REVIEWS The role of CD22 and Siglec‑G in B-cell tolerance and autoimmune disease Jennifer Müller and Lars Nitschke Abstract | A high proportion of peripheral human B cells produce polyreactive or autoreactive antibodies, which indicates that they have escaped the elimination of self-reactive B cells in the bone marrow. CD22 and Siglec‑G are two inhibitory receptors of the sialic-acid-binding immunoglobulin-like lectin (Siglec) family that inhibit the B‑cell antigen receptor (BCR) signal. The ability of these two receptors to bind sialic acids is crucial for regulating inhibition and inducing tolerance to self-antigens. Sialylated glycans are usually absent on microbes (although several pathogenic microorganisms have evolved strategies to mimic self by decorating their surfaces with sialic acids) but abundant in higher vertebrates and might, therefore, provide an important tolerogenic signal. Combined Siglec‑G deficiency and CD22 deficiency leads to spontaneous autoimmunity in mice, and mutations in an enzyme that modifies Siglec ligands are directly linked to several autoimmune diseases in humans. New data show that high-affinity ligands for CD22 and Siglec‑G can be used to induce antigen-specific B-cell tolerance, which might be one strategy for the treatment of autoimmune diseases in the future. Müller, J. & Nitschke, L. Nat. Rev. Rheumatol. advance online publication 29 April 2014; doi:10.1038/nrrheum.2014.54
Introduction
Chair of Genetics, Department of Biology, University of Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany (J.M., L.N.). Correspondence to: L.N. [email protected]
B cells have an important role in the protection of organisms from pathogens by producing pathogen-specific antibodies. To avoid an immune response against selfantigens, several tolerance mechanisms quickly sense and remove autoreactive B cells from the bone marrow or periphery.1 However, there are some ‘self-recognizing’ B cells that escape the guards of the immune system, resulting in autoantibody production and potentially in autoimmune disease.2 The sialic-acid-binding immunoglobulin-type lectins (Siglecs) CD22 and Siglec‑G (or the human orthologue Siglec‑10) are the only Siglecs expressed on B-cell surfaces.3 These molecules are constitutively expressed on B cells and act as inhibitory co-receptors of the B‑cell antigen receptor (BCR) by dampening the calcium (Ca2+) response, resulting from BCR crosslinking by its speci fic antigen.4,5 Mouse Siglec‑G and human Siglec‑10 are orthologues with high sequence homology. Both Siglec‑G and Siglec‑10 are expressed on B cells, suggesting a simi lar function.6,7 CD22, Siglec‑G and Siglec‑10 are transmembrane proteins with a cytoplasmic signalling domain and an extracellular ligand-binding domain that exclusively recognizes sialylated carbohydrates. Via conserved tyrosine residues in immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular tail, CD22 can activate phosphatases, which dephosphory late positive components of the BCR signalling cascade to cause a dampening of the BCR signal. 8,9 A similar Competing interests L.N. has received grants for research from UCB Pharma. J.M. declares no competing interests.
function has been shown for Siglec‑10.7 Deficiency in one or both of these molecules results in increased Ca2+ signalling and a hyper-responsive phenotype of B cells.4,5 The results from studies on B cells with other defective inhibitory receptors, or defective inhibitory pathways, revealed that increased basal activation can lead to autoimmune disease.10,11 Siglec‑G deficiency or CD22 deficiency alone does not lead to the production of highly reactive autoantibodies, whereas Siglec‑G and CD22 double-deficient mice develop a severe autoimmune phenotype at old age.12 In mice and humans, the biological function of CD22 and Siglec‑G/10 as negative regulators of the BCRmediated Ca2+ response has been examined in detail by many groups. However, the contribution of both Siglecs to the maintenance of tolerance and prevention of autoimmunity is only now becoming understood—new data show how important CD22 and Siglec‑G/10 are for the prevention of autoimmune diseases. In this Review, we recapitulate the role of the Siglec family members CD22 and Siglec‑G. In particular, we outline the participation of CD22 and Siglec‑G in maintaining B‑cell tolerance by suppressing antibody responses, and we outline their contribution to the prevention of autoimmune diseases, such as systemic lupus erythematosus (SLE). We also discuss therapeutic targeting of CD22, with antibodies and synthetic CD22 ligands, in autoimmune diseases.
B-cell Siglecs’ inhibitory functions CD22 and Siglec‑G are inhibitory co-receptors of the BCR on B cells. CD22 is the prominent inhibitor of the BCR signal in conventional (B2) cells, whereas Siglec‑G
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REVIEWS Key points ■■ On B cells, CD22 and Siglec‑G mediate inhibition of B‑cell antigen receptor-induced signalling; Siglec‑G is an inhibitory receptor for B1 cells ■■ Ligand-binding of CD22 and Siglec‑G to other membrane glycoproteins in cis regulate their association to the B‑cell receptor and the degree of inhibition ■■ CD22-deficiency or Siglec‑G-deficiency does not result in autoimmunity, whereas CD22 and Siglec‑G double-deficient mice develop a systemic lupus erythematosus (SLE)-like disease ■■ The immunomodulatory CD22-specific antibody, epratuzumab, reduces disease activity in patients with SLE, although the exact mechanism is unknown ■■ Attaching CD22-specific or Siglec‑G-specific sialic acids with antigens on liposomes induces antigen-specific tolerance in mice and could be a new autoantigen-specific treatment strategy
seems to have its main role as an inhibitory co-receptor on B1 cells, as Siglec‑G-deficient mice show a B1-cellspecif ic phenotype. 5 B1 cells, which are most clearly defined in the mouse, are a subpopulation of B cells that secrete natural IgM antibodies that are important for first-line defense against bacteria. Antigen-mediated BCR crosslinking causes rapid tyrosine phosphory lation of CD22 by the tyrosine kinase Lyn, thereby recruiting and activating the SH2-domain-containing tyrosine phosphatase, SHP‑1.13,14 SHP‑1 subsequently dephosphorylates positive activators of the Ca2+ response, a
such as CD19 or B-cell linker protein (also known as SLP65), and promotes Ca 2+ efflux from the cell 9,15,16 (Figure 1). The results from studies in mice with mutated CD22–ITIM sequences indicate that efficient inhibition by CD22 requires all three ITIM sequences.17 However, the mutation of the three ITIMs does not reach the same level of Ca2+ elevation as in CD22-deficient mice, sug gesting a role for the other three tyrosines of the CD22 cytoplasmic tail in Ca2+ inhibition to prevent overstimulation of B cells.17 Siglec‑G also mediates the inhibition of BCR signalling in a SHP‑1-dependent manner.18 Simi larly, Siglec‑10 binds SHP‑1 and SHP‑2 tyrosine phos phatases on its intracellular tail, suggesting a similar inhibitory mechanism as for CD22.7
Ligand binding regulates signalling CD22 and Siglec‑G recognize endogenous sialic acids expressed on glycoproteins of various cellular surfaces. Both Siglecs bind to α2,6-linked sialic acids (2,6Sia), in cis on the same cell or in trans on other cells, with their N‑terminal immunoglobulin-like domain.3,19 Sialic acids with an α2,3 linkage (2,3Sia) can additionally be bound by Siglec‑G. In resting B cells, CD22 is a prominent cis ligand for itself, forming CD22 homo-oligomers20 (Figure 1). The moderately low affinity of CD22 to b
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Figure 1 | Regulation of BCR signalling by CD22 and Siglec‑G binding to cis ligands. a | CD22 mediates the inhibition of BCR signalling in conventional B2 cells. CD22 forms homo-oligomers by binding to 2,6Sia on neighbouring CD22 molecules. Upon antigen engagement, CD22 homo-oligomers are recruited to the BCR (indicated by black arrow), which induces phosphorylation of the ITIMs at the intracellular tail of CD22. SHP‑1 binds two of the three phoshorylated ITIMs, becomes activated and inhibits calcium signalling. b | Siglec‑G is the main inhibitor of Ca2+ signalling in B1 cells. A conclusive mechanism is not known, but studies with Siglec‑G ligand-binding mutants indicate a direct Siglec‑G recruitment to 2,3Sia or 2,6Sia on IgM after antigen stimulation. Both Siglec-G and Siglec-10 bind SHP-1. Abbreviations: 2,3Sia, α2,3-linked sialic acids; 2,6Sia, α2,6-linked sialic acids; BCR, B-cell receptor; Ca2+, calcium ion; ITIM, immunoreceptor tyrosine-based inhibitory motifs; P, denotes tyrosine-phosphorylation of ITIMs; SHP-1, tyrosine-protein phosphatase non-receptor type 6; Siglec, sialic-acid-binding immunoglobulin-like lectin; Y, denotes tyrosine within CD22 or Siglec‑G tail.
