The family of B cell subsets results from an evolutionary process of embryonic cells expressing different surface markers (especially CD19, CD20, and CD38) through their lifespan, in different organs, until they reach the state of antibody secreting cells (ASC), thus culminating the evolution process with the presence of effector B cells. ⁶ The change of stage from membrane-linked antibody cell to ASC represents the terminal differentiation toward B cells that do not proliferate ^{7, 8} ( Figure 1). The B cell lineage begins in fetal life from pluripotent hematopoietic stem cells (SC), located in the fetal liver and in the postnatal bone marrow (BM). Henceforth, they evolve into multipotent myeloid/lymphoid progenitors (MPP), which continue their evolution towards the common lymphoid progenitors (CLP). ⁶ CLP from the BM evolve to a pro-B state in which they express the CD19 marker and then transform into pre-B cells; those, in addition to expressing CD19, begin to express CD20, and later progress to immature B cells that express IgM as a surface marker. ⁶ While transiting from the BM to the secondary lymphoid organs (SLO), the B cells express the B cell receptor (BCR) surface markers IgM and IgD, thus evolving into transitional B cells; this step requires a checkpoint that entails clonal deletion and receptor editing before entering the SLO (spleen, lymphoid node, tonsils, and mucosa-associated lymphoid tissue [MALT]) where they become mature naïve B cells. ⁶ The mature naïve B cells, at this point, can have three possible destinations: a) they enter the marginal zone of the spleen where they may become short-lived plasma cells (SLPC) that produce IgM and rapidly enter apoptosis (since these are B cells involved in rapid and transitory defense); b) move to the intestine and the pulmonary epithelium (B1 cells); c) migrate to splenic follicles and lymphoid nodules, becoming follicular B cells. ^{9, 10} Naïve B cells that carry the BCR IgD go through early class switch recombination (CSR) in the extrafollicular zone, with support from T cells, then enter the germinal center (GC) where they undergo somatic hypermutation (SHM), after which they express BCR IgG. ¹¹ Subsequently, the resulting memory B cells, PB and PC will have the ability to secret high-affinity antibodies for decades, or for the lifespan of an individual, and most of them will be able to migrate to the bone marrow to establish as long-lived plasma cells (LLPC). ^{11, 12, 13} Dysregulation of the GC has been associated with autoimmune disease. ¹⁴ Evidence suggests that the origin of B cell autoreactivity occurs in the GC due to dysfunction of thymus-derived follicular T helper cells and follicular regulatory T cells. ¹⁵ PB may develop from any type of activated B cell (including naïve, marginal zone, follicular, and memory B cells), but it is not clear if PB that originated from these cells (except for memory B cells) are competent to mature to LLPC. ⁶ PB will carry the CD19 ⁺CD20 ^-CD27 ⁺⁺CD38 ⁺⁺IgG ^+/- markers, and will express the chemokine receptor CXCR4, which will help them get attracted to the chemokine CXCL12 in the BM niches. As an alternative, PB expressing the receptor CXCR3 will become LLPC in the spleen and lymph nodes (assisted by the chemokine CXCL12) or in inflamed tissue (assisted by the chemokines CXCL9-CXCL12), and subsequently undergo apoptosis upon resolution of inflammation. ⁷ Hereafter, memory B cells access the CNS through the disrupted blood brain barrier (BBB). They are identified in perivascular spaces, demyelinating lesions in the brain and spinal cord, and disperse in the meninges where they can form aggregates known as tertiary lymphoid organs (TLO). These TLO emulate GC function, supporting the formation and persistence of cortical lesions. ^{16, 17, 18, 19} In addition, they are a local source of class-switch IgG that contribute to the immune process and are subsequently determined as OCB in the CSF of patients with MS. ²⁰ In the meninges, the inflammatory infiltrates are composed of CD3 ⁺ T cells, CD20 ⁺ B lymphocytes and PC. ¹⁶ In the white matter lesions, the inflammatory infiltrates are localized in the perivascular spaces containing T and B lymphocytes and PC. ¹⁶ In the diffuse infiltrates and normal-appearing white matter, CD8 ⁺ T lymphocytes predominate almost exclusively. ¹⁶ The presence of PB in CSF has been reported. ^{21, 22} Despite the knowledge accumulated to this date, the complete understanding of the evolution of the B cell lineage is still in progress. Activated lymphocytes are able to access the CNS, both in health and disease, through the BBB, the blood meningeal barrier, and the blood-CSF barrier. ¹² In normal conditions the amount of B cells that access the CNS is very low. ²³ These cells primarily exit the CNS via lymphatic drainage through nasal blood vessels, or via meningeal lymphoid vessels to the lymphoid cervical nodules. ²⁴

