Inhibitory B cell co-receptors and autoimmune diseases

Abstract B cells express various inhibitory co-receptors including CD22 (also known as Siglec-2), Siglec-10 (Siglec-G in mice), CD72, LILRB (PIR-B in mice) and FcγRIIB that contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and negatively regulate BCR signaling by recruiting phosphatases to the ITIMs. Some of the inhibitory B cell co-receptors suppress development of SLE. Among these, CD72 most strongly regulates SLE. CD72 recognizes Sm/RNP, a lupus self-antigen and an endogenous TLR7 ligand, as a specific ligand, and suppresses B cell response to this TLR7 ligand. This suppression may inhibit development of SLE because TLR7 is indispensable in multiple mouse SLE models. In contrast, inhibitory B cell co-receptors such as CD22 and CD72 inhibit expansion of regulatory B cells that are known to regulate development of autoimmune diseases including type 1 diabetes (T1D) and multiple sclerosis. CD72 strongly exacerbate development of T1D in NOD mice probably by limiting expansion of regulatory B cells. Thus, inhibitory B cell co-receptors especially CD72 regulates distinct autoimmune diseases either positively or negatively. As B cell depletion therapy clearly reveals crucial roles of B cells in the regulation of various autoimmune diseases, CD72 may be a novel therapeutic target for treatment of autoimmune diseases.


Introduction
Studies in the past two decades markedly advanced our understanding of the mechanisms for development of autoimmune diseases. As a consequence, it is well established that B cells play crucial roles in regulation of various autoimmune diseases. First, B cell depletion by anti-CD20 antibody ameliorates various autoimmune diseases including those traditionally viewed as T cell-mediated diseases such as type 1 diabetes (T1D) and multiple sclerosis [1][2][3][4]. Second, a large number of disease-associated genes have been identified by genome-wide association studies (GWAS). Analysis of SLE-associated genes are accumulated in genes expressed in B cells [5], suggesting that functional defects in B cells play an important role in development of SLE probably by abrogating tolerance of self-reactive B cells. Third, accumulating evidence suggests pathogenic roles of autoantibodies specifically associated with a disease or a clinical symptom such as anticitrullinated protein antibodies in rheumatoid arthritis and antineutrophil cytoplasmic antibodies in vasculitis [6,7].
B cells appear to contribute to the pathogenesis of autoimmune diseases by multiple mechanisms. First, B cells produce pathogenic autoantibodies. Second, B cells may play a role in development of autoimmune diseases by presenting self-antigens to T cells and secreting cytokines [4,8,9]. In contrast, inhibitory cytokines such as IL-10 produced by B cells are shown to inhibit development of various autoimmune diseases. A fraction of CD1d hi B cells and plasmablasts are responsible for production of inhibitory cytokines, and are therefore called regulatory B (B reg) cells [10][11][12]. Our recent studies show that inhibitory receptors expressed in B cells especially CD72 inhibit both activation of self-reactive B cells and generation of B reg cells [13,14], thereby regulating autoimmune diseases such as T1D and SLE either positively or negatively depending on the disease [15,16].

Inhibitory B cell co-receptors and SLE
B cells express various inhibitory receptors including CD22 (also known as Siglec-2), Sigle-10 (Siglec-G in mice), CD72, LILRB (PIR-B in mice), PECAM-1 (also known as CD31) and FccRIIB (Table 1) [17,18]. These receptors contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic tails. Upon ligation of B cell receptor (BCR), ITIMs are phosphorylated at the tyrosine residue by the BCR-associated kinase Lyn, thereby recruiting and activating SH2-containing phosphatases such as SH2-contingin protein tyrosine phosphatase 1 (SHP-1, also known as PTPN6), SHP-2 (also known as PTPN11), and SH2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1, also known as INPP5D). These phosphatases then dephosphorylate signaling molecules activated by BCR ligation, thereby negatively regulate BCR signaling. Because of the functional association with BCR, these inhibitory receptors are called inhibitory B cell co-receptors. Expression of CD22, Siglec-10/ G, and CD72 is mostly restricted to B cells, whereas other inhibitory receptors are expressed in various immune cell types as well as B cells (Table 1).
