Glycobiology vol. 24 no. 9 pp. 807–817, 2014 doi:10.1093/glycob/cwu066 Advance Access publication on July 6, 2014 REVIEW CD22 and Siglec-G regulate inhibition of B-cell signaling by sialic acid ligand binding and control B-cell tolerance Downloaded from http://glycob.oxfordjournals.org/ Lars Nitschke1 co-receptors is regulated by extracellular-bound ligands, which Division of Genetics, Department of Biology, University of Erlangen, determine the association of the co-receptors to the BCR. B-cell Erlangen, Germany immunity depends on recognition of foreign antigens and a lack of responsiveness to self-antigens. Although central B-cell toler- Received on April 29, 2014; revised on June 26, 2014; accepted on June 27, 2014 ance is induced in the bone marrow by several mechanisms, many peripheral B cells in the periphery escape these tolerance CD22 and Siglec-G are two B-cell expressed members of the mechanisms, produce self-reactive antibodies and need to be con- Siglec (sialic acid-binding immunoglobulin (Ig)-like lectin) trolled by peripheral tolerance mechanisms in order to prevent family and are potent inhibitors of B-cell signaling. Genetic autoimmunity (Wardemann et al. 2003). Inhibitory co-receptors approaches have provided evidence that this inhibition of on B cells contribute to this control of B-cell tolerance. at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 B-cell antigen receptor (BCR) signaling by Siglecs is depend- CD22 (Siglec-2) and Siglec-G (or its human ortholog ent on ligand binding to sialic acids in specific linkages. The Siglec-10) are two B-cell expressed members of the Siglec (sialic cis-ligand-binding activity of CD22 leads to homo-oligomer acid-binding immunoglobulin (Ig)-like lectin) family and have formation, which are to a large extent found in membrane been shown to negatively regulate BCR signaling (Jellusova and domains that are distinct from those containing the BCR. In Nitschke 2012). Both are associated to some extent with the BCR contrast, Siglec-G is recruited via sialic acid binding to the (Peaker and Neuberger 1993; Zhang and Varki 2004; Müller et al. BCR. This interaction of Siglec-G with mIgM leads to an in- 2013) and are inhibitory co-receptors. Both Siglecs carry intracel- hibitory function that seems to be specific for B-1 cells. Both lular inhibitory ITIMs (immunoreceptor tyrosine-based inhibition CD22 and Siglec-G control B-cell tolerance and loss of these motifs) and recruit the tyrosine phosphatase SHP-1 that inhibits proteins, its ligands or its inhibitory pathways can increase signaling (Doody et al. 1995; Pfrengle et al. 2013). The inhibitory the susceptibility for autoimmune diseases. CD22 is a target functions of both Siglecs are regulated by ligand binding (Hutzler protein both in B-cell leukemias and lymphomas, as well as et al. 2014). Both Siglecs bind to sialic acids in specific linkages, in B-cell mediated autoimmune diseases. Both antibodies which are attached to glycoproteins and which are present on and synthetic chemically modified sialic acids are currently cellular surfaces, including the B-cell surface. The presence or tested to target Siglecs on B cells. absence of these ligands regulates the association of these two Siglecs to the BCR and thus influences the strength of inhibition. Keywords: autoimmunity / B lymphocytes / inhibitory Since sialic acids are abundantly expressed in vertebrates, but signaling / Siglecs often not found on microorganisms they are a part of “self- structures” and there is evidence that the sialic acid binding by B-cell Siglecs regulates B-cell tolerance and prevents autoimmun- Introduction to B-cell Siglecs ity (Crocker et al. 2007). B cells are an important part of the adaptive immune system and generate the humoral immune response by secretion of antigen- Regulation of B-cell signaling specific antibodies. The signaling by the B-cell antigen receptor (BCR), induced by antigen binding is crucial for triggering B-cell CD22 and Siglec-G are the only two Siglecs which are development, B-cell activation, as well as differentiation to expressed on murine B cells. Human B cells express CD22, antibody-secreting plasma cells. Accessory transmembrane mole- Siglec-10, as well as Siglec-6 (Crocker et al. 2007). CD22 has a cules or co-receptors on the B-cell surface modulate BCR signal- very B-cell restricted expression pattern (Torres et al. 1992). An ing. These B-cell co-receptors can be activatory or inhibitory and expression and function outside of the B-cell lineage has only fulfil these functions by binding additional signaling proteins been shown on intestinal eosinophils and on a DC subpopula- with their intracellular tails and thereby recruiting these effectors tion (Wen et al. 2012; Ma et al. 2013). Also Siglec-G shows the into the vicinity of the BCR. Very often the activity of highest expression pattern on B cells; however, it is also expressed on dendritic cells (DCs) and eosinophils as shown by use of newly generated monoclonal anti-Siglec-G antibodies 1To whom correspondence should be addressed: Tel: +49-9131-85-28453; (Pfrengle et al. 2013; Hutzler et al. 2014). Both Siglecs are able Fax: +49-9131-85-28526; e-mail: [email protected] to associate to the BCR on B cells and inhibit BCR-mediated

© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 807 L Nitschke signaling. While both Siglecs can inhibit BCR signaling on B 1996; Otipoby et al. 1996; Sato et al. 1996; Nitschke et al. cells, Siglec-G deficient mice show that Siglec-G is mainly a 1997). A second phosphatase, the inositol phosphatase SHIP, non-redundant inhibitory receptor on B-1 cells, a B-cell subpo- can also be bound to the tyrosine phosphorylated CD22 tail in a pulation with special functions (Ding et al. 2007; Hoffmann complex with the adaptor proteins Grb2 and Shc, but has a so et al. 2007). In contrast, CD22 seems to be the dominant inhibi- far unknown function (Poe et al. 2000). The CD22 ITIM motifs tory Siglec for conventional B cells (also called B-2 cells). are critical for the inhibitory function of CD22, as has been However, when transfected into a chicken B-cell line, which shown in B-cell lines, but also recently in mice in which the resembles immature conventional B cells, Siglec-G