Naïve and memory B cells are crucial within the B cell lineage and they have been shown to negatively correlate in their function: increased memory B cells and decreased naïve B cells have been linked with a worsening of the disease, and vice-versa. ⁵ In fact, when the presence of memory B cells induce the auto-proliferation of CD4 T cells, which tend to home in the brain, naïve B cells are decreased. ⁵ Memory B cells can be heterogeneous, i.e., originate from different cells or express different phenotypes, including class-switched (CD19 ⁺CD27 ⁺IgM ^-IgD ^-) and class-unswitched (CD19 ⁺CD27 ⁺IgM ⁺IgD ^-). ²⁵ Inhibition of memory B cells prevents relapsing MS. ²⁵ In a study in naïve patients with relapsing remitting MS (RRMS), the interaction between T and B cells was documented, highlighting that B cells act as antigen presenting cells (APC) to auto proliferating CD4 ⁺ T cells. ⁵ Increased amounts of T cell co-stimulatory proteins and major histocompatibility complex (MHC) class II molecules are expressed by B cells in the periphery (blood and SLO), and in the CSF and CNS of patients with MS. ¹² Patients in remission carrying the HLA-DR15 ⁺ marker showed an increase in auto-proliferative B and T cells and a decrease in naïve B cells. ⁵ On the other hand, treatment with anti-CD20 was associated with a decrease in auto-proliferative memory T and B cells and increased presence of naïve B cells. ⁵

Within the B cell lineage, the regulatory B cells subset (Bregs) stands out, since there is still no agreement on its origin and classification. Bregs are not a specific subtype of B cells, but represent a regulatory functional state resulting from inflammation. ²⁶ Although it has been assumed that interleukin 10 (IL10) is the hallmark of Bregs, other factors such as IL35, transforming growth factor (TGF) β, and direct cell-to-cell contact are also mechanisms of Bregs function. ²⁶ Immature B cells, mature B cells, PB and PC are believed to function as Bregs. ²⁶ The Bregs can express the following markers: IL10, CD27, CD5, CD25, CD86, CD24 and CD28. ²⁷ Based on the production of IL10, three important subtypes of regulatory Bregs have been identified, including the transitional (CD19 ⁺CD24 ^highCD38 ^high), naïve (CD19 ⁺CD24 ⁺CD38 ⁺), and memory (CD19 ⁺CD24 ^highCD38 ^-) subtypes, among which the transitional cells are the main producers of IL10. ²⁸ Transitional B cells are capable of suppressing differentiation of naïve T cells into Th1 and Th17, which are dependent on the co-stimulatory molecules CD80 and CD86. ²⁹ Survival of Bregs is linked to the B cell activating factor (BAFF) and to a proliferation inducing ligand (APRIL). ²⁰ Under normal conditions, the population of Bregs is kept low in order to maintain immune homeostasis. ²⁶ In newborns, 50% of umbilical cord blood B cells correspond to the transitional B cell subtype whereas, in adults, it represents only 4% of the cell population in peripheral blood. ²⁸ It is estimated that, in human peripheral blood, the Bregs subtype represents only 1-2% of all B cells. ²⁹ In an experimental allergic encephalitis (EAE) animal model, it was documented that IL10 contributed to the reduction of the inflammatory response mediated by microglia and astrocytes in the CNS. ³⁰ Bregs transfer reversed the increase in Th1 and Th17 cells in an arthritis model lacking IL10. ³¹ In mice with low expression of the IL35 subunit p35, or EBi3 in B cells, an inability to recover from EAE was detected, with evidence of activation of macrophages and inflammatory T cells, and an increased activity of B cells such as APC. ³² IL10 regulates the differentiation of the lineage from IL10-secreting B cells to PB that secrete IgG or IgM. ³³ In a post-mortem analysis of MS patients with high levels of meningeal inflammation and cortical demyelination, Magliozzi et al., reported an increase in IL10 expression, among other proinflammatory cytokines and molecules related to B cell activity and lymphogenesis in meninges and CSF. Additionally, an increase in IL10 was found in the CSF of MS patients with high cortical involvement at the time of diagnosis. ³⁴ Early development of MS in individuals with the clinically isolated syndrome (CIS), or radiologically isolated syndrome (RIS), seems to correlate with a reduced production of IL10 by B cells. ³⁵ In a cohort of MS patients followed for 10 years, Farian et al. found that patients with positive OCB at diagnosis advanced, more frequently and earlier in the course of the disease, to a progressive phase. ³⁶ This can be explained by the presence of a greater intrathecal inflammatory component that causes greater cortical involvement. ³⁶ Besides, the authors reported an over-expression of inflammatory molecules, including IL10. ³⁶ PB and PC inside the MS lesions presented a high IL10 expression. ³⁷