Most of the inhibitory co-receptors activate SHP-1 but not SHIP1, whereas FccRIIB activates SHIP1 but not SHP-1 [17,18]. B cell-specific SHP-1 deletion causes defects in B cell development, marked expansion of B-1 cells and development of severe lupus-like disease in mice [19]. Mice deficient in SHIP1 in B cells show similar phenotypes [20]. Thus, both of these phosphatases regulate multiple phenotypes in B cells probably by globally regulating BCR signaling. In contrast, inhibitory co-receptors regulate distinct phenotypes depending on the receptors. CD22 -/mice show defects in B cell development [21][22][23] similar to those in B cell specific SHP-1 -/mice whereas Siglec-G -/mice show marked B-1 cell expansion [24] as is the case for B cell specific SHP-1 -/mice. CD22 -/mice and PIR-B -/mice do not develop lupus-like disease [25,26]. However, mice that are deficient in PIR-B and carry Fas lpr , a loss-of-function mutant of Fas, develop renal disease [26], and PECAM1 -/mice and Siglec-G -/mice develop mild lupus-like nephritis in a fraction of mice over 12 months of age [25,27]. Interestingly, CD72 -/mice develop severe lupuslike disease similar to B cell-specific SHP-1-deficient mice [16,28]. CD72 -/mice produce autoantibodies such as anti-DNA antibodies, and develop nephritis in almost all the mice already at 6-month of age. CD72 -/-Fas lpr/lpr mice develop more severe lupuslike disease comparable to MRL-Fas lpr/lpr mice [16] ( Figure 1). Thus, many of the inhibitory B cell coreceptors regulate development of SLE though weakly, but CD72 strongly inhibits development of this autoimmune disease. A crucial role of CD72 in development of SLE is supported by the finding that CD72 is associated with lupus nephritis in patients with SLE by candidate gene approach [29], although association of CD72 with SLE has not yet been shown by GWAS probably because of the absence of CD72 alleles with significant functional difference. Because the severity of the lupus-like disease in CD72 -/mice is comparable to that in B cell-specific SHP-1-deficient mice [16,19], CD72 appears to be the major activator of SHP-1 in self-reactive B cells. Likewise, CD22 and Siglec-G are the major activators of SHP-1 in the regulation of B cell development and B-1 cell homeostasis, respectively. Thus, different inhibitory B cell co-receptors regulate distinct phenotypes of B cells probably by regulating BCR signaling under specific conditions [18]. FccRIIB is the only one inhibitory B cell coreceptor known to activate SHIP1 [30]. Although B cell-specific SHIP1 deletion causes phenotypes similar to B cell-specific SHP-1 -/mice including development of severe lupus-like disease [20], FccRIIB -/mice do not develop lupus-like disease at least on C57BL/6 background, the same genetic background as the B cell-specific SHIP1 -/mice. Initially, FccRIIB -/mice were reported to develop lupus-like disease [32]. Later, development of the disease turned out to require the 129 mouse strain-derived autoimmune-prone SLAM haplotype, which is closely located to the FccRIIB locus on the chromatin, and FccRIIB -/mice on the C57BL/6 background do not develop autoimmune disease [33].
Thus, SHIP1 appears to be activated by a mechanism independent of inhibitory receptors to regulate autoimmune disease. Indeed, SHIP1 is recruited and activated by Iga/Igb in the BCR complex upon phosphorylation [34]. SHIP1 may be activated by Iga/Igb in B cells including self-reactive B cells, leading to protection from autoimmunity.

CD72 recognizes the lupus self-antigen Sm/RNP as a specific ligand
It is already established that development of SLE involves nucleic acid (NA) sensors [35][36][37][38]. NA sensors activate various immune cells including B cells by recognizing microbial nucleic acids as pathogenassociated molecular patterns (PAMPs) thereby contributing host defense against pathogens. However, aberrant responses of NA sensors to self-NAs appear to play a central role in the pathogenic processes of SLE. GWAS have shown that SLE is associated with various genes encoding NA sensors and signaling molecules involved in signaling through NA sensors [39,40]. Moreover, mice deficient in various genes involved in NA metabolism develop lupus-like disease [41]. These findings strongly suggest that excessive responses to NAs are involved in development of lupus. Responses of B cells to self-NAs induce autoantibody production to various NAs and NA-related nuclear self-antigens that characterizes SLE. Responses of plasmacytoid dendritic cells (pDCs) to self-NAs cause type 1 interferon (IFN I) production, resulting in expression of various IFN-responsible genes known as interferon signature, which is the most significant changes in gene expression in SLE patients.