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REVIEWS Table 1 | The effect of mutations and treatments on signalling and B-cell phenotypes Mutation or treatment
Signalling
B-cell phenotype
Cd22 –/– mice
B2-cell Ca2+
Normal B-cell numbers
Siglecg –/– mice
B1-cell Ca2+
B1-cell population expansion
Cd22-Arg130Glu mice
B2-cell Ca
Normal B-cell numbers
Siglec-G-Arg120Glu mice
B1-cell Ca2+
Mutation
Cd22
x Siglecg
–/–
–/–
2+
mice
B1-cell population expansion
B1-cell and B2-cell Ca
2+
B1-cell population expansion Autoimmunity
Treatment Epratuzumab
B-cell Ca2+
Slightly reduced blood B-cell numbers in patients with SLE
Synthetic sialic acids
B-cell Ca2+
Normal B-cell numbers (in mice)
Sialic acid-liposomes (STALs)
B-cell Ca2+ inhibition
B-cell tolerance induction Apoptosis (in mice)
CD22-Arg130Glu mice are CD22-knockin mice with a mutated ligand binding domain. Siglec-G-Arg120Glu mice are Siglecg-knockin mice with a mutated ligand binding domain. Epratuzumab is an anti-human CD22 antibody. Synthetic sialic acids are chemically modified sialic acids as synthetic Siglec ligands. Sialic acidliposomes are Siglec-engaging tolerance-inducing STALs. Abbreviations: , denotes increased Ca2+ responses; , denotes strongly increased Ca2+ responses; , denotes decreased Ca2+ responses; Siglec, sialic-acid-binding immunoglobulin-like lectin; STALs, Siglec-engaging tolerance-inducing antigenic liposomes.
its 2,6Sia ligand, but high sialyation on cell surface proteins, causes a limited availability to trans ligands, hence CD22 is ‘masked’ by self-ligands.21 The disruption of CD22 homo-oligomers, either by deletion of the sialyltransferase St6gal1, or with a mutated CD22 ligandbinding domain (CD22 Arg130Glu) in mice, results in increased CD22–BCR association and enhanced Ca2+ inhibition upon anti-IgM stimulation 17,22 (Table 1). This effect indicates a crucial cis-ligand binding function of CD22 in regulating the B‑cell activation status. Siglec‑G-deficient mice have substantially increased B1 cell numbers and strongly increased BCR signalling in B1 cells, but not in B2 cells.5 It seems that Siglec‑G has a B1-cell restricted inhibitory function, although good evidence exists that, upon high-affinity ligand binding, this Siglec is also capable of inhibiting BCR activation in B2 cells and of inhibiting T‑cell-dependent antibody responses.18 Siglec‑G-knockin mice expressing a mutated ligand-binding domain (Siglec‑G Arg120Glu) revealed that Siglec‑G cis-ligand-binding is decisive for its inhibitory function on B1 cells, as an elevation of Ca2+ flux in B1 cells has been observed (Hutzler, S. et al., unpublished work) (Table 1). Trans-ligand-binding of CD22 and Siglec‑G induces B‑cell signal inhibition when target cells co-express antigen and sialic acids.19,23 Mechanistically, by recog nizing sialylated structures on target cells, ligation of Siglec‑G or CD22 to the BCR is enforced and suppresses a response to this antigen (Figure 2a). As Siglecs can be engaged both by cis ligands, as well as by trans ligands, and both depend on ligand affinity and accessibility, there will always be competition between both mechanisms.
Pathogen exploitation of Siglecs Sialic acids are commonly expressed in humans and higher vertebrates, and are recognized as ‘self ’ by Siglecs.
Microorganisms do not usually express sialic acids on their surfaces, although some pathogens (including viruses, bacteria and parasites) have acquired sialic acids through different mechanisms. Pathogens disguised with sialic acids can not only mimic self, but can potentially engage Siglecs and induce inhibitory signals in human cells.24 On B cells, Siglecs can be recruited to the BCR when pathogen-expressed sialic acids and antigens are simultaneously recognized. The resulting dampening of the B‑cell response could induce tolerance to the pathogen. One strain of group B Streptococcus, for example, has been shown to suppress neutrophil responses by expressing sialylated capsular polysaccharides, which bind to the inhibitory Siglec‑9 on neutrophils. 25 This mechanism has not been proven for B-cell Siglecs, although circumstantial evidence24 exists to suggest that pathogen exploitation of Siglec inhibitory mechanisms takes place. Humans, in contrast to other mammals (such as mice and rats), have a high number of Siglec genes, and pairwise expressed inhibitory and activating Siglecs, as well as allelic variants with inactivating mutations in activatory Siglec genes.24,25 This variability suggests a strong ongoing evolution in response to infectious pathogens.
