B cell exchange across the blood-brain barrier in multiple sclerosis

Abstract

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.

Figure 1. Clusters of B cells expressing identical and/or related IgG-VH are shared between CSF and BP.

Closely related IgG-VH sequences from (A) patients with MS and (B) patients with ONDs were clustered and colored according to their compartment (blue circles or arrowheads indicate CSF; red circles or arrowheads indicate PB) and depicted as networks. Only clusters containing at least one CSF node (i.e., IgG-VH clone) are shown. Directly connected nodes differ in their H-CDR3 by 1 amino acid; node sizes are proportional to the total number of identical H-CDR3 sequences identified. Clusters shaded in gray are presented as lineage trees in Figures 2–5, as indicated by labels with figure number and panel designation. Overall, IGHV4 germline segments are preferentially used in MS CSF; round nodes (circles) indicate clusters of related IgG-VH using IGHV4; v-shaped nodes (arrowheads) indicate clusters using all other IGHV subfamilies. The smallest nodes represent 2 IgG-VH sequences; the largest node (patient MS-6) represents 4,278 IgG-VH sequences.

Figure 2. In MS, B cell receptors are subject to extensive intrathecal SHM.

Nucleotide sequences, represented by clusters shown in Figure 1, were selected from the IgG-VH sequence database and used to generate lineage trees using IgTree software (see Methods). (A–E) Representative trees for CSF-restricted clusters for patients (A) MS-1, (B) MS-3, (C) MS-4, (D) MS-6, and (E) MS-5. The corresponding IGHV, IGHJ, and most common H-CDR3 AA sequences are listed in Supplemental Table 2. In the lineage trees, each round node represents at least one unique IgG-VH sequence ranging from at least the 5′ end of H-CDR1 to the 3′ end of H-CDR3; larger nodes represent up to hundreds of identical sequences. Putative germline sequences were determined using SoDA ( https://dulci.org/soda/; ref. 36) and are labeled as black, and hypothetical intermediates calculated by IgTree are labeled as beige. The numbers represent mutational steps between nodes; only mutational steps >1 are indicated; thus, unlabeled branches represent a single mutation. Triangular nodes contain 2 or more singleton sequences in leaves.

Figure 3. Select B cells undergoing SHM in the periphery of patients with MS are also present in the CSF.

Shown are IgG-VH lineages with predominantly PB-derived IgG-VH, (A and B) suggestive of B cell migration from the CNS to the PB or (C–E) seeding from the PB into the CNS. Blue nodes represent CSF-derived IgG-VH sequences, red nodes represent PB-derived IgG-VH sequences, and green nodes represent identical sequences found in both compartments. For additional information, see the legend of Figure 2.

Figure 4. B cells undergoing intrathecal SHM in patients with MS can also be found in the periphery.

IgG-VH lineage trees with predominantly CSF-derived IgG-VH sequences for patient (A and C) MS-5 and (B) MS-6. These lineages are suggestive of B cell migration from the PB into the CNS with traces of the clusters remaining in the PB and with extensive intrathecal B cell SHM. Blue nodes represent CSF-derived IgG-VH sequences, red nodes represent PB-derived IgG-VH sequences, and green nodes represent identical sequences found in both compartments. For additional information, see the legend of Figure 2.

Figure 5. Lineage trees of MS IgG-VH suggest ongoing B cell exchange across the BBB.

IgG-VH lineages suggestive of ongoing B cell exchange across the BBB for patient (A and C) MS-1, (B) MS-5, and (D) MS-6. These lineages could also reflect affinity maturation occurring in both compartments in parallel. Blue nodes represent CSF-derived IgG-VH sequences, red nodes represent PB-derived IgG-VH sequences, and green nodes represent identical sequences found in both compartments. For additional information, see the legend of Figure 2.

Figure 6. Preferential usage of IGHV4 germline segments by IgG-expressing CSF B cells in MS.

(A) Averages of IGHV germline segment usage in (A) MS CSF compared with that in OND CSF and (B) B cell clusters in MS CSF compared with MS PBMCs. (A) IgG-VH using IGHV4-39 (P = 0.04, resampling-based permutation test) and IGHV4-59 or IGHV4-61 (P = 0.01) are significantly overrepresented in MS CSF (also see Supplemental Table 3) compared with OND CSF. (B) Comparisons of IGHV usage in clusters of MS and OND B cells contributing to the CSF reveal overrepresentation of IGHV4-59 or IGHV4-61 in MS (P = 0.02)(also see Supplemental Table 4). IGHV4-39 and IGHV4-4 are marginally overrepresented (P = 0.06 and P = 0.07, respectively). IGHV3-53/66, IGHV3-53 or IGHV3-66; IGHV3-30/33rn, IGHV3-30 or IGHV3-33; IGHV4-59/61, IGHV4-59 or IGHV4-61; IGHV4-30-4/31, IGHV4-30-4 or IGHV4-31. Values shown in the graph are mean ± SEM. *P < 0.05.

Figure 7. Direct comparisons of IGHV germline segment usage in CSF and PB visually confirm overrepresentation of IGHV4 germline segments in MS CSF.

IGHV segment usage in PB (expected) plotted against usage in CSF (observed) in (A) MS and (B) OND. Proportions (%) of IGHV segments in PB were plotted against relative proportions of CSF IGHV. Each graph represents an individual patient. IGHV4-39 frequencies are in light red and IGHV4-59 or IGHV4-61 frequencies are in purple. IGHV most frequently observed in the CSF are labeled if different from IGHV4-39 and IGHV4-59 or IGHV4-61.