In multiple sclerosis (MS4) B cell depleting therapy using monoclonal anti-CD20 antibodies, including rituximab (RTX) and ocrelizumab (OCR), effectively reduces disease activity. Based on indirect evidence, it is generally believed that elimination of the antigen presenting capabilities and antigen non-specific immune functions of B cells underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T cells) was shown to also express CD20, but controversy prevails surrounding the true existence of this T cell subpopulation. Using single-cell imaging flow cytometry and expression profiling of sorted lymphocyte subsets, we unequivocally demonstrate the existence of CD3+CD20dim T cells. We show that in MS patients increased levels of CD3+CD20dim T cells are effectively depleted by RTX. The pathological relevance of this T cell subset in MS remains to be determined. However, given their potential pro-inflammatory functionality, depletion of CD20-expressing T cells may also contribute to the therapeutic effect of RTX and other monoclonal antibodies targeting CD20.

Transitional cells represent a crucial step in the differentiation and selection of the mature B cell compartment. Human transitional B cells have previously been variably identified based on the high level of expression of CD10, CD24, and CD38 relative to mature B cell populations and are expanded in the peripheral blood following rituximab-induced B cell-depletion at reconstitution. In this study, we take advantage of the gradual acquisition of the ABCB1 transporter during B cell maturation to delineate refined subsets of transitional B cells, including a late transitional B cell subset with a phenotype intermediate between T2 and mature naive. This late transitional subset appears temporally following the T1 and T2 populations in the peripheral compartment after rituximab-induced B cell reconstitution (and is thus termed T3) and is more abundant in normal peripheral blood than T1 and T2 cells. The identity of this subset as a developmental intermediate between early transitional and mature naive B cells was further supported by its ability to differentiate to naive during in vitro culture. Later transitional B cells, including T2 and T3, are found at comparatively increased frequencies in cord blood and spleen but were relatively rare in bone marrow. Additional studies demonstrate that transitional B cells mature across a developmental continuum with gradual up-regulation of mature markers, concomitant loss of immature markers, and increased responsiveness to BCR cross-linking in terms of proliferation, calcium flux, and survival. The characterization of multiple transitional B cell subpopulations provides important insights into human B cell development.

Objective. Transient B cell depletion with the monoclonal anti-CD20 antibody rituximab has resulted in favorable clinical responses in patients with rheumatoid arthritis (RA). However, little is known about the regeneration profile of different peripheral B cell subpopulations. The aim of this study was to delineate the regeneration profile of different B cell subsets in the peripheral blood after selective anti-CD20-mediated B cell depletion.Methods. Seventeen patients with RA refractory to standard therapy were treated with rituximab. Patients 1-6 received 4 weekly infusions of rituximab at a dose of 375 mg/m 2 , and patients 7-17 received 2 infusions of rituximab (1,000 mg), 2 weeks apart. Four-color staining was performed at several time points, using CD38, IgD, and CD27 in addition to other cell surface markers. In one patient, the mutational status of the immunoglobulin receptor was examined.Results

In multiple sclerosis (MS), an exchange of lymphocytes, in particular B cells, between the central nervous system (CNS) and periphery is believed to be required for the maintenance of active disease. Therapeutic monoclonal antibodies that prevent lymphocytes from crossing the blood-brain barrier (BBB) or induce near-complete peripheral B cell depletion rapidly mitigate MS disease activity. Using next-generation sequencing technology, we recently found that clonally related B cells exist in the cerebrospinal fluid (CSF) and peripheral blood (PB) of MS patients, establishing the existence of an immune axis across the BBB. However, it remains unclear which subpopulations of the highly diverse peripheral B cell compartment share antigen-specificity with intrathecal B cell repertoires, and whether their antigen stimulation occurs on both sides of the BBB. To address these questions, we combined flow cytometry sorting of PB B cell subsets with deep immune repertoire sequencing of CSF and PB B cells. Immunoglobulin (IgM and IgG) heavy chain variable (VH) region repertoires of five PB B cell subsets from MS patients (n=8) were compared with their CSF Ig-VH transcriptomes. In 6 of 8 patients, we identified peripheral CD27+IgD−memory B cells, CD27hiCD38hi plasma cells/plasmablasts, or CD27−IgD− B cells providing an immune connection to the CNS compartment. Pinpointing Ig class-switched B cells as key component of the immune axis thought to contribute to ongoing MS disease activity strengthens the rationale of current therapeutic strategies and may lead to more targeted approaches.

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.