Peripheral B Cell Activation May Drive CNS Autoimmunity in MS

Pathological B lymphocytes are most likely activated in the periphery in patients with multiple sclerosis (MS) and then travel to the central nervous system (CNS), where they do damage, according to two studies reported in the August 6, 2014, issue of Science Translational Medicine. Finding B cells sharing antigen specificity in both compartments suggests to the researchers that peripheral antigen-driven B cell activation can lead to CNS autoimmune reactions.

This “trafficking” or migration of B cells appears to be a two-way street, with the lymphocytes circulating back and forth between the CNS and peripheral compartments. One therapeutic implication is that agents such as the monoclonal antibodies rituximab and ocrelizumab that deplete peripheral B cells may also limit them in the CNS. Similarly, CNS damage may be prevented through the use of a monoclonal antibody such as natalizumab that prevents lymphocyte migration across the blood-brain barrier (BBB).

A study by Stern et al. (2014) from Yale School of Medicine in New Haven, Connecticut, used paired CNS autopsy tissues and draining cervical lymph nodes (CLNs) from five patients with relapsing-remitting, primary progressive, secondary progressive, chronic progressive, and chronic MS. They used high-throughput sequencing of cDNA derived from mRNA from four of the patients to generate about 32 million raw sequence reads to delineate the B cell immunoglobulin heavy-chain repertoire. They found that antigen-driven, clonally expanded B cells were present in both the CLNs and the CNS.

"But there's not a bona fide system within the CNS for B cells to do that," Kevin O'Connor, Ph.D., an assistant professor of neurology, a member of the Human and Translational Immunology Program at Yale, and the senior author of the paper by Stern et al., told MSDF. The researchers therefore looked in CLNs as the closest place for maturation to happen, "and that's where we found they are maturing," he said.

The researchers found clonally expanded B cells in both locations, as indicated by highly mutated sequences of immunoglobulin heavy chains. However, about 90% of the founding members of the clones were detected more often in the draining CLNs, as indicated by less clonal diversity in that compartment, suggesting that initial antigen-dependent B cell activation and maturation occur mainly in the periphery. This concept is in line with previous findings of neuronally derived antigens in draining lymph nodes in animal models and in CLNs of MS patients (de Vos et al., 2002).

Stern and co-workers saw more mature clones in both the CLNs and in the CNS, including in MS lesions. The B cells in CNS lesions were characteristic of postgerminal center reactions, being class-switched with acquired somatic mutations and expanded clonality. "So we suggest that the initial activation is occurring in the cervical lymph nodes, then the cells are migrating to the central nervous system, and then, again, our data suggest that they travel back and forth," O'Connor concluded.

“The model proposes that B cell maturation is not confined to the MS CNS but occurs in both the periphery and CNS and further proposes that antigen-driven maturation originates in the periphery,” the authors stated. O'Connor noted, however, that the model does not exclude the possibility that some initial B cell activation events could occur in the CNS.

CSF B cells activated in the periphery

In the second paper, by Palanichamy et al. (2014), researchers used flow cytometric sorting of peripheral blood B cells based on surface markers to determine which subsets in the periphery share immunoglobulin heavy-chain variable regions with B cells in the cerebrospinal fluid (CSF) from the same patients. Samples were obtained from eight patients with clinically definitive, untreated MS.

By deep sequencing of variable region genes of immunoglobulin heavy chains in the CSF and in the peripheral blood, Palanichamy et al. detected a population of B cells in the blood using the same immunoglobulin genes and complementarity-determining region sequences corresponding to B cells in the CSF.

They found that the greatest degree of sequence overlap was between CSF B cells and peripheral blood class-switched memory B cells and plasma cells, with a total of 46 bicompartmental clusters of clonally related immunoglobulin heavy-chain variable regions in six of the eight patients. Very few (<1%) of the total number of peripheral blood B cell clusters were shared with the CSF. The authors noted that “no connections were identified between CSF and naïve or unswitched memory B cells in PB [peripheral blood].”

Just as Stern and colleagues found, Palanichamy and co-workers, too, suggested that immunoglobulin class-switched B cells form an antigen-experienced immune axis between the periphery and the CNS in MS. B cells undergo somatic hypermutation in both the periphery and in the CSF. Thus, there are probably disease-driving antigens in the periphery, and in addition, intrathecal tissues may support affinity maturation of B cell receptors.

Hans-Christian von Büdingen, M.D., an assistant professor of neurology at the University of California, San Francisco, and senior author of the Palanichamy paper, told MSDF that the finding of class-switched B cells connecting the periphery and the CSF is important because it clearly indicates that MS is not an autoimmune disease sequestered to the CNS.

