B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes

Abstract

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune-mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF), and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS-resident B cells encounter antigen and experience affinity maturation. Paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clones were more often found in the draining CLNs. More mature clonal members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier, with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or affect the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.

Fig. 1. B cell antibody repertoire sequencing demonstrates that clonally expanding B cells populate the MS CNS and draining CLNs

(A) Heat maps showing the sequence overlap between compartments. The number of unique IgG (upper left triangles) and IgM (lower right triangles) sequences that were shared between each pair of tissue compartments in subjects M5, M2 and M3 is displayed. Percentages are calculated as a fraction of sequences from the tissue with the lower number. Values on the transecting axis indicate the total number of distinct sequences collected from each tissue. (B) Column graphs showing how members of clones were distributed between the CNS and CLN. For each IgG (upper y-axis) and IgM (lower y-axis) clone with at least one member found in the CNS, the fraction of distinct mRNA sequences that reside in the CLN is shown.

Fig. 2. The antibody repertoire in the CNS and CLN share features

Usage of both (A) IGHV families and (B) IGHJ genes were compared between the CNS and CLN. For each subject, the odds ratio (log) was calculated by measuring the distribution of the IGHV families in the CNS and normalizing it by the distribution in the periphery for the same subject. Positive values indicate that the observed segment usage is greater in the CNS and negative values indicate greater usage in the periphery. (C) The Hill diversity index (^qD), which measures diversity in a population, was calculated for the set of IgG clones present in the CNS (solid black line curves) and CLN (dotted curves) to determine whether the diversity in the repertoires differed between the compartments. For each subject (shown in different panels), the repertoire was sub-sampled to the number of sequences in the smallest sample and the Hill diversity index was calculated independently in 1,000 equally spaced q values between 0 and 15. As q varies from 0 to infinity the diversity (^qD) depends less on rare species and more on common ones, thus encompassing a range of definitions that can be visualized as a single curve. For q=0, the diversity is defined as the total number of clones, while as q approaches infinity the diversity is given by one over the frequency of the largest clone. At a given value of q (x-axis), lower values of ^qD (y-axis) indicate lower diversity. The gray bands indicate the middle 95% percentiles of the sampled distribution, thus when both lines are separated and fall outside of the gray band the difference between the two is significant. The analysis shows that the CNS repertoire had lower ^qD values than those in the CLN demonstrating lower diversity in the CNS (i.e., a more focused repertoire).

Fig. 3. Multi-compartment lineage trees illustrate that B cells traffic between the CLN and CNS

Representative lineage trees are shown for subject M2 (A), M3 (B) and M5 (C–F). Lineage trees are shown with the germline sequence at the root (black square). The number of somatic mutations accumulated from one node to the next is shown on each edge (branch) of the tree; an unlabeled edge corresponds to either 1 mutation (solid line) or 0 mutations (dotted line). Observed unique B cell sequences (nodes of the tree) are each annotated with text representing the tissue in which they were observed. The size (area) of each node is proportional to the number of unique mRNA sequences (number of UIDs) identified with the same nucleotide sequence.

Fig. 4. Trafficking between CNS compartments and the CLN often occurs early in clonal expansion

Sequences were classified according to where they resided; CNS, CLN or CLN/CNS (multi-compartment) and lineage trees were constructed for all clones that contained at least one CNS or multi-compartment sequence. Two separate analyses demonstrate that multi-compartment B cells are more often observed as ancestors than those that resided in a single compartment. (A) For each lineage, the fraction of somatic mutation events accumulated by progeny of CNS, CLN or multi-compartment sequences was determined. The distribution over all lineages, along with the mean fraction (black horizontal bar), is shown as a violin plot for each subject. Increasing values on the Y-axis represents the propensity of a clonal family member to produce offspring. The width of the plot for the CNS, CLN or multi-compartment sequences is proportional to the fraction of clonal members that produced descendants. A column that is wide only on the bottom indicates that few ancestors were among the clones. A column that is wide through its height indicates that many of the clonal members were ancestral as they produced further ancestors. (B) The set of all expanded (>10 unique sequences) multi-compartment lineage trees from each subject was analyzed to identify direct parent-child relationships among sequences from different compartments (numbers in each box). Statistically significant relationships, determined through permutation of node compartment labels (n=2000), are indicated in bold text. Numbers in parentheses indicate the effect strength, defined as the base 2 logarithm of the number of observed over the number of expected edges (branch in a lineage tree) of each type. These values designate whether the observation is more than (positive) or less than (negative) expected. Each edge type is shaded according to this effect strength, with darker colors indicating more edges (branches) than expected. Thus, the positive effect strength values observed only in the cases in which the parent node resided in two compartments indicates that these shared sequences more often gave rise to daughters than those restricted to a single compartment.

Fig. 5. Proposed model of B cell maturation and migration observed between the CLN and CNS

A reconstruction of an immunoglobulin lineage from a hypothetical clone is shown on both sides of the blood brain barrier (BBB). GL refers to germline cells and letters refer to different antibody sequences (clonal variants represented as nodes) in a single clonal family. Empty nodes represent sequences that are not empirically observed in a particular compartment. Mature clonally expanded (IgM, IgG or IgA) B cells traffic between the CLN and the CNS compartments by crossing the BBB indicated by the dashed arrow. Cells originate (node GL) and clonally expand (node A) in the CLN before trafficking into the CNS. However, this process begins early in the clonal expansion process as less mature offspring (e.g. node C and H) from the CLN can reside in the CNS. Several possibilities exist where the B cells can migrate back and forth through the BBB. The proposed scenario suggests that periphery B cell clones migrate from the CLN to the CNS while undergoing additional clonal expansion. Clonally expanded (IgM, IgG and/or IgA) B cells within the CNS can then traffic back into the peripheral tissue and undergo additional clonal expansion.