In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses

Abstract

Objective: To determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients.

Methods: Mass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients.

Results: We find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood-brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related.

Interpretation: Our data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier.

Figure 1

Bicompartmental B-cell lineage contributing to oligoclonal band (OCB) production in Patient MS-2. Shown are the (A) lineage tree (hierarchic layout) and (B) alignment of immunoglobulin G (IgG) heavy chain variable region (IgG-VH) amino acid sequences from cerebrospinal fluid (CSF) and peripheral blood (PB) mononuclear cells. Lineage trees are calculated using nucleotide sequences IgTree software and displayed using Cytoscape (see Patients and Methods). In the lineage tree, each round node represents at least 1 unique IgG-VH sequence ranging from at least the 5′ end of heavy chain CDR1 region (H-CDR1) to the 3′ end of H-CDR3; larger nodes represent up to hundreds of identical sequences. CSF-derived IgG-VHs are represented by blue nodes, PB-derived IgG-VHs are red. Numbers between nodes are numbers of nucleotide mutations; unlabeled connections between nodes represent single nucleotide mutations. Putative germline sequences were determined using SoDA and are labeled black; hypothetical intermediates calculated by IgTree are beige. Numbers on lines between nodes (edges) represent mutational steps (nucleotides) between nodes. Triangular nodes contain 2 or more singleton sequences in leaves. The OCB peptide (YFAWSAGK) identified from isoelectric focusing band D (see Fig 4) maps to the H-CDR3 and was only found in a CSF-restricted sublineage. Additional sublineages in PB and CSF suggest that B-cell affinity maturation and plasma cell maturation (OCB production) occur in parallel in both compartments. IGHV = Ig heavy chain variable germline gene.

Figure 2

B-cells involved in oligoclonal band (OCB) production are present in the peripheral blood. Shown are (A–D) examples of bicompartmental IgG heavy chain variable region (IgG-VH) lineages (hierarchic layout) from Patient MS-5, and (E) a IgG-VH lineage from Patient MS-1 in which OCB peptides mapped to peripheral blood mononuclear cellimmunoglobulin G (IgG) sequences exclusively. Cerebrospinal fluid–derived IgG-VHs are represented by blue nodes, peripheral blood–derived IgG-VHs are red, and identical sequences found in both compartments are green. Numbers between nodes are numbers of nucleotide mutations; unlabeled connections between nodes represent single nucleotide mutations. Numbers in nodes are numbers of OCB peptides mapping to a specific node. Triangular nodes contain 2 or more singleton sequences in leaves. Ig heavy chain variable germline gene (IGHV) and Ig heavy chain joining (IGHJ) usage, matched peptide, and corresponding representative CDR3 are indicated per lineage. Amino acids differing from germline segments are underlined; in E, the detected peptide is entirely located in the non–germline-encoded heavy chain CDR3 region.

Figure 3

B-cell clusters participating in oligoclonal band (OCB) production undergo immune stimulation in the periphery. (A) Lineage tree (organic layout) and (B) alignment of representative immunoglobulin G (Ig) heavy chain variable region (IgG-VH) amino acid sequences from Patient MS-5 are shown. Germline node (black arrow), node comprised of cerebrospinal fluid (CSF) and peripheral blood (PB) mononuclear cell (PBMC) IgG-VH (green arrow), and PB-derived subcluster identified by OCB peptide searches (gray arrow) are indicated. In the alignment, matched OCB peptides are shaded in gray; IgG-VH is derived from the Ig heavy chain variable germline gene (IGHV)3–9 germline segment. Only representative IgG-VHs are shown. Organic layout was chosen over hierarchic layout because the latter generates very extensive horizontal images. For additional information, please refer to the legends of Figures 1 and 2.

Figure 4

Oligoclonal bands (OCBs) represent diverse and partially related immunoglobulin G (IgG) populations. Shown are isoelectric focused (IEF) cerebrospinal fluid and serum IgG from Patients MS-1 to MS-5. IEF gels were stained and processed for mass spectrometry as described in Patients and Methods; analyzed gel slices are labeled with letters. *Gel slices yielding patient-specific OCB bands. **Gel slices yielding peptides linking to peripheral blood mononuclear cell–only clusters or subclusters. IEF bands yielding IgG OCB peptides belonging to the same cluster are connected by gray brackets; dashed and solid lines were used for better visual separation.

Figure 5

(A) Immunoglobulin G (IgG) heavy chain variable germline gene (IGHV) usage and (B) heavy chain CDR3 region (H-CDR3) length distribution of IgG heavy chain variable region clusters associated with oligoclonal band (OCB) peptides. AA = amino acids.

Figure 6

Oligoclonal band (OCB) peptides reveal signs of affinity maturation. Shown is an immunoglobulin G (IgG) heavy chain variable germline gene 4–39–derived, truncated IgG heavy chain variable region with OCB peptides identified by mass spectrometry in isoelectric focusing gel slices A, B, D, J, and K (see Fig 4; Supplementary Table S3) in Patient MS-3’s cerebrospinal fluid. Shaded in light gray are aligned amino acid sequences of identified OCB peptides reflective of ongoing affinity maturation in a B-cell cluster contributing to OCB production in Patient MS-3. Heavy chain CDR2 region (H-CDR2) and H-CDR3 are in gray frames. Dots indicate identity with the consensus sequence (top).