Tuning Antigen Receptor Signaling Review by CD22: Integrating Cues from Antigens and the Microenvironment

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Immunity, Vol. 6, 509–517, May, 1997, Copyright 1997 by Cell Press Tuning Antigen Receptor Signaling Review by CD22: Integrating Cues from Antigens and the Microenvironment

Jason G. Cyster* and Christopher C. Goodnow† not arrest development but may desensitize the signal- *Department of Microbiology and Immunology ing complex, provided a threshold receptor occupancy University of California, San Francisco is achieved (Goodnow et al., 1995). In mature B cells, San Francisco, California 94143–0414 potent antigen receptor cross-linking, for example by † Howard Hughes Medical Institute polyclonal anti-IgM antibodies or by T-independent anti- Beckman Center gens, can induce cells to enter the cell cycle, whereas Stanford University School of Medicine less multivalent antigens, including most T-dependent Stanford, California 94305 antigens, transmit signals that are insufficient to initiate the cell cycle. Cells engaging these antigens are highly receptive to T cell–derived signals, and the T cell and antigen receptor signals synergize to promote B cell Cell fate within multicellular organisms is often deter- proliferation or differentiation into antibody-secreting mined by the concerted action of several extracellular cells or germinal center cells. As well as regulating cell signals. In lymphocytes, a signaling complex that con- cycle entry and responsiveness to T cell help, antigen tributes to many cell fate decisions is the antigen recep- receptor triggering in mature B cells also affects cell tor, with different extents of antigen receptor engage- migration within lymphoid tissues, causing rapid exit of ment leading to such outcomes as cell death, survival, B cells from follicles or marginal zones and promoting or proliferation. Great strideshave beenmade in the past their accumulation in outer T cell zones and other sites two decades in understanding how antigen receptor (Liu etal., 1991; Cyster et al., 1994; Cyster and Goodnow, signals are transmitted within cells. It is only in the past 1995b; Shokat and Goodnow, 1995; Bachmann et al., few years, however, that an understanding has begun 1996; Fulcher et al., 1996). Signaling from the antigen to develop of what regulates the strength and quality of receptor is likely to be controlled at multiple levels, some the signal. Most excitingly, this regulation involves the molecules acting at the antigen receptor complex itself, action of multiple transmembrane molecules, leading to regulating signaling via multiple pathways, and others the recognition that the strength and quality of signal acting further downstream within specific signaling transmitted by the antigen receptor in response to an pathways. It is becoming increasingly apparent that sev- antigen may be regulated both by properties of the anti- eral of the proximal regulators of signal strength and gen and the microenvironmental context of the cell. quality are transmembrane molecules such as CD22. In this review we discuss the most recent studies on a transmembrane regulatory molecule on B cells, CD22, CD22: A Negative Regulator of Antigen Receptor Signaling and attempt to put these studies into the context of The B lineage molecule, CD22, is a member of the immu- other transmembrane molecules that regulate antigen noglobulin superfamily (Stamenkovic and Seed, 1990; receptor signal strength in B cells. Finally we speculate Wilson et al., 1991; Torres et al., 1992) and was sug- on how these molecules may function to relate microen- gested to participate in antigen receptor signaling fol- vironmental information to intracellular signaling from lowing the observation that treatment of cells with anti- the antigen receptor and cell fate. CD22 antibodies synergized with antigen receptor cross-linking in promoting B cell proliferation (Pezzutto B Cell Antigen Receptor Signaling Thresholds et al., 1987; Pezzutto et al., 1988; Tuscano et al., 1996a). The notion that CD22 contributed positively to antigen The core units of the B cell antigen receptor are transmembrane immunoglobulin (Ig) and the associated mol- receptor signaling