can also three ITIM tyrosines were mutated (Müller et al. 2013). These Downloaded from inhibit surface (s)IgM-triggered signaling there (Hoffmann CD22 ITIM-mutated knockin mice (called CD22-Y2,5,6F et al. 2007). Also when mouse B cells are treated with high- mice, for mutations in the 2nd, 5th and 6th tyrosine of the three affinity Siglec-G ligands, BCR signaling can be suppressed in ITIMs) had a similar phenotype as CD22-deficient mice. conventional B cells (Pfrengle et al. 2013). The inhibitory sig- Siglec-G is a negative regulator of Ca2+ signaling specifically naling by CD22 and Siglec-G has been the subject of several in B-1 cells, as Siglecg−/− mice have a B-1-cell restricted pheno- recent reviews and will therefore be covered here only briefly type and Siglecg−/− B-1 cells show increased anti-IgM-induced http://glycob.oxfordjournals.org/ (Nitschke 2009; Jellusova and Nitschke 2012). Ca2+ signaling, in contrast to B-2 cells of these mice (Hoffmann The signaling function of CD22 has been dissected in detail et al. 2007). Siglec-G and the human ortholog Siglec-10 both by several groups. The intracellular tail of CD22 carries six tyr- have one ITIM and one ITIM-like sequence and can bind SHP-1 osines, which are the only known signaling modules of CD22. (as shown for Siglec-G) (Pfrengle et al. 2013)orSHP-1andthe Three tyrosines are found within conventional ITIMs (Y783, related phosphatase SHP-2 (as shown for Siglec-10) (Whitney Y843 and Y863), one is part of an ITIM-like motif (Y817) and et al. 2001). How these SHP phosphatases regulate downstream one is a Grb-2 binding motif (Y828) (Doody et al. 1995; signaling in B-1 cells is not known in detail as crucial signaling Otipoby et al. 2001) (Figure 1). Upon BCR cross-linking CD22 molecules which are part of the BCR signalosome, such as is rapidly phosphorylated by the Src kinase Lyn (Smith et al. PLCγ, Btk or SLP65/BLNK showed unchanged tyrosine phos- at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 1998). The phosphorylated ITIM residues of the CD22 tail are phorylation in Siglecg−/− B-1 cells (Hoffmann et al. 2007). The bound by the tyrosine phosphatase SHP-1, a central inhibitory transcription factor NFATc1 has elevated expression levels in signaling molecule (Doody et al. 1995). SHP-1 is the main Siglecg−/− B-1 cells, which could be an autoregulatory process downstream effector of CD22 and CD22-deficient B cells show due to increased Ca2+ signaling (Jellusova and Nitschke 2012). increased tyrosine phosphorylation of several proteins which One study also reported increased NF-kB activation in Siglecg−/− are involved in triggering proximal BCR signaling responses, B-1 cells, a finding that was not confirmed by another group such as SLP65/BLNK, Vav1 and CD19 (Fujimoto et al. 1999; (Ding et al. 2007; Jellusova and Nitschke 2012). Gerlach et al. 2003). Correspondingly, BCR-induced Ca2+ sig- Both CD22 and Siglec-G also modulate TLR signaling in B naling is strongly enhanced in Cd22−/− B cells (O’Keefe et al. cells. TLRs are pattern recognition receptors for bacterial or viral

Fig. 1. Regulation of BCR signaling by CD22 and Siglec-G binding to sialic acids in cis.(A) CD22 is an inhibitor of BCR signaling on conventional B cells (B-2 cells). CD22 forms homo-oligomers by binding to 2,6Sia (α2,6-linked sialic acids) on neighboring CD22 molecules. These CD22 homo-oligomers are normally separated from the BCR. Upon antigen binding, CD22 homo-oligomers are recruited to the BCR (indicated by red arrow), this leads to phosphorylation of ITIMs (immunoreceptor tyrosine-based inhibitory motifs) (indicated by Y in green boxes in the tail) in the intracellular tail of CD22. SHP-1 is recruited to the phosphorylated ITIMs and inhibits Ca2+ signaling. (B) Siglec-G mainly inhibits Ca2+ signaling in B-1 cells. Studies with Siglec-G ligand-binding mutants mice indicate that Siglec-G binds directly to glycosylated IgM via 2,6Sia or 2,3Sia (α2,3-linked sialic acid) binding. Siglec-G or the human ortholog Siglec-10 recruits SHP-1 to its phosphorylated ITIM. BCR: B-cell receptor, Y denotes a tyrosine residue in the Siglec tail. P denotes phosphorylation of tyrosines in ITIMs. 808 CD22 and Siglec-G on B cells

products. TLRs are expressed on cells of the innate immune ↑ system, but are also present on B cells. Both Siglec-G-deficient and CD22-deficient B cell showed increased proliferative ↑ n.d. ↑ n.d. responses when stimulated with TLR4, TLR7 or TLR9 ligands norm (3/4) (Kawasaki et al. 2011; Jellusova and Nitschke 2012). The exact mechanism of the negative regulation of TLR responses is not norm n.d. ↑ known; however, CD22 could inhibit TLR-4-dependent NF-kB ↓ norm n.d. activation and could reduce TLR-induced expression of SOCS1 Downloaded from and SOCS3 (Kawasaki et al. 2011). SOCS1 and SOCS3 are inhi- bitors in TLR-induced signaling. The negative regulation by normnorm norm norm norm IgM n.d. Siglecs on TLR signaling was also found for other Siglecs that )norm are expressed on cells of the innate immune system. Siglec-E has ↑ been shown to bind to SHP-1 and SHP-2 after LPS stimulation http://glycob.oxfordjournals.org/ ↑↓ norm norm norm n.d. n.d. n.d. ↑ and to inhibit TLR-mediated NF-kB activation after antibody- norm,( induced Siglec-E cross-linking (Blasius et al. 2006; Boyd et al. , increased; norm, normal; n.r., not relevant; n.d., not 2009). This indicates a possible signaling pathway that may also ↑ apply for CD22 and Siglec-G. Since upregulated TLR-7 signal- ing or co-engagement of TLR-7 and BCR has been linked to n.d. induction of autoimmunity (Viglianti et al. 2003; Deane et al. norm↑↑↓ n.d.