Siglecs preventing autoimmunity Maintaining tight regulation of peripheral B‑cell tolerance is of utmost importance for an organism, as 30–40% of B cells that emerge from the bone marrow can recognize self-antigens. 2 The three main mechanisms of B‑cell tolerance—namely receptor editing, deletion and anergy—are always dependent on induction of BCR signalling by self-antigens. CD22 and Siglec‑G are mediators of BCR signal inhibition and their role in preventing dysregulation of tolerance mechanisms has been analysed in gene-targeted mice. Three sets of independently generated CD22-deficient mice do not show an autoimmune phenotype. 4,26,27 However, the loss of CD22 can contribute to autoimmunity when CD22-deficient mice are crossed to autoimmune-prone strains, such as Yaa (Y-linked autoimmune accelerator), or certain genetic backgrounds (C57BL/6 × 129).28,29 This contributory effect demonstrates that loss of CD22 alone is not sufficient for development of autoimmune diseases, but that CD22 deficiency might increase the susceptibility to autoimmune disorders. Lupus-prone mouse strains, such as the NZW or BXSB strains, express an aberrant form of CD22—namely CD22a—which has an impaired ability to bind cis ligands.29,30 Expression of CD22a in B6-congenic mice results in constitutively activated B cells, suggesting that alterations in CD22 ligand binding contribute to autoimmunity.30 Another factor linking B‑cell Siglecs to autoimmunity is the sialic acid acetylesterase (SIAE), which can modify sialic acids on glycosylated membrane proteins by removing an inhibitory acetyl group from the 9‑OH position on sialic acid ligands and enabling CD22 and Siglec‑G binding.31,32 SIAE is an important regulator of B‑cell tolerance, as mice lacking SIAE develop an SLE-like autoimmune disease with high titres of autoantibodies and IgG containing glomerular deposits.31 On SIAE-deficient
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REVIEWS B cells, an enhancement of 9‑O-acetylation of sialic acid can be observed, which limits the accessibility of sialic acids to both Siglecs.32 In addition, SIAE-deficient B cells have increased Ca2+ signalling, similar to CD22-deficient or Siglec‑G-deficient B cells. Thus, circumstantial evidence exists that SIAE defects directly affect CD22 and Siglec‑G functions, indicating that the enzymatic activity of SIAE could be crucial for prevention of autoimmunity in a CD22–Siglec‑G-dependent fashion. This mechanism could also be involved in disease susceptibility in humans, as functionally defective germline variants of the human SIAE gene are associated with several autoimmune diseases.33 Increased numbers of B1 cells are thought to be associated with development of autoimmunity.34 Siglec‑Gdeficient mice, despite having hyper-responsive B cells and an enlarged B1a-cell population, do not develop autoimmune disease.5 However, Siglec‑G x CD22 d oubledeficient mice develop a lupus-like phenotype with high levels of IgG autoantibodies (with specificities including a
ssDNA, dsDNA and RNA) and develop glomerulo nephritis.12 This work demonstrates a redundancy of Siglec‑G and CD22 on B cells in maintaining B-cell tolerance, as loss of both molecules, or dysregulation of their ligands by loss of SIAE, results in autoimmunity (Figure 2b). Genome-wide association studies (GWAS) have not identified CD22 or SIGLEC10 as SLE-linked loci so far. However, the CD22 signalling pathway was identified by GWAS, as LYN (coding for the tyrosinekinase phosphorylating CD22) and the locus coding for the related kinase Blk were shown to be associated with SLE in humans.35,36 Thus, the results from studies in mice and humans strongly suggest genetic links of the CD22 and Siglec‑G/10 pathways to SLE.
Siglecs as therapeutic targets B cells have an important role in the progression of SLE, as demonstrated by the successful clinical trial and approval of belimumab, the anti-BAFF antibody that blocks a crucial B‑cell survival factor. 37 Surprisingly, b
Other cell
2,3Sia Autoantigen
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Figure 2 | CD22 and Siglec‑G maintain tolerance to self-antigens. a | CD22 and Siglec‑G can induce tolerance to cellbound autoantigens, as surfaces of many other cells contain abundant sialic acids (2,3Sia and 2,6Sia). The trans binding of Siglecs to sialic acids recruits Siglecs to the vicinity of the BCR (indicated by a black arrow), dampening autoantigeninduced BCR signalling by activating SHP‑1. b | In CD22-deficient and Siglec‑G-deficient B cells, this inhibition of BCR signalling cannot take place, resulting in hyper-responsiveness of B cells owing to increased calcium flux. This constant activation of the cell potentially results in production of autoantibodies directed against self-structures. Abbreviations: 2,3Sia, α2,3-linked sialic acids; 2,6Sia, α2,6-linked sialic acids; BCR, B-cell receptor; ITIM, immunoreceptor tyrosinebased inhibitory motifs; P, denotes tyrosine-phosphorylation of ITIMs; Siglec, sialic-acid-binding immunoglobulin-like lectin; Y, denotes tyrosine within CD22 or Siglec‑G tail.
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REVIEWS a
b STAL
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CD22 Siglec-G/10
IgM 2,6Sia
2,3Sia
Y Y P Y Y Y Y Y YY Y Y Y Y P Y Y Y P Y Y Y Y Y
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Y Y Y P Y Y Y Y SHP-1 Y Y Y Y P Y Y Y Y P Y Y Y
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Y Y P Y
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P Y Y Y Y Y Y Y Y Y Y Y P Y Y Y P Y Y Y Y Y Y Y Y Y Y YY Y Y
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Antigen-specific cell death
Siglec-G/10
Figure 3 | Targeting CD22 by antibodies and liposomes. a | Epratuzumab is a therapeutic anti-CD22 antibody, which induces CD22 phosphorylation and inhibition of BCR signalling in B cells. Thus, this antibody induces signal modulation, rather than B-cell depletion. b | STALs consisting of high-affinity Siglec-ligands (modified 2,3Sia or 2,6Sia ligands) and specific antigen attached to liposomes can suppress antigen-specific antibody responses. If an autoantigen is attached to the STAL, this process can suppress autoantigen-specific antibody responses. The mechanism is probably enhanced recruitment of Siglec molecules to the BCR, leading to inhibition of responses and eventually to apoptosis of the selfreactive B cells. Abbreviations: 2,3Sia, α2,3-linked sialic acids; 2,6Sia, α2,6-linked sialic acids; BCR, B-cell receptor; ITIM, immunoreceptor tyrosine-based inhibitory motifs; P, denotes tyrosine-phosphorylation of ITIMs; Siglec, sialic-acidbinding immunoglobulin-like lectin; STALs, SIGLEC-engaging tolerance-inducing antigenic liposomes; Y, denotes tyrosine within CD22 or Siglec‑G tail.