gained further support with the dis- coveries that a fraction of CD22 within B cells can be ecules CD79␣ and CD79␤ (Ig␣ and Ig␤) (Reth, 1992). coprecipitated with the antigen receptor (Leprince et Antigen receptor cross-linking leads to activation of Src- al., 1993; Peaker and Neuberger, 1993) and that CD22 family kinases, including Lyn, Fyn and Blk, and phos- becomes rapidly tyrosine phosphorylated following anti- phorylation of tyrosines within ITAM motifs in the CD79 gen receptor engagement (Schulte et al., 1992; Leprince cytoplasmic domains. Protein tyrosine kinase Syk asso- et al., 1993; Peaker and Neuberger, 1993; Williams et ciates with the phoshorylated CD79 ITAMs and is acti- al., 1994). More recently it has been observed that CD22 vated by phosphorylation. The concerted action of Syk, can associate with the tyrosine kinases Lyn and Syk Src-family kinases, and other downstream kinases leads and with PI3-kinase and phospholipase C-␥ (PLC␥) (Law to the rapid phosphorylation of multiple substrates and et al., 1996; Tuscano et al., 1996b). to the activation of calcium pathways, the Ras/Erk path- The positive regulatory activity of CD22 was brought way, additional mitogen-activated protein–family ki- into question, however, when it was observed that the nases, and possibly other signaling pathways (Gold and SHP-1 protein tyrosine phosphatase (PTPase; pre- DeFranco, 1994). Antigen receptor signaling in B cells viously PTP1C), which negatively regulates antigen re- has different consequences depending on the stage of ceptor signaling (Cyster and Goodnow, 1995a), rapidly B cell maturation and the strength and duration of the associates with CD22 following antigen receptor en- signal. In immature B cells, extensive antigen receptor gagement (Campbell and Klinman, 1995; Doody et al., cross-linking reversibly arrests development and pro- 1995; Law et al., 1996). Characterization of the SHP- motes clonal elimination or receptor editing, whereas 1–binding sites in CD22 identified three of the six cyto- engagement of less potently cross-linking antigens does plasmic tyrosines, each in the motif V/IXYXXL, that when Immunity 510

Figure 1. Role of CD22 and SHP-1 in Negatively Regulating Signaling in Resting and Activated B Cells Spontaneous firing and antigen-induced firing of the antigen receptor (sIg) may normally be counteracted by CD22/SHP-1 such that in cells lacking either of these molecules, basal as well as antigen-induced signaling is elevated.

phosphorylated were able to bind and activate SHP-1 for SHP-1 and negatively regulate antigen receptor sig- (Doody et al., 1995). This tyrosine-based motif was pre- naling (Figures 1 and 2). Candidate targets for SHP- viously observed as a SHP-1–binding site in Fc receptor 1–mediated dephosphorylation are CD79␣ and ␤, Lyn, (FcR) Fc␥RIIb (D’Ambrosio et al., 1995) and has now Syk, CD19, and PLC␥, and a goal of future work will be been identified in the killer-inhibitory receptors on natu- to identify the specific targets of this phosphatase within ral killer (NK) cells that also recruit SHP-1 (Burshtyn et the cell after it is recruited to CD22. al., 1996). Down-modulation of sIgM on B lymphocytes occurs The potential role of CD22 as a negative regulatory rapidly following antigen engagement, the extent of molecule was supported by an experiment in which it modulation increasing with the dose and valency of anti- was cross-linked independently of surface immuno- gen (Goodnow et al., 1989). The exaggerated down- globulin (sIg) using anti-CD22 coated beads: an en- modulation of sIgM reported in mature CD22-deficient hanced anti-immunoglobulin response was observed, and SHP-1–deficient B cells compared to wild-type and it was suggested that drawing CD22 away from an cells, in the absence of increased antigen engagement, interaction with sIg limited the extent to which it could suggests that basal or constitutive signaling may be elevated in the absence of CD22/SHP-1–mediated nega- negatively regulate antigen receptor signaling (Doody tive regulation (Cyster and Goodnow, 1995a; O’Keefe et et al., 1995). This experiment could not fully exclude al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke the possibility that CD22 was transmitting a positive