↑ n.d. n.d. n.d. , decreased; 2007), enhanced TLR B-cells responses of Siglec-deficient B ↓ cells may contribute to occurrence of autoimmunity. at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 n.d. ↑↑ ↓ ↓↑↑↑ norm↓↑↑ norm norm norm norm n.d. n.d. n.d. ↓↑ B-cell development and immune responses in gene-targeted mice with mutated Cd22 or Siglecg genes Siglec-G is expressed from the pro-B-cell stage throughout the whole B-cell lineage with slightly higher levels on immature, B-1 or Marginal zone B cells than on follicular mature B cells, as n.r.n.r. n.d. n.d. n.d. norm ↓ found with new monoclonal antibodies (Pfrengle et al. 2013; n.r. Hutzler et al. 2014). Similarly, CD22 is expressed from pre-B cells onwards throughout the B-cell development (Torres et al. (B1) n.r. 1992). Both molecules seem not to be important for early B-cell (B1) ↑ ↑ ↑ n.d. n.d. development, as neither CD22- nor Siglec-G-deficient mice ↓↑ ↓↑ ↓ ↓ ↓ ↓↓ show impairment of B-cell differentiation in the bone marrow. ) norm n.d. CD22-deficient mice have a lower percentage of immature ↑ transitional B cells and a higher percentage of mature B cells in ↑ ↑↑ ↑↑ norm, ( ↓↑ the spleen. Splenic B cells also express higher levels of MHC ↓↑ class II, which is an activation marker on B cells (Otipoby et al. 1996; Nitschke et al. 1997; Gerlach et al. 2003). Both pheno- types indicate that Cd22−/− B cells are preactivated due to rm n.d. n.r.n.r. n.d. n.d. no norm n.r. stronger BCR signaling. Although mature follicular B cells are ↓ ) found in normal numbers in the spleen or lymph nodes of ↓ Cd22−/− mice, they show an increased apoptosis rate in vitro ↓ ↓ norm n.r. norm n.r. and a higher turnover in vivo, as indicated by BrdU incorpor- ↓

ation (Otipoby et al. 1996; Nitschke et al. 1997). Recently, CD22 ) knockin mice were generated in which Arg 130 was mutated, a ↑ critical residue for sialic acid binding (CD22-R130E) or where norm norm norm norm↑ normnorm norm norm norm n.d. ↑ norm ( norm norm, ( the three ITIM motifs were mutated (CD22-Y2,5,6F) (Table I). CD22-Y2,5,6F mice showed increased B-cell turnover and ↑↑ ↓ ↓ norm norm norm norm ↓↓ ↓ MZ B-1 Recirc CD22 expr BrdU incorp BCRCa2+ Siglec-IgM assoc. anti-IgM prolif TLR-ind prolif Serum IgM TD resp TI resp Auto-AB ↓ ↓ norm ↓ ↓ decreased survival, similar to KO mice, while both was un- ↓ changed in CD22-R130E mice (Müller et al. 2013). Also previ- ously generated ligand-binding mutant mice with mutated Arg130 and Arg137 (CD22-R130,137A) showed unchanged B-cell turnover, while mice with deleted first and second Ig-domains of CD22 (Cd22-D1-2), which are also defective in − − / 2, CD22R130,137A Sia binding surprisingly had increased B-cell turnover (Poe / − – Summary of phenotypes of mice with mutated CD22 or Siglec-G genes or with mutated genes for ligand-modifying enzymes − − − − 1 / / et al. 2004). Overall, these data show that the impaired B-cell / − Δ − survival and increased B-cell turnover is dependent on the − CD22x Siglec-G;double-KO ST6GalI ST6GalI × CD22;double-KO determined. If two phenotypes are given, two or more mouse strains show variations. References for these phenotypes are given in the text. Cmah MZ, B-1, recirc, number of MZB-cell B turnover; cells, BCR B-1 Ca2+, cells BCR-induced or Ca2+proliferation of recirculating signaling; B B Siglec-IgM cells cells; assoc., in TD association the of resp, bone TI CD22 marrow, or resp, respectively; response Siglec-G CD22 to to expr, T-cell CD22 mIgM; dependent expression Anti-IgM or level prolif, T-cell on anti-IgM-induced independent B proliferation; antigens; TLR-ind cells; respectively: prolif, BrdU auto-AB, TLR-induced incorp, autoantibodies; BrdU incorporation as measurement of CD22 Table I. Mice B-cell populations B-cell phenotype Signaling Immune responses CD22-Y2,5,6F CD22-R130E CD22 Siglec-G SIAE ITIM-signaling domain, but not on ligand binding (Table I). Siglec-G-R120E norm 809 L Nitschke

Despite these changes in B-cell turnover and despite changes B-1 cells are a special B-cell population, which is found at in B-cell signaling, CD22-deficient mice can mount normal unique locations, such as in the peritoneal and pleural cavity, is T-cell dependent immune responses (Otipoby et al. 1996; largely derived from fetal liver precursors and produces Igs with Sato et al. 1996; Nitschke et al. 1997). This is probably due to a restricted Ig repertoire. B-1 cells are responsible for secreting T-cell help, which can overcome the changed BCR signaling and the so-called natural antibodies of the class IgM which are im- apoptosis sensitivity. In support of this anti-IgM-induced prolif- portant for early anti-bacterial responses and are also thought to eration of Cd22−/− B cells is impaired, while anti-CD40 stimula- fulfill house-keeping functions, e.g., in clearance of apoptotic tion is normal (Table I). However, one study showed in a cells (Baumgarth 2011). In the mouse, two populations of B-1 Downloaded from BCR-transgenic mouse line that germinal centers (GC) formed cells can be found, the B-1a cells which are CD5+ and the B-1b earlier and GC B cells proliferated stronger in a T-cell dependent cells which are CD5−. Siglec-G-deficient mice have increased response, if CD22 was absent (Onodera et al. 2008). This shows populations of both B-1 cell subsets, but particularly strongly that under some conditions the loss of CD22 and increased BCR increased B-1a cell numbers. This B-1-cell expansion is B-1-cell signaling can drive B cells into earlier immune responses. intrinsic, as has been shown by mixed chimaera experiments Although CD22-deficient mice have normal mature follicular with adoptive transfers of bone marrow cells (Hoffmann et al. http://glycob.oxfordjournals.org/ B cells in spleen and lymph nodes, these cells are strongly 2007). B-1 cells are a population that has self-renewing capacity reduced in the bone marrow (Nitschke et al. 1997). Since the and the maintenance of this population depends on BCR signal- majority of mature B cells in the bone marrow are recirculating ing. Therefore, Siglecg−/− B-1 cells, which show stronger B cells, i.e., cells that have matured in the spleen and migrate BCR-induced Ca2+ signaling obtain a stronger survival/prolifer- back to the bone marrow, this indicated a migration or a sur- ation signal which partly explains their higher numbers. This vival defect. First experiments indicated a migration defect of was also directly demonstrated as Siglecg−/− B-1 cells show Cd22−/− B cells, as bone marrow endothelium expresses the increased survival in vitro and after adoptive transfer to RAG1−/− CD22 ligands: 2,6-linked sialic acids (2,6Sia) and transfer mice in vivo, when compared with WT B-1 cells (Jellusova, experiments of Cd22−/− B cells showed a migration defect to Duber, et al. 2010). The exact signaling pathway leading to at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 