the B‑cell-depleting antibody, rituximab, failed in two randomized clinical trials of SLE.38 CD22 and Siglec‑G, expressed during all B-cell stages, are quickly internalized upon ligand binding, and can modulate BCR responses.3 SLE B cells exhibit high early signal trans duction events after BCR crosslinking and, therefore, both Siglecs are potential B‑cell targets for modulation of BCR signalling.39 Epratuzumab is a humanized monoclonal antibody that recognizes the extracellular domain of CD22 and induces depletion of circulating B cells in the blood of patients to ~65%.40 In clinical trials, epratuzumab improved disease activity in patients with SLE compared with placebo.41 The exact mechanism of B‑cell regulation by this therapeutic antibody is not fully understood, although a new mouse model with humanized CD22 could help to reveal the exact mechanism in epratuzumab–CD22-mediated B-cell immunomodulation.42 Epratuzumab has an immunomodulatory role in patients rather than B‑cell depleting function.37 The results from in vitro studies have revealed an inhibitory effect of epratuzumab on B cells resulting in CD22
phosphorylation and CD22 internalization upon epratu zumab binding.43 Accordingly, activation of the posi tive mediators of BCR signalling (Syk and PLCγ2 and subsequently Ca 2+ flux) are diminished after BCR engagement in the presence of epratuzumab (Table 1).44 Another possible mechanism of action is the modulation of epratuzumab-opsonized B cells by trogocytosis of BCR-modulating cell-surface proteins, such as CD19 and CD21, to effector cells.45 The use of high-affinity synthetic CD22 ligands that are derived from sialic acids is another approach to target CD22 on human B cells in systemic autoimmune diseases.46 These synthetic ligands, as dimers or oligomers, can bind and crosslink CD22 on the B‑cell surfaces away from BCRs, and thereby induce enhanced Ca2+ signalling.47 The release from CD22 inhibition could, theoretically, induce an excessively strong BCR response, which could then result in apoptosis of B cells, as has been shown for CD22-deficient B cells in vitro.4 Additionally, B‑cell lymphoma cells can efficiently be killed in vitro, when these dimeric or oligomeric sialosides are coupled to toxins.47,48
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REVIEWS Synthetic CD22 or Siglec‑G ligands have also been used to induce tolerance to antigens when both are attached to carriers. This effect was first shown when synthetic T-cell‑independent type 2 (TI‑2) antigens were decorated with Siglec ligands on polyacrylamide carriers.19,49 Furthermore, STALs (SIGLEC-engaging toleranceinducing antigenic liposomes) were developed, which are liposomes displaying CD22 or Siglec‑G ligands and protein antigens together to induce B-cell tolerance. Treatment of mice with these STALs induced antigenspecific suppression of immune responses, caused by CD22-mediated and Siglec‑G-mediated suppression of BCR signalling, and eventually apoptosis of specific B cells.50,18 This effect can be explained by the hypothesis that more Siglec molecules are recruited into the vicinity of the BCR by STAL engagement and thereby cause strong signal inhibition (Figure 3).51 These findings have great therapeutic potential, as Siglec-specific liposomes decorated with identified autoantigens could be used to silence self-recognizing B cells in autoimmune diseases in an autoantigen-specific manner. This concept was dem onstrated in a mouse model of haemophilia, in which STALs were able to prevent the formation of inhibitory antibodies to factor VIII.50 Newly designed synthetic sialic acid analogues, which bind CD22 and Siglec‑G with higher affinity than their original ligands, will help to improve this approach.47,52
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prevention of autoimmunity, as mutations in one or more of these molecules can cause a break in tolerance owing to enhanced BCR signal strength. However, in addition, trans-ligand binding of both Siglecs can be exploited to induce B‑cell tolerance. Liposomic STALs displaying CD22 or Siglec‑G ligands together with antigens are powerful suppressors of antigen-specific B‑cell responses, and have a great potential as a new therapeutic approach to silence autoreactive B cells. Furthermore, Siglecs have been shown to be suitable targets for therapeutic antibodies targeting autoreactive B cells. Epratuzumab is a promising antibody for treatment of SLE that targets CD22. In contrast to other therapeutic antibodies, epratuzumab seems to be nondepleting, but rather modulates B‑cell signalling. B cells also express other inhibitory coreceptors, such as FcγRIIb, PIR‑B or CD72, which mediate their BCR signal inhibition in a SHP‑1-dependent or SHIP (SH2 domain-containing inositol 5‑phosphatase)dependent manner, and respective knockout mutants also develop autoimmune syndromes.53 These receptors might also be targets for therapeutic antibodies, but to our knowledge there are no therapeutic antibodies in clinical trials yet that target these other B-cell inhibitory receptors. Targeting of Siglecs and other inhibitory receptors on B cells is a promising new therapeutic approach for autoimmune diseases.
Conclusions
Review criteria
The cis-ligand-binding ability of Siglec‑G (in B1 cells) and CD22 (in B2 cells) is highly relevant for the regulation of BCR-mediated signalling, and also for the maintenance of B-cell tolerance. The B-cell inhibitory pathway, consisting of Siglecs, SIAE, Lyn and SHP‑1, contributes to the
We searched for original articles focusing on the subject in PubMed without time restriction. The search terms we used were “CD22”, “Siglec‑G”, “B-cell autoimmune disease” and “epratuzumab”. All papers identified were English-language full text papers.
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autoreactivity? Eur. J. Immunol. 32, 348–355 (2002). Cao, H. & Crocker, P. R. Evolution of CD33related siglecs: regulating host immune functions and escaping pathogen exploitation? Immunology 132, 18–26 (2011). Angata, T., Margulies, E. H., Green, E. D. & Varki, A. Large-scale sequencing of the CD33related Siglec gene cluster in five mammalian species reveals rapid evolution by multiple mechanisms. Proc. Natl Acad. Sci. USA 101, 13251–13256 (2004). Otipoby, K. L. et al. CD22 regulates thymusindependent responses and the lifespan of B cells. Nature 384, 634–637 (1996). Sato, S. et al. CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice. Immunity 5, 551–562 (1996). O’Keefe, T. L., Williams, G. T., Batista, F. D. & Neuberger, M. S. Deficiency in CD22, a B cellspecific inhibitory receptor, is sufficient to predispose to development of high affinity autoantibodies. J. Exp. Med. 189, 1307–1313 (1999). Mary, C. et al. Cd22a PRE-mRNA dysregulated expression of the Cd22 gene as a result of a short interspersed nucleotide element insertion in Cd22a lupus-prone mice. J. Immunol. 165, 2987–2996 (2000). Nitschke, L. et al. Expression of aberrant forms of CD22 on B lymphocytes in Cd22a lupus-prone mice affects ligand binding. Int. Immunol. 18, 59–68 (2006). Cariappa, A. et al. B cell antigen receptor signal strength and peripheral B cell development are regulated by a 9‑O‑acetyl sialic acid esterase. J. Exp. Med. 206, 125–138 (2009). Sjoberg, E. R., Powell, L. D., Klein, A. & Varki, A. Natural ligands of the B cell adhesion molecule CD22β can be masked by 9‑O‑acetylation of sialic acids. J. Cell Biol. 126, 549–562 (1994). Surolia, I. et al. Functionally defective germline variants of sialic acid acetylesterase in autoimmunity. Nature 466, 243–247 (2010).