Among various NA sensors TLR7 plays a central role in development of SLE at least in mice because mice deficient in the RNA sensor TLR7 no longer develop the autoimmune disease in multiple mouse SLE models such as MRL-Fas lpr/lpr mice and pristine-induced lupus [42,43]. In contrast, the DNA sensor TLR9 suppresses development of lupus by competing transport of TLR7 to endosome [42,44] where NA-sensing TLRs are converted to functional receptors by proteolytic processing [45], indicating that immune responses to RNA-related self-antigens but not DNA play a central role in development of SLE. Sm/RNP is an RNA-containing nuclear selfantigen, whose major component is U1-snRNP Figure 1. Lupus-like nephritis in CD72 -/and CD72 -/-Fas lpr/lpr C57BL/6 mice at 6 months of age. Representative PAS staining and immunohistochemistry for IgG and C3 of kidney sections (A), and scores of the severity of glomerulonephritis (B) in female wild-type (WT), CD72 -/-, Fas lpr/lpr and CD72 -/-Fas lpr/lpr C57BL/6 mice at 6 months of age are shown. Severity of glomerular damage was scored as described previously [31]. Grade 0, no involvement; grade 1, 2, and 3, changes in 0-25%, 25-50%, 50-75% of total glomeruli, respectively; grade 4, sclerosis or crescent formation in greater than 90% of glomeruli. Ã p < .05; ÃÃÃ p < .001. Note that both CD72 -/and CD72 -/-Fas lpr/lpr mice develop nephritis in all the mice at 6 months of age. Originally published in The Journal of Immunology. Xu et al. 2013 [16]. Copyright # [2013] The American Association of Immunologists, Inc.
involved in RNA splicing. Anti-Sm/RNP antibody is known as a disease-specific autoantibody in SLE [46]. Sm/RNP was shown to be an endogenous TLR7 ligand [47]. Although most of the endogenous ligands of innate receptors including TLRs induce inflammation as damage-associated molecular patterns (DAMPs) [48], endogenous ligands of TLR7 such as Sm/RNP appear to be involved in development of SLE by activating self-reactive B cells to produce autoantibodies to NA-related self-antigens and activating pDCs to produce IFN-I.
CD72 contains a C-type lectin-like domain (CTLD) in the extracellular region. Previously, we demonstrated that CD72 CTLD specifically recognizes Sm/RNP [13]. By recognizing Sm/RNP as a ligand, CD72 negatively regulates BCR signaling induced by Sm/RNP. As is the case for other inhibitory B cell co-receptors, CD72 is phosphorylated at Figure 2. CD72 specifically inhibits BCR signaling in Sm/RNP-reactive B cells by recognizing Sm/RNP as a ligand. When Sm/ RNP binds to Sm/RNP-reactive BCR, CD72 is recruited to the close proximity of BCR by binding to Sm/RNP bound to BCR (A). CD72 ITIM is then phosphorylated by the BCR-associated kinase Lyn and recruits SHP-1. This causes suppression of BCR signaling in Sm/RNP-reactive B cells thereby inhibiting production of anti-Sm/RNP antibody crucial for development of lupus. When BCR is ligated by other antigens that do not bind to CD72, CD72 is kept away from BCR and does not inhibit BCR signaling (B). Originally published in Frontiers in Immunology, Tsubata (2018) [18]. . CD72 maintains self-tolerance to NAs. Among self-NAs, free NAs are rapidly degraded by nucleases in the body fluid after release from dead cells before they reach endosome. In contrast, NAs complexed with proteins are resistant to nucleases and are able to stimulate endosomal NA sensors. Antibody responses to the complexes of DNA and proteins are nonpathogenic. The pathogenic complexes of RNA and proteins such as Sm/RNP are recognized by CD72. This recognition inhibits activation of B cells reactive to the self-RNA/protein complexes, and inhibits production of pathogenic autoantibodies to these self-antigens. Originally published in Proceedings of the Japan Academy, Series B, Physical and Biological Sciences, Tsubata (2018) [49].
an ITIM by the BCR-associated kinase Lyn, thereby recruiting and activating SHP-1. Thus, CD72 needs to be located in the proximity of BCR to negatively regulate BCR signaling. When BCR is ligated by Sm/RNP, CD72 is recruited to BCR by recognizing BCR-bound Sm/RNP, and is phosphorylated by Lyn associated with BCR. In contrast, CD72 is kept away from BCR when BCR is ligated by other antigens (Figure 2). By this mechanism, CD72 appears to negatively regulate BCR signaling when BCR is ligated by Sm/RNP but not other antigens. We further demonstrated that CD72 inhibits proliferation of Sm/RNP-reactive B cells upon stimulation with Sm/RNP [13]. Proliferation of B cells stimulated by Sm/RNP was shown to depend on TLR7 [47]. It is not yet known whether CD72 inhibits TLR signaling. However, CD72-mediated inhibition of BCR signaling generated by Sm/RNP may inhibit B cell proliferation induced by Sm/RNP as dual signaling through BCR and TLR7 may be required for proliferation of these B cells. Thus, CD72 antagonizes TLR7 in autoantibody production by suppressing BCR signaling induced by the endogenous TLR7 ligand Sm/RNP (Figure 3).