et al., 1997). Recruitment of SHP-1 to CD22 may not signal that synergized with sIg signaling, and this is be restricted to the period following antigen receptor where gene knockout experiments have filled an impor- engagement by antigen but may occur at a low level tant gap. B cells from mice made deficient in CD22 by continuously, counteracting the “spontaneous firing” of targeted gene disruption, established independently by the receptor (Figure 1). Following antigen receptor en- four groups (O’Keefe et al., 1996; Otipoby et al., 1996; gagement, CD22 phosphorylation is increased, and a Sato et al., 1996; Nitschke et al., 1997), show two charac- much larger battery of SHP-1 molecules is recruited and teristic and striking properties: exaggerated elevation activated (Figure 1). in intracellular calcium in response to antigen receptor SHP-1 is recruited to the cell membrane by multiple cross-linking, and spontaneous modulation of sIgM on receptor systems and in each case it appears to play a peripheral B cells. The similarity of this phenotype with negative role (Scharenberg and Kinet, 1996). Within B that previously observed for B cells deficient in SHP-1 lymphocytes, SHP-1 is recruited to the cytoplasmic do- (Cyster and Goodnow, 1995a) supports the view that a main of Fc␥RII following co–cross-linking of sIg and major function of CD22 in B cells is to serve as a dock Fc␥RII (D’Ambrosio et al., 1995), and SHP-1 recruitment Review 511

Figure 2. Model of Relationship among CD45, Lyn, CD22, and SHP-1 in B Cell Antigen Re- ceptor Signaling CD45-mediated dephosphorylation of Lyn (or related Src-family kinase) activates the kinase domain (red), which then phosphory- lates (yellow dots) multiple substrates, including CD22 (green). Ig␣ and Ig␤ also become tyrosine phosphorylated. Syk associates with phosphorylated Ig␣/␤, and SHP-1 with phosphorylated CD22. Additional associations of these molecules may also occur. The strength and quality of signals transmitted depend on the balance between recruitment of Syk (and other kinases) and SHP-1. The transition to an active complex is promoted by antigen receptor cross-linking but may also occur spontaneously. CD22/SHP-1 promotes inacti- vation of the signaling complex. may account for the down-regulation of signaling that reduced proliferative responses depending on the rela- occurs upon immunoglobulin–FcR co–cross-linking. tive amount of cell death, which might be influenced by However, recent studies suggest that SHP-1 cannot ac- such features as the type of anti-immunoglobulin cross- count for all of the negative regulatory activity of Fc␥RII linking agent used and the duration of the in vitro assay. and that the lipid phosphatase, SHIP, that is also re- The delicate balance between proliferation and death cruited to the receptor participates in the negative func- may also account for the reduced in vivo antibody tion (Ono et al., 1996). Because SHIP is recruited to response to type II T-independent antigens but the the same tyrosine-phosphorylated motif in Fc␥RIIb as normal or slightly elevated response to T-dependent SHP-1, it is possible that SHIP may also be recruited antigens in CD22-deficient animals. Type II T-indepen- by other molecules that have been found to bind SHP-1, dent antigens, such as the trinitrophenol-Ficoll used in such as CD22. This possibility warrants further investi- studying the CD22-deficient mice (Otipoby et al., 1996; gation since it bears on the issue of whether transmem- Sato et al., 1996; Nitschke et al., 1997), are multivalent brane regulatory molecules control the quality as well polymers and are thought to induce B cell responses as strength of antigen receptor signaling (Scharenberg by extensively cross-linking the antigen receptor. The and Kinet, 1996). However, the similar albeit less severe reduced response in some of the studies (Otipoby et phenotype of B cells deficient in CD22 compared to al., 1996; Nitschke et al., 1997) might therefore reflect SHP-1 deficiency argues that CD22 negatively regulates exaggerated signaling in TNP-specific B cells causing antigen receptor signaling largely by recruiting SHP-1. their elimination. T-dependent antigens, by contrast, The identification of a negative function