“It is clearly a disease that is also driven by the peripheral immune system,” he said. “The ideal outcome at some point will be that we will be able to clearly identify, track, and hopefully also then destroy the culprit B cells in multiple sclerosis.”

In an accompanying editorial (Lu and Robinson, 2014), Ph.D. student Daniel Lu and William Robinson, M.D., Ph.D., both of the Stanford University School of Medicine, wrote that clinical studies have shown that B cell–depleting therapies and therapies that block immune cell trafficking across the BBB reduce MS disease activity. What has not been known are the sites of B cell activation, affinity maturation, and antigen-dependent selection that produce B cells populating the CNS.

But now the findings by Stern et al. of antigen-experienced B cells maturing in peripheral lymph nodes and their bidirectional migration into and out of the CNS provides “insight into the trafficking of the presumed pathogenic B cells in MS and a framework by which peripheral deletion or modulation of specific B cell subsets could provide therapeutic benefit,” Lu and Robinson wrote.

B cell activation and affinity maturation appear to occur largely in the periphery, but the editorialists note that affinity maturation may also occur in CNS tertiary lymphoid structures. Taken together with the bidirectional exchange of B cells found by both research groups, these findings provide a mechanistic basis for the efficacy in MS of therapies that limit B cell transport. First, the cells may be prevented from reaching the CNS at all. Furthermore, interrupting the iterative process of trafficking back and forth between the CNS and periphery may moderate affinity maturation between the compartments.

Because affinity-matured B cells provide the continuum between the peripheral lymphoid tissue and the CNS, and because their formation requires prior exposure to antigen and maturation in germinal centers, treatments that reduce the number and magnitude of these events may avert the development of clinical MS in at-risk individuals, Lu and Robinson proposed.

“Important frontiers”

Amit Bar-Or, M.D., an associate professor of neurology at McGill University and scientific director of the Clinical Research Unit at the Montreal Neurological Institute in Canada, who was not involved with either study, told MSDF that both B and T cell trafficking and the crosstalk that occurs between cells in different compartments “are very important frontiers.”

Any work shedding light on these mechanisms “is likely to give us insights that ultimately will be important in recognizing that the predominant processes contributing to injury in MS likely change over time. They may coexist and overlap for the most part, but the predominant ones, where you get the biggest bang for the buck of therapy, may be different in the course of a treatment potentially in an individual.”

Molecular biology techniques can now pick up very subtle changes in B cell repertoires, and comparing B cell profiles “is allowing people to start talking about the relationships of particular B cells and their history in the different compartments and hence make inferences about the dynamics between the compartments,” Bar-Or said.

Bar-Or says that although overactivation or insufficient regulation of immune cells in the periphery is thought to contribute to CNS injury by lymphocyte migration across the BBB, “we also recognize that in MS there is an injury that can percolate along and continue to progress relentlessly even when we do a pretty good job at limiting the ability of the peripheral cell activation and trafficking into the CNS. This is what one would refer to as the biology of progressive MS.”

He postulates that one possibility is that progressive MS reflects the presence of immune cells that “set up shop within the central nervous system” and then act as part of intra-CNS compartmentalized processes involving inflammation and possibly degeneration, or likely, a combination of the two.

Key open questions

• Where does the initial antigen exposure of naïve B cells that then mature to become memory B cells and plasma cells in both the CNS and the periphery occur: in the periphery or in the CNS?
• What is/are the antigens to which naïve B cells become activated?
• Do the antigens originate in the CNS, and if so and B cells become activated in the periphery, how do these antigens reach the peripheral lymphoid tissues?
• May there be other antigens (e.g., viral or bacterial) in the periphery that mimic CNS antigens and raise an immune response to them?
• Do particular antibody repertoires correlate with the diverse array of clinical features of MS?
• Can therapies be developed that target particular antibody specificities, repertoires, or heavy-chain variable region families if they are found to be over-represented or pathogenic in MS?

Disclosures and sources of funding

Kevin O'Connor, Ph.D., has received speaking fees from EMD Serono. William Robinson, M.D., Ph.D., owns equity in, is a consultant to, and is a member of the board of directors of Atreca. Hans-Christian von Büdingen, M.D., has received research funding from Pfizer Inc. and F. Hoffmann-La Roche and consulting fees from Novartis. Amit Bar-Or, M.D., has participated as a speaker at meetings sponsored by and has received consulting fees and/or grant support from Biogen Idec, Diogenix, Genentech, GSK, EMD Serono, Novartis, Ono Pharma, Sanofi/Genzyme, Receptos, Roche, and Teva Neuroscience. He is also a scientific advisory board member of the Accelerated Cure Project, the parent organization of MSDF.