Although recognition of microbial NAs especially viral NAs is crucial in immune responses to microbes, recognition of self NAs causes inflammation and autoimmunity [38]. Recognition of self NAs is suppressed by multiple mechanisms in normal immune system. First, many of NA sensors recognize structure of NAs specific to microbial NAs such as double-stranded RNA and 5 0 -triphosphate RNA [50], although some NA sensors such as TLR7, TLR9 [47,51,52], and the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) recognize endogenous NAs [53,54] as well as microbial NAs. Second, NA sensors are located in either cytosol or endosome but not on the cell surface. Self-NAs derived from dead cells are mostly free NAs, which are rapidly degraded by nucleases in the body fluid. In contrast, microbial NAs located inside of microbes are released from microbes either in endosome or cytosol only after they are taken up by host cells. Thus, NA sensors in cytosol and endosome efficiently sense microbial NAs but not self NAs. A role of NA sensor localization in discrimination of microbial vs. host NAs is underlined by the finding that aberrant expression of TLR9 on the cell surface causes autoimmune disease [55]. However, complexes composed of NAs and nuclear proteins such as Sm/RNP are relatively resistant to nucleases. When Sm/RNP is released from dead cells, Sm/RNP may be able to access endosomal NA sensors after it is taken up by self-reactive B cells, and activate B cells to produce autoantibodies. CD72 inhibits responses of B cells to Sm/RNP, thereby constituting a mechanism that discriminates self NAs complexed with proteins from microbial NAs (Figure 3). CD72 appears to recognize the protein part of Sm/RNP but not RNA. This recognition may avoid impairment of immune responses to microbes.

Regulation of B reg cells and autoimmune diseases by inhibitory co-receptors
It is already established that a subset of B cells called B reg cells regulate development of autoimmune diseases such as MS and T1D by secreting inhibitory cytokines such as IL-10 and IL-35 [10][11][12]. B reg cells were initially shown to be accumulated in CD1d hi B cells [56]. Later studies demonstrated that the majority of B reg cells are present in plasmablasts especially LAG3 þ plasmablasts [11,14]. More recently, IgA þ plasma cells are also shown to function as B reg cells [57]. Defect in IL-10 production from B cells was reported in patients with various autoimmune diseases such as MS, T1D, and SLE [58][59][60][61], suggesting a role of B reg cells in the regulation of autoimmune diseases. However, effect of B cell-specific IL10 deficiency depends on the diseases. B cell specific IL-10 deficiency exacerbates experimental allergic encephalomyelitis (EAE) [62], a mouse model of MS. In contrast, global IL-10 deficiency but not B cell-specific IL-10 deficiency exacerbates lupus-like disease in MRL-Fas lpr/lpr mice [63,64], suggesting that SLE is regulated by IL-10 from cell types other than B cells. Thus, B reg cells suppress development of some but not all autoimmune diseases. These findings do not contradict with the findings that autoimmune diseases including T1D and MS are ameliorated by B cell depletion therapy using anti-CD20 antibody [1][2][3][4]. Because plasmablasts are CD20 -, majority of B reg cells may not be depleted by rituximab.
Inhibitory B cell co-receptors such as CD22 and CD72 have been shown to regulate expansion of B reg cells. CD22 -/mice show expansion of CD1d hi B cells and B cells that produce IL-10 upon stimulation with LPS, PMA, and ionomycin [65]. CD72 -/mice show expansion of LAG-3 þ plasmablasts, and B cells that produce IL-10 upon Salmonella infection [14]. These findings clearly demonstrate that both CD22 and CD72 inhibit B reg expansion. TLR signaling has been shown to induce IL-10 production and expansion of B reg cells [65,66]. Because CD22 negatively regulates B cell activation induced by TLR ligands such as LPS, CpG and imiquimod [67], CD22 may inhibit expansion of B reg cells by negatively regulating B cell responses to TLR ligands. It is not yet known whether CD72 regulates B cell activation induced by TLR ligands. However, CD72 recognizes the endogenous TLR7 ligand Sm/RNP as a specific ligand, and inhibits B cell responses to Sm/RNP [13]. Suppression of responses to Sm/RNP might be involved in inhibition of B reg expansion by CD72.