34. Murakami, M., Yoshioka, H., Shirai, T., Tsubata, T. & Honjo, T. Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells. Int. Immunol. 7, 877–882 (1995). 35. Harley, I. T. W., Kaufman, K. M., Langefeld, C. D., Harley, J. B. & Kelly, J. A. Genetic susceptibility to SLE: new insights from fine mapping and genome-wide association studies. Nat. Rev. Genet. 10, 285–290 (2009). 36. Cui, Y., Sheng, Y. & Zhang, X. Genetic susceptibility to SLE: recent progress from GWAS. J. Autoimmun. 41, 25–33 (2013). 37. Dörner, T. & Lipsky, P. E. B cells: depletion or functional modulation in rheumatic diseases. Curr. Opin. Rheumatol. 26, 228–236 (2014). 38. Duxbury, B., Combescure, C. & Chizzolini, C. Rituximab in systemic lupus erythematosus: an updated systematic review and metaanalysis. Lupus 22, 1489–1503 (2013). 39. Liossis, S. N., Kovacs, B., Dennis, G., Kammer, G. M. & Tsokos, G. C. B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events. J. Clin. Invest. 98, 2549–2557 (1996). 40. Jacobi, a M. et al. Differential effects of epratuzumab on peripheral blood B cells of patients with systemic lupus erythematosus versus normal controls. Ann. Rheum. Dis. 67, 450–457 (2008). 41. Wallace, D. J. et al. Efficacy and safety of epratuzumab in patients with moderate/severe active systemic lupus erythematosus: results from EMBLEM, a phase IIb, randomised, doubleblind, placebo-controlled, multicentre study. Ann. Rheum. Dis. 73, 183–190 (2014). 42. Wöhner, M., Born, S. & Nitschke, L. Human CD22 cannot fully substitute murine CD22 functions in vivo, as shown in a new knockin mouse model. Eur. J. Immunol. 42, 3009–3018 (2012). 43. Carnahan, J. et al. Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties. Clin. Cancer Res. 9, 3982S–3990S. (2003). 44. Sieger, N. et al. CD22 ligation inhibits downstream B cell receptor signaling and
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Ca2+ flux upon activation. Arthritis Rheum. 65, 770–779 (2013). Rossi, E. a. et al. Trogocytosis of multiple B-cell surface markers by CD22 targeting with epratuzumab. Blood 122, 3020–3029 (2013). Kelm, S. The ligand-binding domain of CD22 is needed for inhibition of the B Cell receptor signal, as demonstrated by a novel human CD22-specific inhibitor compound. J. Exp. Med. 195, 1207–1213 (2002). Schweizer, A., Wöhner, M., Prescher, H., Brossmer, R. & Nitschke, L. Targeting of CD22positive B-cell lymphoma cells by synthetic divalent sialic acid analogues. Eur. J. Immunol. 42, 2792–2802 (2012). Collins, B. E. et al. High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J. Immunol. 177, 2994–3003 (2006). Courtney, A. H., Puffer, E. B., Pontrello, J. K., Yang, Z.‑Q. & Kiessling, L. L. Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation. Proc. Natl Acad. Sci. USA 106, 2500–2505 (2009). Macauley, M. S. et al. Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis. J. Clin. Invest. 123, 3074–3083 (2013). Nitschke, L. Suppressing the antibody response with Siglec ligands. N. Engl. J. Med. 369, 1373–1374 (2013). Kelm, S. et al. C-4 modified sialosides enhance binding to Siglec-2 (CD22): towards potent Siglec inhibitors for immunoglycotherapy. Angew. Chem. Int. Ed. Engl. 52, 3616–3620 (2013). Nitschke, L. The role of CD22 and other inhibitory co-receptors in B-cell activation. Curr. Opin. Immunol. 17, 290–297 (2005).
Acknowledgements The authors are supported by grants from the Deutsche Forschungsgemeinschaft (SFB643, TRR130). Author contributions J.M. and L.N. contributed equally to researching data for the article and writing the article. L.N. reviewed and edited the manuscript before submission.
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Introduction
Chair of Genetics, Department of Biology, University of Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany (J.M., L.N.). Correspondence to: L.N. [email protected]
B cells have an important role in the protection of organisms from pathogens by producing pathogen-specific antibodies. To avoid an immune response against selfantigens, several tolerance mechanisms quickly sense and remove autoreactive B cells from the bone marrow or periphery.1 However, there are some ‘self-recognizing’ B cells that escape the guards of the immune system, resulting in autoantibody production and potentially in autoimmune disease.2 The sialic-acid-binding immunoglobulin-type lectins (Siglecs) CD22 and Siglec‑G (or the human orthologue Siglec‑10) are the only Siglecs expressed on B-cell surfaces.3 These molecules are constitutively expressed on B cells and act as inhibitory co-receptors of the B‑cell antigen receptor (BCR) by dampening the calcium (Ca2+) response, resulting from BCR crosslinking by its speci fic antigen.4,5 Mouse Siglec‑G and human Siglec‑10 are orthologues with high sequence homology. Both Siglec‑G and Siglec‑10 are expressed on B cells, suggesting a simi lar function.6,7 CD22, Siglec‑G and Siglec‑10 are transmembrane proteins with a cytoplasmic signalling domain and an extracellular ligand-binding domain that exclusively recognizes sialylated carbohydrates. Via conserved tyrosine residues in immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular tail, CD22 can activate phosphatases, which dephosphory late positive components of the BCR signalling cascade to cause a dampening of the BCR signal. 8,9 A similar Competing interests L.N. has received grants for research from UCB Pharma. J.M. declares no competing interests.
function has been shown for Siglec‑10.7 Deficiency in one or both of these molecules results in increased Ca2+ signalling and a hyper-responsive phenotype of B cells.4,5 The results from studies on B cells with other defective inhibitory receptors, or defective inhibitory pathways, revealed that increased basal activation can lead to autoimmune disease.10,11 Siglec‑G deficiency or CD22 deficiency alone does not lead to the production of highly reactive autoantibodies, whereas Siglec‑G and CD22 double-deficient mice develop a severe autoimmune phenotype at old age.12 In mice and humans, the biological function of CD22 and Siglec‑G/10 as negative regulators of the BCRmediated Ca2+ response has been examined in detail by many groups. However, the contribution of both Siglecs to the maintenance of tolerance and prevention of autoimmunity is only now becoming understood—new data show how important CD22 and Siglec‑G/10 are for the prevention of autoimmune diseases. In this Review, we recapitulate the role of the Siglec family members CD22 and Siglec‑G. In particular, we outline the participation of CD22 and Siglec‑G in maintaining B‑cell tolerance by suppressing antibody responses, and we outline their contribution to the prevention of autoimmune diseases, such as systemic lupus erythematosus (SLE). We also discuss therapeutic targeting of CD22, with antibodies and synthetic CD22 ligands, in autoimmune diseases.
