Comparison of immunoglobulin G heavy-chain sequences in MS and SSPE brains reveals an antigen-driven response.

OBJECTIVE: To better understand B-cell activation in MS by analyzing the immunoglobulin (Ig)G heavy chain variable region (VH) repertoire found in MS brains and comparing it with brain VH sequences in individuals with subacute sclerosing panencephalitis (SSPE)--a chronic encephalitis produced by measles virus (MV)-and characterized by an antigen-driven oligoclonal IgG response to MV antigens. BACKGROUND: The specificity of oligoclonal IgG in MS CSF and plaques, and their relevance to the pathogenesis of MS is unknown. METHODS: Nested PCR was used to amplify and sequence the rearranged IgG heavy-chain VH repertoire in plaques of three acute MS brains and in three SSPE brains. A representative population of VH sequences from each tissue was aligned to the known 51 functional VH germline segments. From this the authors determined the closest VH family germline segment, and the degree and location of somatic mutations for each unique IgG. RESULTS: As expected for an antigen-driven response against MV antigens, most VH sequences from the SSPE brains were mutated extensively compared with their closest germline segments. Furthermore, SSPE VH sequences accumulated replacement mutations preferentially in the complementary-determining regions (CDRs) relative to framework regions-features normally observed during antigen-driven selection. A comparison of VH family and germline usage also demonstrated that each SSPE brain had its own unique IgG response. When the authors compared the VH response in MS plaques with SSPE, MS VH sequences were also mutated extensively, displayed a preferential accumulation of replacement mutations in CDRs, and were unique in each MS brain. CONCLUSION: The presence of an antigen-driven response in MS, rather than a nonconventional mechanism of B-cell activation, warrants additional analysis of the specificity of IgG in MS brain and CSF.

Cloning the antibody response in humans with inflammatory central nervous system disease: analysis of the expressed IgG repertoire in subacute sclerosing panencephalitis brain reveals disease-relevant antibodies that recognize specific measles virus antigens.

The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands (OGBs) in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV) or Cryptococcus neoformans, respectively. Using SSPE as a model system, we have developed a PCR-based strategy to analyze the repertoire of IgG V region sequences expressed in SSPE brain. We observed abnormal expression of germline V segments, overrepresentation of particular sequences that correspond to the oligoclonal bands, and substantial somatic mutation of most clones from the germline, which, taken together, constitute features of Ag-driven selection in the IgG response. Using the most abundant or most highly mutated gamma H chain and kappa or lambda L chain sequences in various combinations, we constructed functional Abs in IgG mammalian expression vectors. Three Abs specifically stained MV-infected cells. One Ab also stained cells transfected with the MV nucleoprotein, and a second Ab stained cells transfected with the MV-fusion protein. This technique demonstrates that functional Abs produced from putative disease-relevant IgG sequences can be used to recognize their corresponding Ags.

Restricted use of VH4 germline segments in an acute multiple sclerosis brain.

Multiple sclerosis (MS) cerebrospinal fluid and brain contain increased IgG and oligoclonal bands. Whether this oligoclonal and polyclonal IgG is directed against a disease-relevant antigen remains unknown. To distinguish between random activation versus a targeted B-cell response, we analyzed the IgG heavy chain variable region (VH) repertoire expressed in different lesions of an acute MS brain. To obtain a representative sample of the VH repertoire, we constructed directional complementary DNA libraries from plaque-periplaque messenger RNA and amplified VH regions from the library by nested polymerase chain reaction. When MS VH sequences were aligned to germline segments, about 60% of different VH sequences in the acute MS brain were VH4 germline segments, significantly greater than the known approximately 20% VH4 germline prevalence. Specific VH sequences were overrepresented and expressed at multiple plaque sites. Within some overexpressed populations, there were distinct sequence differences (clonal variants) indicative of clonal expansion. Alignment of VH sequences to their closest germline counterparts revealed extensive somatic mutation and the preferential accumulation of amino acid replacement mutations in complementarity determining regions. These observations suggest the limited B-cell response found in this acute MS brain was antigen driven.