the bone marrow (Nitschke et al. 1999). However, mouse increased survival of Siglec-G-deficient B-1 cells is not known, models with mutated CD22 ligand-binding domains did not but might include the overexpression of NFATc1. NFATc1 is generally show the defect of mature B cells in the bone marrow. crucial for B-1-cell survival as has been shown in NFATc1- Furthermore, transfer experiments of B cells with mutant CD22 deficient mice (Berland and Wortis 2003). ligand-binding domains did not lead to impaired migration to Siglec-G-deficient mice have an enlarged B-1-cell population the bone marrow (Poe et al. 2004; Müller et al. 2013). In con- and these B-1 cells also show shifts in the produced Ig repertoire. trast, ITIM-mutated CD22-Y2,5,6F mice showed a reduced B-1 cells have a restricted VDJ segment usage producing recirculating B-cell population in the bone marrow and also a antibodies dominated by specificities for common bacterial struc- migration defect upon transfer (Table I). Since B-cell survival tures and also self-antigens. Siglecg−/− B-1 cells showed inter- is impaired in CD22-Y2,5,6F mice, it is likely that the defect of esting changes in the Ig repertoire (Jellusova, Duber, et al. 2010). bone marrow recirculating B cells in CD22-deficient mice is When B-1 cells of Siglec-G-deficient mice were stained with due to impaired B-cell survival, but not to ligand interactions to typical antigens recognized by mIgM on B-1 cells, such as phos- bone marrow endothelium (Müller et al. 2013). porylcholine (PC) or phosphatidylcholine, a lower percentage of CD22-deficient mice have a strong reduction of marginal zone these cells was found. However, IgM serum levels of Siglecg−/− (MZ) B cells in the spleen (Samardzic et al. 2002). These MZ B mice are
10-fold increased, so overall typical secreted anti-PC cells are found outside B-cell follicles and are important for antibodies or antibodies against oxidized lipoproteins, which are T-cell independent antigen responses. These responses are beneficial during artherogenic responses are normal or even reduced in CD22-deficient mice. Both CD22 knockin mice with increased in frequency (Jellusova, Duber, et al. 2010). mutated ITIM motifs or mutated ligand-binding domains show a Since Siglec-G does not show a B-1-cell restricted expression comparable MZ B-cell defect (Poe et al. 2004; Müller et al. within the B-cell lineage, it was unclear why Siglec-G-deficient 2013). This suggests that the BCR signaling response, which is mice show a B-1-cell restricted phenotype. Recently, mice with a normally modulated by CD22, as well as on possible ligand mutated ligand-binding domain of Siglec-G were generated interactions of CD22 to 2,6Sia-expressing cells in the spleen (Siglec-G R120E mice) and these mice showed a very similar regulate generation, maintenance or correct location of MZ B phenotype to Siglec-G-deficient mice: Increased B-1 cell cells. Altered chemokine responsiveness of CD22-deficient B numbers, increased Ca2+ responses just in B-1 cells, increased cellsmayplayaroleinthisMZB-celldefect(Samardzic et al. serum IgM levels and similar changes in the Ig repertoire of B-1 2002), but further mechanistic details are unclear. cells (Hutzler et al. 2014)(TableI). These data suggested that the Recently, the first monoclonal antibodies against Siglec-G ligand-binding domain of Siglec-G is crucial for the functions of were developed. Studies with both of these antibodies showed a this protein on B-1 cells. The mechanistic details of this are quite uniform expression of Siglec-G on the membrane of all discussed in the following. types of B cells, with just marginally higher levels on B-1 cells in the peritoneum than on B-2 cells (Pfrengle et al. 2013; Hutzler et al. 2014). Therefore, it is quite surprising that Siglecg−/− mice Ligand interactions of CD22 and Siglec-G show a B-1-cell restricted phenotype, not affecting the popula- tion of conventional B-2-cells. The population of B-1 cells is While it is well established that CD22 and Siglec-G are both in- strongly enlarged in Siglecg−/ − mice (Hoffmann et al. 2007). hibitory receptors on B cells, the important questions remain,

810 CD22 and Siglec-G on B cells why does this inhibitory function exist, under which physiologic- and a strongly impaired mIgM-triggered Ca2+ response al situation is the inhibitory signaling needed and how is this in- (Müller et al. 2013) (Table I). Surprisingly, two other mouse hibitory signaling regulated? Recent studies with new genetic models with mutated CD22 ligand-binding domains (CD22- mouse models show that ligand binding plays an important role R130,137A and CD22-Δ1-2) did not show this impaired Ca2+ in this regulation. The first Ig-domain of both CD22 and signaling (Poe et al. 2004). One possibility to explain this Siglec-G mediates binding to sialic acids. CD22 binds sialic difference is that in the latter two mouse strains the surface acids in the α2,6 linkage (2,6Sia) to galactose that is further expression of CD22 was reduced to 50%, maybe due to the attached to the core glycans of glycoproteins (Powell et al. 1993; introduced mutations. So two effects (stronger association to Downloaded from Kelm et al. 1994; Engeletal.1995). In contrast, Siglec-G binds the BCR and reduced surface expression) may compensate both 2,6Sia and α2,3-linked sialic acids (2,3Sia) (Duong et al. each other in this case. 2010). These sialic acids occur on many soluble glycoproteins in For Siglec-G much less is known concerning possible blood plasma, such as haptoglobulin or IgM (Hanasaki et al. cis-ligands. This is due to the fact that less tools, i.e., antibodies 1995; Adachi et al. 2012). Sialic acids are also abundantly for immuno-precipitations, etc., exist. However, recently, a mouse expressed on the surface of many human or murine cells. CD22 model with a mutated ligand-binding domain of Siglec-G http://glycob.oxfordjournals.org/ and Siglec-G can therefore potentially engage in many interac- (Siglec-G R120E mice) was published (Hutzler et al. 2014). This tions and bind sialylated glycoproteins on other cells (in trans) mouse model showed that apparently the association of Siglec-G or on the B-cell surface itself (in cis). Which of these interactions to mIgM