B-cell Siglecs’ inhibitory functions CD22 and Siglec‑G are inhibitory co-receptors of the BCR on B cells. CD22 is the prominent inhibitor of the BCR signal in conventional (B2) cells, whereas Siglec‑G
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REVIEWS Key points ■■ On B cells, CD22 and Siglec‑G mediate inhibition of B‑cell antigen receptor-induced signalling; Siglec‑G is an inhibitory receptor for B1 cells ■■ Ligand-binding of CD22 and Siglec‑G to other membrane glycoproteins in cis regulate their association to the B‑cell receptor and the degree of inhibition ■■ CD22-deficiency or Siglec‑G-deficiency does not result in autoimmunity, whereas CD22 and Siglec‑G double-deficient mice develop a systemic lupus erythematosus (SLE)-like disease ■■ The immunomodulatory CD22-specific antibody, epratuzumab, reduces disease activity in patients with SLE, although the exact mechanism is unknown ■■ Attaching CD22-specific or Siglec‑G-specific sialic acids with antigens on liposomes induces antigen-specific tolerance in mice and could be a new autoantigen-specific treatment strategy
seems to have its main role as an inhibitory co-receptor on B1 cells, as Siglec‑G-deficient mice show a B1-cellspecif ic phenotype. 5 B1 cells, which are most clearly defined in the mouse, are a subpopulation of B cells that secrete natural IgM antibodies that are important for first-line defense against bacteria. Antigen-mediated BCR crosslinking causes rapid tyrosine phosphory lation of CD22 by the tyrosine kinase Lyn, thereby recruiting and activating the SH2-domain-containing tyrosine phosphatase, SHP‑1.13,14 SHP‑1 subsequently dephosphorylates positive activators of the Ca2+ response, a
such as CD19 or B-cell linker protein (also known as SLP65), and promotes Ca 2+ efflux from the cell 9,15,16 (Figure 1). The results from studies in mice with mutated CD22–ITIM sequences indicate that efficient inhibition by CD22 requires all three ITIM sequences.17 However, the mutation of the three ITIMs does not reach the same level of Ca2+ elevation as in CD22-deficient mice, sug gesting a role for the other three tyrosines of the CD22 cytoplasmic tail in Ca2+ inhibition to prevent overstimulation of B cells.17 Siglec‑G also mediates the inhibition of BCR signalling in a SHP‑1-dependent manner.18 Simi larly, Siglec‑10 binds SHP‑1 and SHP‑2 tyrosine phos phatases on its intracellular tail, suggesting a similar inhibitory mechanism as for CD22.7
Ligand binding regulates signalling CD22 and Siglec‑G recognize endogenous sialic acids expressed on glycoproteins of various cellular surfaces. Both Siglecs bind to α2,6-linked sialic acids (2,6Sia), in cis on the same cell or in trans on other cells, with their N‑terminal immunoglobulin-like domain.3,19 Sialic acids with an α2,3 linkage (2,3Sia) can additionally be bound by Siglec‑G. In resting B cells, CD22 is a prominent cis ligand for itself, forming CD22 homo-oligomers20 (Figure 1). The moderately low affinity of CD22 to b
2,6Sia
Antigen
2,3Sia IgM
IgM
Y
Y
Y
B2 cell
Y Y Y Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y
Y
Y
Ca2+ Y Y Y
Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y P Y Y Y Y Y Y SHP-1 Y Y Y P Y Y Y P Y Y Y
Y
Y
Siglec-G/10 YY YY
Y Y Y P SHP-1
Y
Y
Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Ca2+
Y
YY YY
CD22 homooligomers
B1 cell
Siglec-G/10
Figure 1 | Regulation of BCR signalling by CD22 and Siglec‑G binding to cis ligands. a | CD22 mediates the inhibition of BCR signalling in conventional B2 cells. CD22 forms homo-oligomers by binding to 2,6Sia on neighbouring CD22 molecules. Upon antigen engagement, CD22 homo-oligomers are recruited to the BCR (indicated by black arrow), which induces phosphorylation of the ITIMs at the intracellular tail of CD22. SHP‑1 binds two of the three phoshorylated ITIMs, becomes activated and inhibits calcium signalling. b | Siglec‑G is the main inhibitor of Ca2+ signalling in B1 cells. A conclusive mechanism is not known, but studies with Siglec‑G ligand-binding mutants indicate a direct Siglec‑G recruitment to 2,3Sia or 2,6Sia on IgM after antigen stimulation. Both Siglec-G and Siglec-10 bind SHP-1. Abbreviations: 2,3Sia, α2,3-linked sialic acids; 2,6Sia, α2,6-linked sialic acids; BCR, B-cell receptor; Ca2+, calcium ion; ITIM, immunoreceptor tyrosine-based inhibitory motifs; P, denotes tyrosine-phosphorylation of ITIMs; SHP-1, tyrosine-protein phosphatase non-receptor type 6; Siglec, sialic-acid-binding immunoglobulin-like lectin; Y, denotes tyrosine within CD22 or Siglec‑G tail.
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REVIEWS Table 1 | The effect of mutations and treatments on signalling and B-cell phenotypes Mutation or treatment
Signalling
B-cell phenotype
Cd22 –/– mice
B2-cell Ca2+
Normal B-cell numbers
Siglecg –/– mice
B1-cell Ca2+
B1-cell population expansion
Cd22-Arg130Glu mice
B2-cell Ca
Normal B-cell numbers
Siglec-G-Arg120Glu mice
B1-cell Ca2+
Mutation
Cd22
x Siglecg
–/–
–/–
2+
mice
B1-cell population expansion
B1-cell and B2-cell Ca
2+
B1-cell population expansion Autoimmunity
Treatment Epratuzumab
B-cell Ca2+
Slightly reduced blood B-cell numbers in patients with SLE
Synthetic sialic acids
B-cell Ca2+
Normal B-cell numbers (in mice)
Sialic acid-liposomes (STALs)
B-cell Ca2+ inhibition
B-cell tolerance induction Apoptosis (in mice)
CD22-Arg130Glu mice are CD22-knockin mice with a mutated ligand binding domain. Siglec-G-Arg120Glu mice are Siglecg-knockin mice with a mutated ligand binding domain. Epratuzumab is an anti-human CD22 antibody. Synthetic sialic acids are chemically modified sialic acids as synthetic Siglec ligands. Sialic acidliposomes are Siglec-engaging tolerance-inducing STALs. Abbreviations: , denotes increased Ca2+ responses; , denotes strongly increased Ca2+ responses; , denotes decreased Ca2+ responses; Siglec, sialic-acid-binding immunoglobulin-like lectin; STALs, Siglec-engaging tolerance-inducing antigenic liposomes.
its 2,6Sia ligand, but high sialyation on cell surface proteins, causes a limited availability to trans ligands, hence CD22 is ‘masked’ by self-ligands.21 The disruption of CD22 homo-oligomers, either by deletion of the sialyltransferase St6gal1, or with a mutated CD22 ligandbinding domain (CD22 Arg130Glu) in mice, results in increased CD22–BCR association and enhanced Ca2+ inhibition upon anti-IgM stimulation 17,22 (Table 1). This effect indicates a crucial cis-ligand binding function of CD22 in regulating the B‑cell activation status. Siglec‑G-deficient mice have substantially increased B1 cell numbers and strongly increased BCR signalling in B1 cells, but not in B2 cells.5 It seems that Siglec‑G has a B1-cell restricted inhibitory function, although good evidence exists that, upon high-affinity ligand binding, this Siglec is also capable of inhibiting BCR activation in B2 cells and of inhibiting T‑cell-dependent antibody responses.18 Siglec‑G-knockin mice expressing a mutated ligand-binding domain (Siglec‑G Arg120Glu) revealed that Siglec‑G cis-ligand-binding is decisive for its inhibitory function on B1 cells, as an elevation of Ca2+ flux in B1 cells has been observed (Hutzler, S. et al., unpublished work) (Table 1). Trans-ligand-binding of CD22 and Siglec‑G induces B‑cell signal inhibition when target cells co-express antigen and sialic acids.19,23 Mechanistically, by recog nizing sialylated structures on target cells, ligation of Siglec‑G or CD22 to the BCR is enforced and suppresses a response to this antigen (Figure 2a). As Siglecs can be engaged both by cis ligands, as well as by trans ligands, and both depend on ligand affinity and accessibility, there will always be competition between both mechanisms.