CD72 augments T1D in NOD mice
There are three allelic forms of CD72, i.e., CD72 a , CD72 b , and CD72 c [68]. CD72 a and CD72 b are highly homologous each other. In contrast, CD72 c contains a number of amino acid substitutions and a 7 amino-acid deletion compared to the other allelic forms. MRL-Fas lpr/lpr mice carries CD72 c , and CD72 c plays a role in development of lupus-like disease in these mice [16,69,70]. Although Fas lpr , a loss-of-function mutant of Fas, is required for development of severe lupus-like disease in MRL-Fas lpr/lpr mice, the MRL background is also required because mice carrying Fas lpr/lp on other background such as C57BL/6 and C3H do not develop lupus-like disease [71]. First, genetic analysis demonstrated that CD72 c is associated with the disease in MRL-Fas lpr/lpr mice [69]. Later, introduction of CD72 b was shown to ameliorate the disease in MRL-Fas lpr/lpr mice [16,70]. These results clearly indicate that CD72 c in the MRL background is involved in development of lupus-like disease in MRL-Fas lpr/lpr mice. Recently, we demonstrated that binding of CD72 c to Sm/RNP is weaker than that of CD72 a [13], indicating that CD72 c is a hypomorphic allele. CD72 c is involved in development of lupus-like disease probably because CD72 c may inhibit B cell responses to Sm/RNP only weakly.
NOD mice that spontaneously develop T1D also carry CD72 c [72]. To address whether CD72 regulates T1D, we generated NOD mice carrying CD72 b by backcrossing C57BL/6 mice to NOD mice. Although introduction of CD72 b into MRL-Fas lpr/lpr mice ameliorates lupus-like disease [16], CD72 b markedly exacerbates T1D in NOD mice [15]. Our study showed that NOD.CD72 b mice become diabetic in significantly higher frequency and at younger age than NOD mice that carry CD72 c (Figure 4). Incidence and severity of insulitis in NOD.CD72 b mice are much higher than those in NOD mice. Because CD72 c is a hypomorphic allele, these results strongly suggest that CD72 accelerates development of T1D. It is not yet elucidated how CD72 regulates T1D. However, CD72-mediated inhibition of B reg expansion may augment T1D because development of T1D is suppressed by B reg cells [59]. week-old female NOD (CD72 c ) (n ¼ 9) and NOD.CD72 b mice (n ¼ 12). Severity scale: 0, normal islet; 1, peri-insulitis or infiltration of less than 25% of the islet surface area; 2, infiltration of 25-50% of the islet surface area; 3, infiltration of more than 50% of the islet surface area. (C and D) Blood glucose levels (C) and incidence of diabetes (D) in NOD (triangle) (open: male, closed: female) and NOD.CD72 b (circle) (open: male, closed: female) mice at indicated age. Each symbol represents data of each mouse (C). Diabetes was diagnosed when the blood glucose level was greater than 250 mg/ml. Reprinted from Hou et al. [15]. Copyright 2009, with permission from Elsevier.

Conclusions
B cells play a role in development of autoimmune diseases by producing autoantibodies, and also contribute to T cell-mediated autoimmunity probably by activating self-reactive T cells as APCs and secreting cytokines [4,8,9]. In contrast, B reg cells inhibit development of autoimmune diseases by producing inhibitory cytokines such as IL-10 [10][11][12]. Thus, B cells regulate autoimmunity either positively or negatively. Inhibitory B cell co-receptors regulate autoimmunity by suppressing B cell activation. Among these inhibitory B cell co-receptors, CD72 regulates development of autoimmune diseases SLE and T1D most strongly probably because of its recognition of Sm/RNP [13], a lupus self-antigen and an endogenous TLR7 ligand, as a specific ligand. As TLR7 is essential for development of SLE at least in mouse models, CD72 appears to inhibit development of SLE by suppressing B cell responses to endogenous TLR7 ligands such as Sm/RNP. B cell responses to endogenous TLR ligands are also crucial in expansion of B reg cells [65,66]. CD72 accelerates T1D probably by suppressing B cell responses to endogenous TLR7 ligands required for B reg expansion. Because CD72 efficiently regulates development of various autoimmune diseases, CD72 appears to be a good therapeutic target for autoimmune diseases.

Disclosure statement
No potential conflict of interest was reported by the author.