is regulated quite differently from the association of can occur in vivo and which of these are physiologically relevant CD22 to IgM. A proximity-ligation experiment showed that is still an important issue of ongoing research. Siglec-G is normally associated with mIgM, an interaction, which The abundant expression of sialic acids on the cell surface of can be further increased upon BCR stimulation. Surprisingly, this cells of the immune system seems to “mask” most Siglec pro- interaction was almost completely gone in Siglec-G R120E B teins (Razi and Varki 1998). This means that the Siglec proteins cells. There was also an interesting difference found between B-1 are bound by cis-ligands, which limits their accessibility to cells and B-2 cells. B-1 cells expressed more 2,3 Sia on the at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 trans-ligands. This seems to be a general property within the surface, thus potential Siglec-G ligands, than B-2 cells (Hutzler Siglec family. The masking of Siglecs by cis-ligands does not et al. 2014). Since also the association of Siglec-G with mIgM mean that trans-interactions are not possible. This is probably a was higher on B-1 cells than on B-2 cells, these data suggest a dynamic process of competitive sialic acid-binding in cis or in direct Siglec-G binding to sialylated IgM, preferentially on B-1 trans, which depends on ligand availability and affinity, steric cells (Figure 1). Accordingly, Siglec-G R120E mice showed accessibility and cell–cell contacts. It has been demonstrated enhanced B-1 cell Ca2+ signaling, but normal B-2 cell Ca2+ sig- that CD22 can bind in cis to IgM or CD45 (Zhang and Varki naling, similar to Siglec-G-deficient mice. Also all other pheno- 2004). The proximity of CD22 to the BCR is crucial for the types affecting only B-1 cells were found similarly in both mouse CD22 inhibitory function. However, only a small fraction of strains (Hutzler et al. 2014). These data suggest that Siglec-G is CD22 molecules on the B-cell surface is associated with the recruited to mIgM by direct sialic acid binding and that sialic BCR (Peaker and Neuberger 1993). As the interaction of CD22 acid-binding positively regulates its function. with mIgM or CD45 on B cells is not lost upon sialidase treat- In addition to cis-binding, CD22 on B cells can also be ment or upon mutation of Arg130, a critical amino acid for engaged in trans-interactions to ligands on other cells. This has sialic acid binding, these interactions are sialic acid independ- been discussed to be relevant for homotypic B-cell–B-cell or ent and are likely protein–protein interactions (Zhang and Varki for B-cell–T-cell interactions. B-cell interactions with other 2004). Instead, CD22 binds to other sialylated CD22 mole- cells that express a cell-bound antigen together with 2,6Sia can cules, forming oligomers on the B-cell surface, as has been lead to suppression of B-cell signaling and B-cell proliferation demonstrated by a photoaffinity cross-linking approach (Han in a CD22-dependent way (Lanoue et al. 2002) (Figure 2). et al. 2005). These CD22 oligomers seem to be distinct from Also, ST6GalI-deficient B cells show an even CD22 distribu- the BCR on distinct membrane domains (Figure 1). An tion on the cells surface; however, when in contact with wild- increased association of CD22 with mIgM after BCR stimula- type B cells, CD22 is distributed to the cell contact side by tion suggests a recruitment of these CD22 oligomers to mIgM binding to trans-ligands (Collins et al. 2004). In a proteomic molecules (Müller et al. 2013). approach, one study identified trans-ligands for CD22 by How does this homo-oligomer formation of CD22 molecules glycan–protein cross-linking. From opposing B cells, mIgM affect the regulation of BCR signaling? Two genetic approaches was detected as a major in situ trans-ligand for CD22 (Ramya show that when CD22–ligand interactions on the B-cell surface et al. 2010). It has also been demonstrated that soluble glycosy- are disrupted a stronger association of CD22 with mIgM occurs lated pentameric IgM can be recognized by CD22 and in and a stronger inhibition of Ca2+ signaling results. The first ap- complex with antigen can suppress B-cell responses (Adachi proach is the knockout of the gene coding for ST6GalI, the et al. 2012). Early studies also suggested that CD45 on T cells enzyme that creates 2,6Sia linkages on glycoproteins. In this could be a trans-ligand for CD22 (Sgroi et al. 1995). It is so far case, a higher CD22-IgM association, a stronger CD22 tyrosine unknown what the physiological role of these natural trans- phosphorylation and an impaired BCR-induced Ca2+ signaling ligands is. However, synthetic high-affinity trans-ligands for was reported (Collins, Smith, et al. 2006; Grewal et al. 2006). CD22 or Siglec-G attached to liposomes or to high-molecular Similarly, CD22-R130E mice with a mutated ligand-binding weight carriers can quite efficiently suppress B-cell responses, domain showed higher IgM-CD22 association, a higher tyro- as will be discussed below (Macauley et al. 2013; Pfrengle sine phosphorylation of CD22, a stronger SHP-1 recruitment et al. 2013).

811 L Nitschke Downloaded from http://glycob.oxfordjournals.org/ at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016

Fig. 2. CD22 and Siglec-G maintain tolerance to self-antigens by binding ligands in trans.(A) When an autoreactive BCR binds a cell-bound autoantigen on another cell, 2,3Sia or 2,6Sia ligands adjacent to the autoantigen may recruit more CD22 and Siglec-G molecules into the vicinity of the BCR (indicated by red arrows), inhibiting autoantigen-induced BCR signaling by recruiting SHP-1. (B) In CD22 × Siglec-G double-deficient mice, this inhibition of BCR signaling by trans-ligand binding cannot take place, resulting in hyper-responsive B cells, generating increased Ca2+ flux. A similar phenotype was found in mice that cannot produce SIAE (sialic acid acetyl esterase), an enzyme that removes blocking acetyl groups from 2,3Sia or 2,6Sia ligands. The constant activation of B cells due to loss of Siglecs or Siglec ligands potentially results in differentiation to plasma cells (not shown here) producing autoantibodies directed against self-structures.

