4.2.1. Marginal Zone B Cells

Marginal zone (MZ) B cells are a class of innate-like lymphocytes positioned in the marginal zone of the spleen, inhabiting a junction between the circulation and lymphoid follicle [178–180]. While some differences exist between human and mouse MZ B development and function, in both organisms the positioning of MZ B cells within the spleen allows them to act as antibody generating first responders against pathogens in the blood [178,181]. While MZ B cells occupy a critical niche between innate and adaptive immunity, MZ B cells expansion has been associated with autoimmunity and previous studies have determined that MZ B cells possess polyreactive BCRs that can be potentially be self-reactive [178,182–185]. Among the most serious complications of SS is the increased incidence

of B cell lymphoma [3]. Investigation into the types of non-Hodgkin Lymphoma (NHL) tumors within a cohort of 58 pSS patients identified that the two most commonly occurring types of tumors were indolent extranodal MZ B-cell lymphoma of the mucosal associated lymphoid tissue (MALT) at 59%, and nodal MZ lymphoma at 15%, indicating the importance MZ B cells within the disease [186]. MZ-like B cells have been found to be increased in the SGs and peripheral blood of pSS patients [187].

Investigations in SS mouse models have provided additional clues regarding the role of MZ B cells in the disease process. Mice transgenic (tg) for BAFF are known to develop autoimmune symptoms similar to SLE and SS [188,189]. In order to study the role of MZ B cells in this model, Fletcher et al. generated BAFF tg mice lacking lymphotoxin-β (LTβ), as mice lacking LTβ will fail to develop MZ B cells [190]. The authors discovered that while the mice still developed nephritis, they did not develop severe sialadenitis associated with the SS phenotype, suggesting a critical role played by MZ B cells in this aspect of disease [190]. A separate study investigated the role of MZ B cells within the IL-14α transgenic mouse model (IL14αtg) [191]. Elimination of MZ B cells within the IL14atg model restored saliva flow rate, removed lymphocytic infiltrations into the SGs, and prevented formation of autoantibodies [191]. Considering that depletion of MZ B cells improves disease in two separate SS mouse models, targeted depletion of MZ B cells has attracted attention as a potential therapy. Ly9 (CD229) is a cell surface receptor highly expressed on MZ B cells, and anti-Ly9 has been demonstrated to selectively target and deplete MZ B cells [192]. Treatment of NOD.H-2h4 SS model mouse with anti-Ly9 reduced both SG and renal infiltration, and also decreased ANAs and RF [193]. Considering the overwhelming involvement of MZ B cells in NHLs of SS patients, anti-Ly9 antibodies likely represent the first of many strategies to target MZ B cells for depletion in SS.

## 4.2.2. Memory B Cells and Plasma B Cells

Memory B cells are the important cell type that is involved in SS pathogenesis due to their ability to maintain memory for a given antigen in the absence of constant antigen stimulation [194,195]. They are characterized by CD27<sup>+</sup> expression and BCR somatic hypermutation [196,197]. SS shows an increase in accumulation of CD27<sup>+</sup> memory B cells and plasma cells within the SGs infiltrate and the peripheral blood of patients. The glands show a distinct cytokine profile that includes adhesion molecules, cytokines, and B-cell chemokines CXCL13 and CXCL12 [88,90]. CXCL13 is the key cytokine responsible for the homing of B cells to the SGs. GC formation in the SGs is facilitated by CXCL13 secreted by Tfh and fDCs. Interaction with its corresponding receptor CXCR5 on B-cells regulates B-cell movement between different tissues. In the SGs, CXCL13 guides B cell entry into the follicles that causes lymphoid organization visualized in SG biopsies of SS [198–200]. CXCL13 over-expression in inflamed glands of patients with pSS plays the primary role in the recruitment of circulatory CXCR5 expressing CD27<sup>+</sup> memory B cells, attracting the subpopulation of peripheral CD27<sup>+</sup> memory B cells into the inflamed glands where they then reside [88,90].

Plasma B cells are terminally differentiated cells of the B lymphocyte lineage, the primary cell type that produces antibodies, and thus are drivers of antibody-mediated immunity [201]. They are maintained for extended periods, making them an essential component of immune memory and thus linking them closely to memory B cells from which they can differentiate [202]. BAFF, a member of the TNF family, is secreted by inflammatory cells and is needed for prolonged plasma cell survival and sustained Ig production by plasma B cells [203]. The glandular microenvironment is generally rich in BAFF, promoting accumulation of CD27<sup>+</sup> B memory cells resulting in more IgG producing plasma cells in the tissue [203–205]. BAFF has a role in B cell maturation, class switching, survival, and proliferation especially in advanced disease and is produced by SGEC, DC, macrophages, activated T cells, and also B cells [204]. Mice transgenic for BAFF possess increased numbers of mature B and effector T cells, and develop high levels of circulating immune complexes, glandular Ig deposition, and anti-DNA antibodies [189]. The patients of pSS have auto-reactive plasma B cell infiltrates that produce anti-SSA/Ro or anti-SSB/La autoantibodies. These infiltrates appear from the differentiation of CD27<sup>+</sup> memory B cells recruited from circulating blood and from B cells generated in ectopic SG GCs. Other notable cytokines include CXCR3 and CXCR4, whose expression on activated B cells leads to the migration of the plasma cells to the site of inflammation and causes the attraction of lymphocytic cells to the SGs [206–211].

B-cell-targeted mAbs (see below), largely rely on two mAb Fc-dependent mechanisms: antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Rituximab, a mouse/human chimeric IgG1 mAb, was the first B-cell targeting therapeutic antibody approved by the US Food and Drug Administration [212,213]. Ocrelizumab a second generation CD20 mAb(rhumAb 2H7v.16) is a humanized CD20 mAb, which binds a different but overlapping epitope from rituximab [214]. Furthermore B-cell survival, differentiation, and functional properties are tightly regulated by a variety of cytokines and chemokines. Targeting survival and differentiation factors with specific mAbs or fusion proteins is an alternative approach to targeting B-cell surface antigens for active cell depletion [215].