for CD22 does generally have a lower valency and might be less likely not exclude the possibility that it may also have a posi- to induce apoptosis of CD22-deficient B cells. Further- tive signaling role. Two tyrosine-containing motifs in the more, presentation of the antigen to T cells should per- CD22 cytoplasmic domain are similar to the ITAM motifs mit rescue signals to be transmitted by various costimu- of CD3 and CD79 molecules (Leprince et al., 1993; latory molecules, such as CD40, whose function remains Peaker and Neuberger, 1993), and one or other of these intact in CD22-deficient B cells (O’Keefe et al., 1996; motifs may participate in recruiting Lyn, Syk, PI3-kinase, Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., and PLC␥1 (Law et al., 1996; Tuscano et al., 1996b). 1997). It is striking nevertheless that isotype switching, Complexes of CD22 containing Syk, PLC␥1, and SHP-1 germinal center formation, and somatic mutation con- have been reported(Law et al., 1996).The reduced prolif- tinue to occur in the absence of CD22, because these erative response of CD22-deficient B cells following an- processes are severely disrupted in animals deficient tigen receptor cross-linking may reflect a lack of positive in two other B cell transmembrane regulatory molecules, regulation by CD22 (Otipoby et al., 1996; Sato et al., CD19 and CD21 (Engel et al., 1995; Rickert et al., 1995; 1996). However, reduced proliferation was not a consis- Ahearn et al., 1996; Molina et al., 1996). These observa- tent observation in CD22-deficient cells, with one study tions suggest that CD22 does not play an essential role demonstrating increased proliferation (O’Keefe et al., in B cell–T cell interactions, despite the ability of the 1996), and an alternate explanation may be that CD22- extracellular domain to mediate interactions between deficient B cells transmit exaggerated signals for cell these cell types (Aruffo et al., 1992). death as well as cell cycle entry, a possibility directly demonstrated in one study (Nitschke et al., 1997). The CD22, CD5, and SHP-1 Regulate more rapid turnover of peripheral B cells in CD22-defi- Peritoneal B-1 Cells cient mice (Otipoby et al., 1996; Nitschke et al., 1997) Mice deficient in SHP-1 have an enlarged pool of B-1 and the failure to accumulate normal numbers of mature cells, with most B cells in spleen and lymph nodes bear- B cells in SHP-1–deficient mice (Cyster and Goodnow, ing the B-1 phenotype (Sidman et al., 1986). Many stud- 1995a) are also consistent with elevated signals leading ies indicate that the antibodies expressed by B-1 cells to increased cell death. Therefore the outcome of prolif- are polyreactive and bind weakly to multiple endoge- eration assays may easily be shifted from elevated to nous antigens (Kantor and Herzenberg, 1993), raising Immunity 512

the possibility that the type of antigen receptor signals indicating that tolerance mechanisms are probably not a B cell receives during development regulates entry disrupted in the animals. A more detailed investigation into the B-1 compartment. In immunoglobulin transgenic of B cell tolerance, perhaps using an immunoglobulin- mice it has been found that when immunoglobulin transgenic model, is needed to establish whether CD22 transgenes are derived from conventional (B-2) B cells, participates in setting thresholds of antigen receptor the transgenic mice make B-2 cells but few B-1 cells engagement for B cell elimination. (Kantor and Herzenberg, 1993). However, when SHP-1– deficient mice are crossed to immunoglobulin-trans- Lyn-Deficient and CD22-Deficient B Cells genic mice of the B-2 receptor type there is a marked Have a Similar Phenotype accumulation of peritoneal B-1 cells bearing this recep- Interestingly, increased numbers of B-1 cells, elevated tor specificity (Cyster and Goodnow,1995a), further sug- serum IgM and IgA, and reduced B cell lifespan are also gesting that SHP-1 normally functions to inhibit B cell properties observed in Lyn-deficient mice (Hibbs et al., accumulation in this compartment. B-1 cells express 1995; Nishizumi et al., 1995). Although the primary stud- CD22 (Andersson et al., 1996; Erickson et al., 1996), ies of Lyn-deficient animals showed reduced anti-immu- and it