Another subset of CD19 ⁺ B cells that could be relevant to MS pathogenesis are the double negative (DN) B cells (IgD ^-CD27 ^-) and the CD21 ^low cells, which have been associated with aging and autoreactivity. ³⁸ These cells are believed to develop outside the GC, are independent from T-cells, and display a pro-inflammatory cytokine profile. ³⁸ These cells have been found in healthy subjects, and have also been found at higher levels in the CSF of MS patients younger than 60 years when they were compared to age-matched healthy donors (DN B cells 19.5% against 3.03%, and CD21 ^low 21.95% against 6.06%, respectively). ³⁸ Most DN B cells display an IgG ⁺ phenotype while CD21 ^low B cells originate from a heterogeneous population that includes CD27 ^- naïve, CD27 ⁺ memory, and IgG ⁺ and IgM ⁺ B cells. ³⁸ Both DN and CD21 ^low B cell frequencies were higher in the CSF compared to blood levels for these patients. ³⁸ Fraussen et al. have suggested that the DN B cells may have multiple origins, considering IgG ⁺ cells better linked to the class-switched memory B cells, while IgM ⁺ cells share more similarity with the naïve and the non-class-switched IgD ⁺CD27 ⁺ memory B cells. ³⁹

Inflammation of the CNS is reflected in the presence of B cells in the CSF. ²¹ Cepok et al. evaluated the B cell subtype in CSF in MS patients, finding that the majority of detectable cells were memory B cells (CD19 ⁺CD27 ⁺), whereas a minority were naïve B cells (CD19 ⁺CD27 ^-); those were different from naïve B cells that predominated in peripheral blood. ²¹ In addition, they detected PB (CD19 ⁺ CD27 ⁺⁺CD138 ⁺CD38 ⁺) subtypes representing between 30-50% of cells in CSF, and were present in the course of the disease, without correlation with the level of PB present in peripheral blood, while short lived PB (CD19 ⁺CD27 ⁺⁺CD138 ⁺CD38 ⁺⁺HLADR ⁺⁺) and PC (CD19 ^+/- CD27 ⁺⁺CD138 ⁺CD38 ⁺HLADR ^-) were absent from CSF. ²¹ In contrast, Corcione et al. reported the predominance of both memory B cells and PC in the CSF of MS patients without treatment. ¹⁸ In patients with RRMS and primary progressive MS (PPMS) with positive B cells for G1m1 (IgG1 heavy chain gene), Lossius et al. detected IgG1 ASC with a phenotype compatible with highly proliferating PB (CD19 ^dimCD27 ^hiCD38 ⁺) and with high expression of CD138 ⁺, HLA-DR ⁺ and KI67 ⁺ in CSF. ⁴⁰ In pediatric MS, an increase in memory B cells in CSF, with a predominance of non-switched memory B cells and PB, was found, while in adults with MS, class-switched memory B cells and PC predominated in CSF during relapses of MS. ^{25, 41} Using a deep repertoire sequencing of IgG heavy chain variable genes (IgG-Vh) in paired CSF and peripheral blood from patients with MS, VonBudinghen et al. found that there was a cluster of clonally related B cells involved in a bidirectional cell exchange across the BBB, with some of them being present primarily in the CNS while others were present in the periphery or in both compartments. ⁴² Additionally, using the same protocol, they found evidence of clonally related B cell receptors in a patient’s blood and CSF, after seven years of therapy with rituximab, indicating a prolonged presence in this compartment during the disease span due to recirculating memory B cells or LLPC. ⁴³ Greenfield et al. found that clonally related B cells were present as class-switched IgG and CD27 ⁺ in the CSF of patients with MS, leading to the conclusion that, despite having been on DMT, there were complex patterns of persistence of clonal B cells in CSF and blood. ²² A significant depletion of CD20 ⁺ B cells has been detected in the blood, CSF and perivascular spaces in the CNS after therapy with rituximab and ocrelizumab. ^{44, 45} Table 1 presents a summary of the different surface markers that characterize the B cell lineage through its lifespan.

