Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin

Abstract

We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.

Figure 1

Overlaps of CSF-proteome with the transcriptomes from CNS and CSF of patients MS-4 and MS-B2A. IgG-transcriptomes from CNS lesions and CSF were obtained by cDNA cloning. Proteome data were obtained by analyzing purified IgG antibodies from CSF by mass spectrometry. Repertoires were considered as overlapping when at least one characteristic peptide matched to the corresponding transcript. Characteristic peptides carry somatic hypermutated amino acids, or amino acids introduced by VDJ-recombination. Transcripts fulfilling this condition were attributed to the overlapping populations of CNS lesions (dark bars) and CSF (light bars). Their numbers relative to all detected characteristic peptides are given in percent. Transcripts that were found at different morphologically distinct brain lesions were counted independently. The left panel shows the overlaps of Ig-H and Ig-L chain repertoires from patient MS-4. The right panel shows the overlaps of Ig-H and Ig-L chain repertoires from patient MS-B2A. Here we identified less characteristic peptides, because most of the peptides identified by mass spectrometry derived from germline coded sequences.

Figure 2

Detection of overlapping transcriptome of CSF and brain lesion by clone-specific PCR using a formaldehyde-fixed biopsy sample from patient L-296. We determined clonally expanded B cells from CSF by cDNA cloning and then searched for identical clones in the lesion using clone-specific primers. The putative positions of the CDRs are shown as bars. Nucleotides and the respective amino acids that were exchanged by somatic hypermutation are displayed in red letters. We found identical sequences in CSF and the brain lesion for Heavy chain VH4-59/i (A) and Kappa chain VκI-O2/b (B). In brackets we indicate the degree of clonal expansion: the chain VH4-59/i was identified 17 times out of 106 analyzed IgG1 clones and VκI-O2/b was found 102 times out of 116 analyzed κ-chain clones. Arrows indicate the primer positions of the inner PCR primers. For each chain we amplified two independent sequences at different areas in the chains. The areas were chosen so that the primers ended with nucleotides introduced by somatic hypermutation, and that the amplified sequences contained additional somatically mutated nucleotides. PCR products were separated by gel electrophoresis (right panel), excised from the gels and analyzed by sequencing. All indicated VH4-59/i and VκI-O2/b sequences could be confirmed.