was therefore important to ask whether B-1 cells noglobulin induced proliferative responses, these ani- accumulated to a higher frequency in CD22-deficient mals may have been deficient in conventional B cells mice. A small increase in the size of this compartment by the time of analysis because recent experiments with was seen in two of the gene knockout studies (O’Keefe cells from young mice show exaggerated proliferative et al., 1996; Sato et al., 1996). responses (Wang et al., 1996). These studies together The increase compared to SHP-1–deficient non- highlight similarities between Lyn-deficient, SHP-1– transgenic mice (Sidman et al., 1986) might be limited deficient, and CD22-deficient B cells and raise the pos- because some B-1 cells harbor an additional SHP-1 siblity that a major function of Lyn could be phosphoryla- docking molecule, CD5 (Bikah et al., 1996). CD5 is a tion of transmembrane regulatory molecules to permit member of the scavenger receptor family, and its cyto- recruitment of SHP-1. Strongly supporting this possibil- plasmic domain contains an ITAM-like motif that can ity, Fc␥RII-mediated inhibition of sIg signaling is defec- interact with several SH2 containing proteins, including tive in Lyn-deficient cells (Wang et al., 1996). In Figure SHP-1. In T lymphocytes, CD5 associates with the anti- 2 a model is presented in which Lyn-mediated phos- gen receptor and, as observed in studies with CD22, phorylation of CD22 leads to SHP-1 recruitment from cross-linking CD5 can transmit signals that augment the cytosol, activation, and dephosphorylation of Syk T cell receptor (TCR) signaling. The principal T cell phe- and other positive regulatory molecules. An important notype in CD5 deficient mice, however, is an exagger- task of future studies will be to determine whether Lyn ated signaling response consistent with a negative regu- is required for SHP-1 recruitment to CD22. Equally im- latory role for CD5 (Tarakhovsky et al., 1995). Although portant will be to test whether signaling pathways in most B lymphocytes are CD5-negative, a fraction of addition to calcium elevation are affected by CD22 (and peritoneal B-1 cells express CD5 (also called Ly1). B-1 SHP-1) deficiency in B cells and thereby to learn whether cells normally make an abortive attempt at proliferation CD22/SHP-1 regulates the quality as well as the quantity and undergo apoptosis following antigen receptor of the antigen receptor signaling response. cross-linking, and this has beenassociated with a dimin- The more severe B cell phenotype in SHP-1 deficient ished calcium response and failure to translocate NF-␬B mice compared with that associated with CD22 or Lyn to the nucleus (Morris and Rothstein, 1993). In CD5- deficiency raises the possibility that SHP-1 may also deficient B-1 cells, by contrast, antigen receptor cross- regulate B cells by CD22- and Lyn-independent path- linking induces a more sustained calcium response, nu- ways. The B cell phenotype associated with SHP-1 defi- clear translocation of NF-␬B, and a strong proliferative ciency results in part from trans-acting effects through response (Bikah et al., 1996). Cross-linking CD5 inde- macrophage dysregulation, but when these are con- pendently of sIg on wild-type B-1 cells, as in the studies trolled for the failure of mature B cells to accumulate with CD22, also provoked a strong proliferative re- still appears to be more severe (Cyster and Goodnow, sponse. These studies are consistent with a negative 1995a). This difference might be accounted for by SHP-1 regulatory activity for CD5 in B-1 cells. Although no recruitment to other cell surface receptors in CD22-defi- increase in B-1 cell frequency is detected in CD5Ϫ/Ϫ cient cells, such as Fc␥RIIb (D’Ambrosio et al., 1995) mice, it is possible that CD22 sufficiently compensates and sIg itself (Pani et al., 1995). to prevent their accumulation. B-1 cells are thought to be the major source of natural antibodies in the serum of unimmunized mice (Kantor CD45: A Positive Regulator of Antigen and Herzenberg, 1993), and in animals with excess B-1 Receptor Signaling cells, elevated serum antibody levels are usually ob- Acting upstream of Lyn, and presumably of CD22 and served (Sidman et al., 1986; Kantor and