Sialyltransferases and other enzymes involved in the and many physiological processes, ST6GalI-deficient mice synthesis of CD22 and Siglec-G ligands show a B-cell restricted phenotype, which seems to affect mainly the function of CD22. This CD22-restricted function of Since binding of ligands is important for CD22- and Siglec-G- the enzyme was indicated by generation of ST6GalI × CD22 mediated functions, as shown in the previous paragraph, further double-deficient mice, which show an increased Ca2+ flux, insights can be obtained from mouse models in which the comparable with CD22-deficient mice (Collins, Smith, et al. enzymes creating these carbohydrate structures are deleted. 2006; Ghosh et al. 2006) (Table I). This demonstrates that loss Sialic acids are attached to glycans via α2,3, α2,6 or α2,8 lin- fi of CD22 is dominant and enhances B-cell signaling even in kages and these glycan modi cations are generated by distinct ST6GalI-deficient B cells. In contrast to CD22-deficient and enzymes. There exist four types of β-galactoside α2,3 sialyltras- α CD22 knockin mice with mutated ligand-binding domains, ferases (ST3Gal) in mice and 2,3Sia can be found on a great ST6GalI-deficient mice show strongly impaired immune variety of cells (Kono et al. 1997). 2,6Sia is less common and responses to both T-cell dependent and T-cell independent can be generated by several different enzymes (Takashima α immunizations (Hennet et al. 1998). The mechanism for this 2008). The CD22 ligand is 2,6-linked sialic acid coupled to impairment is unclear because impaired Ca2+ signaling cannot galactose and the ST6GalI sialyltransferase is the only enzyme explain this phenotype. Of note, also CD22 × Siglec-G double- which can produce these ligands and has been found to be deficient mice have a weaker T-cell dependent immune re- expressed in hematopoetic and liver cells (Wang et al. 1993). In sponse (Jellusova, Wellmann, et al. 2010). This could indicate contrast, Siglec-G ligands can be both 2,6Sia and 2,3Sia and that 2,6Sia ligand binding of both Siglecs in germinal centers is are therefore generated by different types of sialyltransferases important for these responses. (Duong et al. 2010). fi An enzyme that is also involved in generating CD22 and ST6GalI-de cient mice lack CD22 ligands and phenocopy Siglec-G ligands is the CMP-Neu5Ac hydroxylase (Cmah). In CD22 knockin mice with mutated ligand-binding domains (CD22-R130E mice) in several ways. They show impaired mice, sialic acids occur in two forms, Neu5Ac and Neu5Gc, BCR-induced Ca2+ signaling and show an increased binding which vary just in one oxygen atom at the C5 position. The fi of CD22 to mIgM. As a consequence, CD22 is higher phos- enzyme Cmah is responsible for this modi cation and transforms phorylated and more SHP-1 is recruited (Collins, Smith, et al. Neu5Ac to Neu5Gc (Naito et al. 2007). Humans have lost this 2006; Ghosh et al. 2006; Grewal et al. 2006). Thus, although enzyme during evolution and therefore exclusively produce the lack of 2,6Sia on cellular surfaces may affect many proteins Neu5Ac. While human CD22 can bind to both forms of sialic 812 CD22 and Siglec-G on B cells acids, murine CD22 prefers binding to Neu5Gc. Both human the interaction of human CD22 to this high-affinity ligand Siglec-10 and mouse Siglec-G prefer Neu5Gc binding. The also plays a role in vivo. monoclonal antibody GL7 has been found to specifically bind to Neu5Ac sialic acids in 2,6 linkage (Naito et al. 2007). This is of importance because GL7 is a widely used marker for murine ger- The role of CD22 and Siglec-G in tolerance minal center B cells. It has also been found that LPS-stimulated and autoimmunity B cells can be stained with GL7. These data indicate that the ligands Neu5Gc, preferred by murine CD22 and Siglec-G, are It is very important to regulate B-cell tolerance, as 30–40% of Downloaded from downregulated upon activation of B cells. Accordingly, it was human peripheral B cells can recognize self-antigens found that the expression of Cmah is strongly reduced in germi- (Wardemann et al. 2003). Self-reactive responses must be nal center B cells and binding of mCD22-Fc protein (a fusion avoided and inhibitory receptors play an important role in this protein consisting of the N-terminal domain of CD22 fused to process. Siglecs bind to sialic acids that can be regarded as human IgG Fc) is diminished. Whether this reduction of Siglec “self-structures” because they are abundantly expressed in ver- ligands in germinal center B cells is functionally relevant for tebrates, but normally lacking on pathogen surfaces. Thus, it http://glycob.oxfordjournals.org/ B-cell antibody responses is still unknown because Cmah-defi- was expected that a loss of inhibitory, self-structure-binding cient mice show normal T-cell dependent immune responses Siglecs on B cells may lead to development of autoimmunity. (Naito et al. 2007). Interestingly, activated T cells also downregu- Three out of four independently generated CD22-defcient late Cmah and lose the Neu5Gc species on the surface. Recently, mouse strains did not show this (Otipoby et al. 1996; Sato et al. published in vitro (non-antigen-specific) binding experiments 1996; Nitschke et al. 1997). However, in one strain, which was between B cells and T cells showed that Cmah−/− B cells could on a mixed 129/ C57BL/6 (B6) background, high-affinity anti- bind better to T cells than WT B cells (Naito-Matsui et al. 2014). bodies were produced (O’Keefe et al. 1999). Similarly, when This effect was CD22 dependent, as was shown by Cd22−/− CD22-deficiency was crossed to autoimmune-prone strains, mice and was explained by a loss of cis-ligand interactions and such as Y-linked autoimmune accelerator an increased auto- at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 the availability of trans-interactions to T cells via CD22. Thus, immunity was observed (Mary et al. 2000). Some lupus-prone activated B cells may be able to bind to naïve T cells by this mouse strains, such as NZW or BXSB strains express the allelic mechanism, but not to activated T cells, such as to Tfh cells in CD22a form, which has an impaired ability to bind to 2,6Sia the germinal center when a non-cognate interaction between B (Lajaunias et al. 1999). Backcrossing the CD22a allele into the and T cells is not favorable. B6 background resulted in preactivated B cells (Nitschke et al. A common sialic acid modification is O-acetylation at the C9 2006). All these experiments showed that loss of CD22 on its position. This modification prevents CD22 binding to sialic acids, own cannot cause autoimmune