE brain plasma cell clones.…”

“…Full-length human IgG1 rAbs were produced in mammalian tissue culture cells (Invitrogen, Carlsbad, Calif.) from V region sequences of expanded SSPE brain plasma cell clones as described previously (3). Briefly, V H regions were cloned into pIgG1Flag, a modified version of the episomal expression vector pCEP4 (Invitrogen) that contains a full-length human IgG1 C region domain and a C-terminal Flag epitope (21).…”

“…Increased CNS IgG synthesis is accompanied by an elevated number of CD19 ϩ B cells and the appearance of post-germinal center reaction plasmablast/plasma cells (4,6,18). We previously used laser capture microdissection and single-cell PCR to identify expanded clones among the CD38 ϩ B cells found within the parenchyma of SSPE brain (3). Five of the eight human IgG1 recombinant antibodies (rAbs) prepared from these expanded plasma cell clones recognized the measles virus (MV) nucleocapsid (N) protein, confirming that infiltrating B cells in SSPE brain target primarily the causative agent of the disease.…”

Cloning the Arterial IgA Antibody Response during Acute Kawasaki Disease

“…Abs to certain microbial Ags can dominate the immune response. For example, Burgoon et al (9,10) prepared synthetic Abs from overrepresented IgG sequences in the brains of patients with subacute sclerosing panencephalitis (SSPE) and demonstrated that all of the synthetic Abs that bound to SSPE brain tissue reacted with measles virus nucleocapsid.…”