Advances in immunotherapy have made it possible to limit the presence and expansion of B cells, thus reducing relapses and the progression of disability. However, the determination of which cells from the lineage could be responsible for clinical deterioration, or improvement, is still to be investigated. In the treatment of other autoimmune diseases, such as pemphigus, the mapping of cell markers has been used to evaluate the response to treatment, finding alterations in the function of CD19 ⁺CD24 ^hiCD38 ^hi Bregs cells, which are present in significantly higher numbers in patients in an active state compared to patients in the remitting state of RRMS. ⁴⁶ Late antibody-mediated rejection continues to be a problem for patients undergoing kidney transplantation and, for many years, it was believed that tolerance and rejection of transplantation were mediated by T cells. ⁴⁷ However, it was recently shown that a population of Bregs may be playing a deleterious role in transplant immunity, and be responsible for the production of alloantibodies. ^{48, 49, 50} Recently, B cells (CD19 ⁺CD24 ^hiCD38 ^hi) dysfunction has been reported in peripheral blood, with decreased production of IL10 in patients with RRMS compared to healthy subjects. ²⁸ In turn, these cells have a less stimulatory effect on naïve CD4 ⁺ T cells, which produce IFNγ and tumor necrosis factor α (TNFα). ²⁸ Additionally, anti-CD19 monoclonal antibody has no effect on Bregs, which are regulators of EAE extension/expression through the secretion of immunosuppressive cytokines. ⁵¹ In addition, Chen et al. determined the presence of autoreactive CD19 ⁺CD20 ^- plasma cells in the CSF of patients with RRMS (20.18%), SPMS (29.58%) and PPMS (31.73%), including patients exposed to DMT. ⁵¹

a.

Interferon B (IFNβ) acts in the periphery, inducing apoptosis of CD27 ⁺ memory B cells through a mechanism that requires FAS receptor/transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) signaling, leading to a specific depletion of these memory B cells (which carry the ability to harbor EBV) and an increase in the CD27 ^- cell subtype that contains naïve B cells secreting IL10. ⁵² Furthermore, it was observed that memory B cell depletion was accompanied by a reduction in EBV markers. ⁵² IFNβ leads to the inhibition of leukocyte proliferation and antigen presentation. ⁵³ It also changes the cytokine profile towards an anti-inflammatory profile in both peripheral blood and the CNS, and reduces T cell migration by inhibiting the activity of the T cell matrix proteinase. ⁵³ IFNβ increases naïve B cells and decreases memory B cells in peripheral blood. ⁵⁴ Ersoy et al. showed that IFNβ induces the production of high amounts of IL10 compared to therapy with azathioprine in patients with RRMS. ⁵⁵ A meta-analysis study in patients with MS who received IFNβ showed that there was a lower proportion of Th17 cells in the peripheral CD4 ⁺ T cell pool and a reduction of IL17 and IL23 levels in serum. ⁵⁶

Burton tyrosine kinase (BTK) inhibitors appear promising as potential therapeutic agents, since evobrutinib has previously been shown to prevent the activation of B cells and improves the clinical course in EAE. ⁸⁹