disease but can increase the as has been shown in vitro (Sjoberg et al. 1994). It is assumed susceptibility. that this acetylation similarly affects Siglec-G binding. The Also Siglec-G-deficient mice did not develop autoimmunity enzyme which creates these modifications, a sialic acid O-acetyl spontaneously. No high-affinity IgG autoantibodies were transferase is still not clearly identified. The human Cas1 protein detected. They just developed autoantibodies of the IgM class was described as a possible sialic acid-specific O-acetyl transfer- including rheumatoid factor and anti-erythrocyte antibodies ase, but it has to be confirmed whether this enzyme modifies (Hoffmann et al. 2007). Several autoimmune-prone mouse CD22- or Siglec-G ligands (Arming et al. 2011). In contrast, the strains, such as NZB/W or NZB, have an enlarged population enzyme sialic acid O-acetyl esterase (Siae), which can remove of B-1 cells and this population produces IgM that is to some acetyl groups from the 9-O position of sialic acids, is well charac- extent self-reactive. Therefore, this enlarged population in terized. Siae activity thus leads to creation of more CD22 and autoimmune-prone mouse strains was discussed to be important Siglec-G ligands. The phenotype of Siae-deficient mice resem- for development of autoimmune disease (Duan and Morel bles the phenotype of CD22-deficient mice (Cariappa et al. 2006). Siglec-G-deficient mice do not support this hypothesis, 2009). Ca2+ signaling is increased, MZ B cells and recirculating as B-1 cells are increased to a similar extent as in the B cells are decreased in these mice. The increase of Ca2+ signal- autoimmune-prone strains, but autoimmune disease does not ing in Siae−/− B cells is surprising, as these mice should pheno- develop (Hoffmann et al. 2007). Instead, the self-reactive copy ST6GalI−/− mice because in both cases CD22 and natural IgM produced by B-1 cells is also regarded to be im- Siglec-G ligands are reduced. ST6GalI mice showed impaired portant for house-keeping functions, such as clearance of apop- Ca2+ signaling due to stronger CD22-IgM interaction (Collins, totic cells (Baumgarth 2011). However, when arthritis was Smith, et al. 2006); however, this interaction was not studied in induced in the collagen-induced arthritis model, Siglec-G-defi- Siae−/− mice (Table I). Furthermore, Siae-deficient mice develop cient mice showed an earlier incidence and more severe arthritis autoimmunity, as will be discussed below. than control mice (Bökers et al. 2014). Similarly, when Another modification of 2,6-linked sialic acids is sulfation Siglec-G deficiency was crossed into the MRL/lpr background at the C6-position of GlcNAc which affects CD22 binding. It (which is a common lupus model), an earlier and more severe was first shown on glycan arrays that human CD22 prefers lupus disease was observed (Bökers et al. 2014). This increased this sulfated ligand, compared with the unsulfated ligand severity was accompanied by an earlier occurrence of high- affinity IgG autoantibodies. Apparently, B-1 cells played no (Blixt et al. 2004). Later, it was described that human B lym- role in this earlier onset and more pronounced autoimmune phocytes and endothelial cells express this sulfated CD22 disease, as this population was found in almost normal ligand (Kimura et al. 2007). This expression pattern of the numbers, compared with control MRL/lpr mice. These results α2,6 sialic acid 6-sulfo-GlcNAc ligand makes it likely that indicate that Siglec-G deficiency can increase the susceptibility 813 L Nitschke and severity of autoimmune diseases. It should be noted that inhibitory function of CD22 and decreasing B-cell Ca2+ Siglec-G is also expressed on the surface of dendritic cells and responses (Sieger et al. 2013). a loss of Siglec-G on these cells has been implicated to increase In addition to anti-CD22 antibodies high-affinity synthetic severity in a hepatocyte inflammation model and in bacterial CD22 ligands have been developed by a structure-based design, sepsis (Chen et al. 2009; Chen et al. 2011). This is not part of based on the known structure of the Siglec-binding first Ig this review, which concentrates on B cells, but is covered by domain of Siglec-1 with its ligand. Biphenyl groups were Chen (accompanying review in this journal). attached to the C-9 position of sialic acids in two different chem-

In contrast to single-deficient mice, CD22 × Siglec-G double- ical linkages and bound with increased affinity (IC50: 4 μM Downloaded from deficient mice develop significant levels of IgG autoantibodies, instead of 1.4 mM of the natural ligand Neu5Ac) and high speci- IgG immune complex kidney deposition and glomerulonephritis fi city to CD22 (Kelm et al. 2002). The compound BPC (bi- indicating a lupus-like disease (Jellusova, Wellmann et al. 2010). phenyl carbonyl)–Neu5Ac showed a high specificity for human This indicates that both Siglecs on B cells have redundant func- (h)CD22, whereas BPA (biphenyl-acetyl)-Neu5Ac or tions. Both are involved in the maintenance of B-cell tolerance, BPA-Neu5Gc was highly specific for murine (m)CD22. Linking but the loss of one B-cell Siglec can still be functionally compen- BPA-Neu5Gc in 2,3 or 2,6 linkage to lactose could then be used http://glycob.oxfordjournals.org/ sated by the presence of the other Siglec. Is the ligand-binding to generate high-affinity ligands with specificities for Siglec-G or function of Siglec-G and CD22 involved in this maintenance of murine CD22, respectively (Chen et al. 2010; Macauley et al. B-cell tolerance? A direct link to autoimmunity has also been 2013; Pfrengle et al. 2013). Several labs in the following time found for Siae, the enzyme which modifies CD22 and Siglec-G modified the hCD22-specific BPC-Neu5Ac ligands further by sialic acid ligands. Siae−/− mice spontaneously develop a lupus- additional modifying the C-2 position of the sialic acid, the C-4 like disease (Cariappa et al. 2009). Also, rare variants in the position or the combined C-2 and C-5 position of sialic acids, human SIAE gene, which lead to functional impairment of the reaching higher affinities (IC 50: 50–100 nM) (Abdu-Allah et al. enzyme, are associated with several autoimmune diseases 2009, 2011; Mesch et al. 2011; Kelm et al. 2013). Recently,

