The Choroid Plexus Is Permissive for a Preactivated Antigen-Experienced Memory B-Cell Subset in Multiple Sclerosis.

The role of B cells in multiple sclerosis (MS) is increasingly recognized. B cells undergo compartmentalized redistribution in blood and cerebrospinal fluid (CSF) during active MS, whereby memory B cells accumulate in the CSF. While B-cell trafficking across the blood-brain barrier has been intensely investigated, cellular diapedesis through the blood-CSF barrier (BCSFB) is incompletely understood. To investigate how B cells interact with the choroid plexus to transmigrate into the CSF we isolated circulating B cells from healthy donors (HC) and MS patients, utilized an inverted cell culture filter system of human choroid plexus papilloma (HIBCPP) cells to determine transmigration rates of B-cell subsets, immunofluorescence, and electron microscopy to analyze migration routes, and qRT-PCR to determine cytokines/chemokines mediating B-cell diapedesis. We also screened the transcriptome of intrathecal B cells from MS patients. We found, that spontaneous transmigration of HC- and MS-derived B cells was scant, yet increased significantly in response to B-cell specific chemokines CXCL-12/CXCL-13, was further boosted upon pre-activation and occurred via paracellular and transcellular pathways. Migrating cells exhibited upregulation of several genes involved in B-cell activation/migration and enhanced expression of chemokine receptors CXCR4/CXCR5, and were predominantly of isotype class switched memory phenotype. This antigen-experienced migratory subset displayed more pronounced chemotactic activities in MS than in HC and was retrieved in intrathecal B cells from patients with active MS. Trafficking of class-switched memory B cells was downscaled in a small cohort of natalizumab-exposed MS patients and the proportions of these phenotypes were reduced in peripheral blood yet were enriched intrathecally in patients who experienced recurrence of disease activity after withdrawal of natalizumab. Our findings highlight the relevance of the BCSFB as important gate for the entry of potentially harmful activated B cells into the CSF.

Alemtuzumab in Multiple Sclerosis: Short- and Long-Term Effects of Immunodepletion on the Peripheral Treg Compartment.

Treatment with alemtuzumab is followed by an early increase in Treg frequencies. Whether naïve and memory subsets are differentially affected and how depletion influences dysfunctional MS-Treg is unclear. In this study, we analyzed the effect of alemtuzumab on regulatory T-cells (Treg) in patients with multiple sclerosis (MS). For this purpose 182 blood samples from 25 MS patients were taken shortly before treatment and serially for up to 24 months after two alemtuzumab cycles. We studied Treg by flow cytometry (quantitation, phenotypical characterization), real-time polymerase chain reaction (T-cell receptor (TCR) excision circles [TREC] content), CDR3-spectratyping (clonal distribution), and proliferation assays (suppressive function). CD52-mediated cytolysis of Treg and conventional T-cells was determined by a complement-dependent cytolysis assay. Our studies revealed that 1 week post-alemtuzumab, Treg were depicted at constant frequencies among CD4+ T-cells. In contrast, Treg frequencies were massively increased at month 1. Post-depletional Treg exhibited a CD45RO+ memory phenotype, a skewed TCR repertoire, and contained minimum TREC numbers. Naïve Treg, thymic markers, and TCR-variability commenced to rise after 6 months but did not attain baseline levels. In vitro, Treg exhibited higher susceptibility to lysis than Tcon. Treg suppressive function constantly increased within 1 year when co-cultured with syngeneic T-cells, but remained stable against allogeneic T-cells from normal donors. Our findings suggest that (1) Treg are not spared from alemtuzumab-mediated depletion and thymopoiesis does not considerably contribute to long-term recovery, (2) either homeostatic proliferation and/or conversion from residual Tcon contributes to Treg expansion during the early post-treatment phase (3) the enhanced inhibitory effect of Treg following alemtuzumab is due to altered composition and reactivity of post-depletional Tcon.