Pathogen exploitation of Siglecs Sialic acids are commonly expressed in humans and higher vertebrates, and are recognized as ‘self ’ by Siglecs.
Microorganisms do not usually express sialic acids on their surfaces, although some pathogens (including viruses, bacteria and parasites) have acquired sialic acids through different mechanisms. Pathogens disguised with sialic acids can not only mimic self, but can potentially engage Siglecs and induce inhibitory signals in human cells.24 On B cells, Siglecs can be recruited to the BCR when pathogen-expressed sialic acids and antigens are simultaneously recognized. The resulting dampening of the B‑cell response could induce tolerance to the pathogen. One strain of group B Streptococcus, for example, has been shown to suppress neutrophil responses by expressing sialylated capsular polysaccharides, which bind to the inhibitory Siglec‑9 on neutrophils. 25 This mechanism has not been proven for B-cell Siglecs, although circumstantial evidence24 exists to suggest that pathogen exploitation of Siglec inhibitory mechanisms takes place. Humans, in contrast to other mammals (such as mice and rats), have a high number of Siglec genes, and pairwise expressed inhibitory and activating Siglecs, as well as allelic variants with inactivating mutations in activatory Siglec genes.24,25 This variability suggests a strong ongoing evolution in response to infectious pathogens.
Siglecs preventing autoimmunity Maintaining tight regulation of peripheral B‑cell tolerance is of utmost importance for an organism, as 30–40% of B cells that emerge from the bone marrow can recognize self-antigens. 2 The three main mechanisms of B‑cell tolerance—namely receptor editing, deletion and anergy—are always dependent on induction of BCR signalling by self-antigens. CD22 and Siglec‑G are mediators of BCR signal inhibition and their role in preventing dysregulation of tolerance mechanisms has been analysed in gene-targeted mice. Three sets of independently generated CD22-deficient mice do not show an autoimmune phenotype. 4,26,27 However, the loss of CD22 can contribute to autoimmunity when CD22-deficient mice are crossed to autoimmune-prone strains, such as Yaa (Y-linked autoimmune accelerator), or certain genetic backgrounds (C57BL/6 × 129).28,29 This contributory effect demonstrates that loss of CD22 alone is not sufficient for development of autoimmune diseases, but that CD22 deficiency might increase the susceptibility to autoimmune disorders. Lupus-prone mouse strains, such as the NZW or BXSB strains, express an aberrant form of CD22—namely CD22a—which has an impaired ability to bind cis ligands.29,30 Expression of CD22a in B6-congenic mice results in constitutively activated B cells, suggesting that alterations in CD22 ligand binding contribute to autoimmunity.30 Another factor linking B‑cell Siglecs to autoimmunity is the sialic acid acetylesterase (SIAE), which can modify sialic acids on glycosylated membrane proteins by removing an inhibitory acetyl group from the 9‑OH position on sialic acid ligands and enabling CD22 and Siglec‑G binding.31,32 SIAE is an important regulator of B‑cell tolerance, as mice lacking SIAE develop an SLE-like autoimmune disease with high titres of autoantibodies and IgG containing glomerular deposits.31 On SIAE-deficient
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REVIEWS B cells, an enhancement of 9‑O-acetylation of sialic acid can be observed, which limits the accessibility of sialic acids to both Siglecs.32 In addition, SIAE-deficient B cells have increased Ca2+ signalling, similar to CD22-deficient or Siglec‑G-deficient B cells. Thus, circumstantial evidence exists that SIAE defects directly affect CD22 and Siglec‑G functions, indicating that the enzymatic activity of SIAE could be crucial for prevention of autoimmunity in a CD22–Siglec‑G-dependent fashion. This mechanism could also be involved in disease susceptibility in humans, as functionally defective germline variants of the human SIAE gene are associated with several autoimmune diseases.33 Increased numbers of B1 cells are thought to be associated with development of autoimmunity.34 Siglec‑Gdeficient mice, despite having hyper-responsive B cells and an enlarged B1a-cell population, do not develop autoimmune disease.5 However, Siglec‑G x CD22 d oubledeficient mice develop a lupus-like phenotype with high levels of IgG autoantibodies (with specificities including a
ssDNA, dsDNA and RNA) and develop glomerulo nephritis.12 This work demonstrates a redundancy of Siglec‑G and CD22 on B cells in maintaining B-cell tolerance, as loss of both molecules, or dysregulation of their ligands by loss of SIAE, results in autoimmunity (Figure 2b). Genome-wide association studies (GWAS) have not identified CD22 or SIGLEC10 as SLE-linked loci so far. However, the CD22 signalling pathway was identified by GWAS, as LYN (coding for the tyrosinekinase phosphorylating CD22) and the locus coding for the related kinase Blk were shown to be associated with SLE in humans.35,36 Thus, the results from studies in mice and humans strongly suggest genetic links of the CD22 and Siglec‑G/10 pathways to SLE.
Siglecs as therapeutic targets B cells have an important role in the progression of SLE, as demonstrated by the successful clinical trial and approval of belimumab, the anti-BAFF antibody that blocks a crucial B‑cell survival factor. 37 Surprisingly, b
Other cell
2,3Sia Autoantigen
CD22
2,6Sia IgM
IgM
Siglec-G/10
Y Y P Y Y
Y
YY YY
SHP-1 Y
Ca2+
YY YY
Y Y
P Y Y Y Y Y Y Y Y Y Y P Y Y Y P Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Stronger signal
Ca2+
B cell
Figure 2 | CD22 and Siglec‑G maintain tolerance to self-antigens. a | CD22 and Siglec‑G can induce tolerance to cellbound autoantigens, as surfaces of many other cells contain abundant sialic acids (2,3Sia and 2,6Sia). The trans binding of Siglecs to sialic acids recruits Siglecs to the vicinity of the BCR (indicated by a black arrow), dampening autoantigeninduced BCR signalling by activating SHP‑1. b | In CD22-deficient and Siglec‑G-deficient B cells, this inhibition of BCR signalling cannot take place, resulting in hyper-responsiveness of B cells owing to increased calcium flux. This constant activation of the cell potentially results in production of autoantibodies directed against self-structures. Abbreviations: 2,3Sia, α2,3-linked sialic acids; 2,6Sia, α2,6-linked sialic acids; BCR, B-cell receptor; ITIM, immunoreceptor tyrosinebased inhibitory motifs; P, denotes tyrosine-phosphorylation of ITIMs; Siglec, sialic-acid-binding immunoglobulin-like lectin; Y, denotes tyrosine within CD22 or Siglec‑G tail.