(Surolia et al. 2010). In genome-wide association studies, the combined modifications at the C-2, C-3, C-4, in addition to the at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 CD22 or SIGLEC10 genes have not been found to be associated BPC-group at C-9 were shown to even further increase the affin- with autoimmune diseases such as SLE. However, SNPs in ity for hCD22 to 2 nM. This synthetic sialoside then reaches an genes of the Siglec downstream inhibitory signaling pathways, affinity that is 7.5 × 105 times higher than the natural monomeric such as LYN or BLK have been associated with SLE (Harley ligand Neu5Ac (Prescher et al. 2014). et al. 2009; Cui et al. 2013). All these data can be summarized in Why is there so much interest in designing and improving a model that CD22 and Siglec-G contribute to B-cell tolerance high-affinity sialoside ligands? The natural sialic acids bind by binding sialic acid self-ligands in trans (Figure 2). This may CD22 in the millimolar affinity range, which is a weak affinity. be relevant when the B cell recognizes a cell-bound or cell The hope is that with nanomolar ligands for CD22 B-cell sig- surface expressed autoantigen on a neighboring cell. Additional naling of normal B cells can be modulated or that toxic agents binding of CD22 and Siglec-G to 2,6Sia and 2,3Sia containing can be introduced into malignant B cells by endocytosis. The glycoproteins in trans may recruit more of these Siglec mole- latter approach has been shown for B-cell lymphoma cell lines cules into the vicinity of the BCR, thereby increasing the inhibi- with oligo- or dimeric variants of BPC-Neu5Ac which were tory signal. Once both Siglec proteins and crucial trans-ligands able to kill these lymphoma cells in a CD22-specificway are lacking a break of B-cell tolerance can occur. (Collins, Blixt, et al. 2006; Schweizer et al. 2013). High-affinity CD22 ligands can enhance B-cell Ca2+ signaling, apparently by blocking the inhibitory function of CD22. The exact mech- CD22 and Siglec-G as therapeutic targets anism for this effect is not known (Kelm et al. 2002). But the higher the affinity the lower concentrations of the synthetic sia- CD22 is as a pan B-cell marker expressed on the surface of losides are needed for this signal modulation (Schweizer et al. most B-cell lymphomas and leukemias. CD22 has therefore 2013; Prescher et al. 2014). Recently, it has been shown that been a target for antibody-based therapies. Upon antibody BPA-Neu5Gc in 2,6 linkage to lactose coupled to liposomes binding, CD22 shows an efficient endocytosis (O’Reilly et al. can suppress mouse antibody responses to protein antigens 2011). Therefore, this can be used to target malignant B cells which were also attached to the same liposomes. This suppres- with antibody-bound bacterial or plant toxins. Such sion was CD22 specific(Macauley et al. 2013). Similarly, when CD22-specific immunotoxins have been successfully used in BPA-Neu5Gc was coupled in 2,3 linkage to liposomes this phase I clinical studies for hairy cell leukemia (Kreitman et al. could suppress antibody responses in a Siglec-G-specificway 2005). Also other agents such as RNases have been coupled to (Pfrengle et al. 2013). This shows that both Siglecs on B cells anti-CD22 antibodies to target B cells (Krauss et al. 2005). can be targeted specifically by liposomes containing both CD22 has also been successfully targeted by the humanized ligands and antigens. This approach has a future therapeutic po- anti-CD22 antibody epratuzumab that has been used in clinical tential for treatment of autoimmune diseases in an autoantigen- trials of B-cell Non-Hodgkin lymphoma patients (Leonard and specific fashion (Nitschke 2013). Goldenberg 2007). Since B cells also play an important role in the progression of SLE, epratuzumab has also been used suc- cessfully in a clinical phase I/II trial for SLE (Wallace et al. Conclusion 2014). In contrast to other B-cell-directed therapies, B cells were hardly depleted by epratuzumab in SLE patients. This CD22 and Siglec-G are two potent inhibitors of B-cell signaling. antibody seems to modulate B-cell signaling, by increasing the Genetic approaches have provided evidence that this inhibition of 814 CD22 and Siglec-G on B cells

BCR signaling is dependent on cis-ligand binding to sialic acids phosphorylated following lipopolysaccharide stimulation in order to limit in specific linkages. The ligand-binding activity of CD22 leads to TLR-driven cytokine production. J Immunol. 183:7703–7709. Cariappa A, Takematsu H, Liu H, Diaz S, Haider K, Boboila C, Kalloo G, homo-oligomer formation, which are to a large extent found in Connole M, Shi HN, Varki N, et al. 2009. B cell antigen receptor signal membrane domains distinct to those of the BCR. In contrast, strength and peripheral B cell development are regulated by a 9-O-acetyl Siglec-G is recruited via sialic acid binding to the BCR. This sialic acid esterase. J Exp Med. 206:125–138. interaction of Siglec-G to mIgM leads to an inhibitory function Chen GY,Chen X, King S, Cavassani KA, Cheng J, Zheng X, Cao H, Yu H, Qu that is much more pronounced in B-1 cells. The molecular details J, Fang D, et al. 2011. Amelioration of sepsis by inhibiting sialidase- mediated disruption of the CD24-SiglecG interaction. Nat Biotechnol. for this interaction are still not completely understood. Both 29:428–435. Downloaded from CD22 and Siglec-G are guards of B-cell tolerance and loss of Chen GY,Tang J, Zheng P, Liu Y.2009. CD24 and Siglec-10 selectively repress these proteins or its inhibitory pathways can increase the suscepti- tissue damage-induced immune responses. Science. 323:1722–1725. bility for autoimmune diseases. CD22 is a target protein both in Chen WC, Completo GC, Sigal DS, Crocker PR, Saven A, Paulson JC. 2010. In vivo targeting of B-cell lymphoma with glycan ligands of CD22. Blood. B-cell leukemias and lymphomas, as well as in B-cell-mediated 115:4778–4786. autoimmune diseases. Both antibodies and synthetic chemically Collins BE, Blixt O, DeSieno AR, Bovin N, Marth JD, Paulson JC. 2004. http://glycob.oxfordjournals.org/ modified sialic acids are currently tested for this purpose. Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact. Proc Natl Acad Sci USA. 101:6104–6109. Collins BE, Blixt O, Han S, Duong B, Li H, Nathan JK, Bovin N, Paulson JC. Acknowledgement 2006. 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. The author thanks the Deutsche Forschungsgemeinschaft for 177:2994–3003. support (through SFB643, TRR130). Collins BE, Smith BA, Bengtson P, Paulson JC. 2006. Ablation of CD22 in ligand-deficient mice restores B cell receptor signaling. Nat Immunol. 7:199–206. Conflict of interest statement Crocker PR, Paulson JC, Varki A. 2007. Siglecs and their roles in the immune system. Nat Rev Immunol. 7:255–266.

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815 L Nitschke

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