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REVIEWS a
b STAL
Autoantigen
Epratuzumab
CD22 Siglec-G/10
IgM 2,6Sia
2,3Sia
Y Y P Y Y Y Y Y YY Y Y Y Y P Y Y Y P Y Y Y Y Y
YY YY
Y Y Y P Y Y Y Y SHP-1 Y Y Y Y P Y Y Y Y P Y Y Y
IgM
CD22 homooligomers
Y Y
Y Y P Y
Y
YY YY
SHP-1
Ca2+
Ca2+
Y Y
P Y Y Y Y Y Y Y Y Y Y Y P Y Y Y P Y Y Y Y Y Y Y Y Y Y YY Y Y
Y Y Y
B cell
B2 cell
Antigen-specific cell death
Siglec-G/10
Figure 3 | Targeting CD22 by antibodies and liposomes. a | Epratuzumab is a therapeutic anti-CD22 antibody, which induces CD22 phosphorylation and inhibition of BCR signalling in B cells. Thus, this antibody induces signal modulation, rather than B-cell depletion. b | STALs consisting of high-affinity Siglec-ligands (modified 2,3Sia or 2,6Sia ligands) and specific antigen attached to liposomes can suppress antigen-specific antibody responses. If an autoantigen is attached to the STAL, this process can suppress autoantigen-specific antibody responses. The mechanism is probably enhanced recruitment of Siglec molecules to the BCR, leading to inhibition of responses and eventually to apoptosis of the selfreactive B cells. Abbreviations: 2,3Sia, α2,3-linked sialic acids; 2,6Sia, α2,6-linked sialic acids; BCR, B-cell receptor; ITIM, immunoreceptor tyrosine-based inhibitory motifs; P, denotes tyrosine-phosphorylation of ITIMs; Siglec, sialic-acidbinding immunoglobulin-like lectin; STALs, SIGLEC-engaging tolerance-inducing antigenic liposomes; Y, denotes tyrosine within CD22 or Siglec‑G tail.
the B‑cell-depleting antibody, rituximab, failed in two randomized clinical trials of SLE.38 CD22 and Siglec‑G, expressed during all B-cell stages, are quickly internalized upon ligand binding, and can modulate BCR responses.3 SLE B cells exhibit high early signal trans duction events after BCR crosslinking and, therefore, both Siglecs are potential B‑cell targets for modulation of BCR signalling.39 Epratuzumab is a humanized monoclonal antibody that recognizes the extracellular domain of CD22 and induces depletion of circulating B cells in the blood of patients to ~65%.40 In clinical trials, epratuzumab improved disease activity in patients with SLE compared with placebo.41 The exact mechanism of B‑cell regulation by this therapeutic antibody is not fully understood, although a new mouse model with humanized CD22 could help to reveal the exact mechanism in epratuzumab–CD22-mediated B-cell immunomodulation.42 Epratuzumab has an immunomodulatory role in patients rather than B‑cell depleting function.37 The results from in vitro studies have revealed an inhibitory effect of epratuzumab on B cells resulting in CD22
phosphorylation and CD22 internalization upon epratu zumab binding.43 Accordingly, activation of the posi tive mediators of BCR signalling (Syk and PLCγ2 and subsequently Ca 2+ flux) are diminished after BCR engagement in the presence of epratuzumab (Table 1).44 Another possible mechanism of action is the modulation of epratuzumab-opsonized B cells by trogocytosis of BCR-modulating cell-surface proteins, such as CD19 and CD21, to effector cells.45 The use of high-affinity synthetic CD22 ligands that are derived from sialic acids is another approach to target CD22 on human B cells in systemic autoimmune diseases.46 These synthetic ligands, as dimers or oligomers, can bind and crosslink CD22 on the B‑cell surfaces away from BCRs, and thereby induce enhanced Ca2+ signalling.47 The release from CD22 inhibition could, theoretically, induce an excessively strong BCR response, which could then result in apoptosis of B cells, as has been shown for CD22-deficient B cells in vitro.4 Additionally, B‑cell lymphoma cells can efficiently be killed in vitro, when these dimeric or oligomeric sialosides are coupled to toxins.47,48
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REVIEWS Synthetic CD22 or Siglec‑G ligands have also been used to induce tolerance to antigens when both are attached to carriers. This effect was first shown when synthetic T-cell‑independent type 2 (TI‑2) antigens were decorated with Siglec ligands on polyacrylamide carriers.19,49 Furthermore, STALs (SIGLEC-engaging toleranceinducing antigenic liposomes) were developed, which are liposomes displaying CD22 or Siglec‑G ligands and protein antigens together to induce B-cell tolerance. Treatment of mice with these STALs induced antigenspecific suppression of immune responses, caused by CD22-mediated and Siglec‑G-mediated suppression of BCR signalling, and eventually apoptosis of specific B cells.50,18 This effect can be explained by the hypothesis that more Siglec molecules are recruited into the vicinity of the BCR by STAL engagement and thereby cause strong signal inhibition (Figure 3).51 These findings have great therapeutic potential, as Siglec-specific liposomes decorated with identified autoantigens could be used to silence self-recognizing B cells in autoimmune diseases in an autoantigen-specific manner. This concept was dem onstrated in a mouse model of haemophilia, in which STALs were able to prevent the formation of inhibitory antibodies to factor VIII.50 Newly designed synthetic sialic acid analogues, which bind CD22 and Siglec‑G with higher affinity than their original ligands, will help to improve this approach.47,52
1.
2.
3.
4.
5.
6.
7.
8.
9.
prevention of autoimmunity, as mutations in one or more of these molecules can cause a break in tolerance owing to enhanced BCR signal strength. However, in addition, trans-ligand binding of both Siglecs can be exploited to induce B‑cell tolerance. Liposomic STALs displaying CD22 or Siglec‑G ligands together with antigens are powerful suppressors of antigen-specific B‑cell responses, and have a great potential as a new therapeutic approach to silence autoreactive B cells. Furthermore, Siglecs have been shown to be suitable targets for therapeutic antibodies targeting autoreactive B cells. Epratuzumab is a promising antibody for treatment of SLE that targets CD22. In contrast to other therapeutic antibodies, epratuzumab seems to be nondepleting, but rather modulates B‑cell signalling. B cells also express other inhibitory coreceptors, such as FcγRIIb, PIR‑B or CD72, which mediate their BCR signal inhibition in a SHP‑1-dependent or SHIP (SH2 domain-containing inositol 5‑phosphatase)dependent manner, and respective knockout mutants also develop autoimmune syndromes.53 These receptors might also be targets for therapeutic antibodies, but to our knowledge there are no therapeutic antibodies in clinical trials yet that target these other B-cell inhibitory receptors. Targeting of Siglecs and other inhibitory receptors on B cells is a promising new therapeutic approach for autoimmune diseases.
Conclusions
Review criteria
The cis-ligand-binding ability of Siglec‑G (in B1 cells) and CD22 (in B2 cells) is highly relevant for the regulation of BCR-mediated signalling, and also for the maintenance of B-cell tolerance. The B-cell inhibitory pathway, consisting of Siglecs, SIAE, Lyn and SHP‑1, contributes to the
We searched for original articles focusing on the subject in PubMed without time restriction. The search terms we used were “CD22”, “Siglec‑G”, “B-cell autoimmune disease” and “epratuzumab”. All papers identified were English-language full text papers.
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Acknowledgements The authors are supported by grants from the Deutsche Forschungsgemeinschaft (SFB643, TRR130). Author contributions J.M. and L.N. contributed equally to researching data for the article and writing the article. L.N. reviewed and edited the manuscript before submission.
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