Herzenberg, SHP-1, is another transmembrane regulatory molecule, 1993). It seems likely therefore that the increased serum CD45. CD45 is a PTPase that positively regulates anti- IgM and IgA levels reported in 4–6-month-old CD22- gen receptor signaling at least in part by dephosphory- deficient mice are a result of the increased numbers of lating the negative regulatory carboxy-terminal tyrosine B-1 cells (O’Keefe et al., 1996; Sato et al., 1996). A small residues on Src-family tyrosine kinases, activating the elevation in the titer of autoantibodies seen in some kinases (Chan et al., 1994; Trowbridge and Thomas, CD22-deficient mice does not appear greater than the 1994). In vivo studies in CD45-deficient mice (Kishihara increase in total antibody titer (O’Keefe et al., 1996), et al., 1993) have confirmed the positive role of CD45 Review 513

in antigen receptor signaling: B lymphocytes lacking between mature B cells and other cells or molecules in CD45 show reduced activation of calcium and Erk sig- peripheral microenvironments. naling pathways and have an elevated threshold for elimination by self-antigen (Cyster et al., 1996). A striking Role of the Ectodomains in Regulating finding in CD45-deficient animals carrying immunoglob- Cytoplasmic Domain Function ulin transgenes specific for the soluble antigen hen egg A question that must be raised in considering the func- lysozyme was that accumulation of mature B lympho- tion of CD22, CD45, and other transmembrane mole- cytes is impaired but is rescued when the cells devel- cules that regulate antigen receptor signaling in lympho- oped in the presence of the soluble hen egg lysozyme cytes is: What is the function of their ectodomains? If autoantigen (Cyster et al., 1996). Just as too much sig- all that these molecules needed to do was to provide naling from the antigen receptor shortens cell lifespan, cytoplasmic docking or catalytic sites, one might have as may occur in SHP-1– or CD22-deficient B cells, insuf- predicted that their ectodomains would be similar to ficient signaling also appears to allow B cell death. The the CD3 or CD79 chains—that is, molecules with small need for an intact antigen receptor signaling complex extracellular domains (or none atall) that associatefirmly for the transition from immature to mature B cell, or with the antigen receptor complex. Instead, CD22, persistence of mature B cells, received further support CD45, and several other regulatory molecules have large from an experiment demonstrating that B cells with a extracellular domains, some of which can exist in multi- mutation in the CD79␣ cytoplasmic domain fail to accu- ple alternatively spliced forms and have well-defined mulate in the periphery (Torres et al., 1996). ligand-binding domains (Trowbridge and Thomas, 1994; In addition to the positive signaling pathways that we Tedder et al., 1997). Current insight into how these do- visualize CD45 to activate, by dephosphorylating and mains may regulate intracellular domain function has activating Lyn, CD45 likely promotes phosphorylation come from studies with antibodies and chimeric mole- of CD22, recruitment of SHP-1, and down-regulation of cules. As we have already discussed, cross-linking the tyrosine kinase–mediated signaling response in a CD22 or CD5 independently of the antigen receptor negative feedback loop. In this sense CD45 is also play- complex can enhance signaling from the receptor, sug- inga negative role. An important parameter in controlling gesting that one function of the ectodomains may nor- the relative intensity of positive and negative pathways mally be to regulate the extent of association with the activated by the antigen receptor is likely to be the rela- antigen receptor. Elegent studies with chimeric mole- tive amount and distribution of CD45 and CD22 (or CD5) cules containing the CD45 cytoplasmic domain linked at the cell surface. to the epidermal growth factor receptor extracellular domain demonstrated that dimerizing this molecule in- activated the positive signaling role of the CD45 cyto- Immature B Cells Express Surface CD22 plasmic domain (Desai et al., 1993). Ligand binding to Many studies have suggested that CD22 is not ex- the intact CD45 molecule might therefore be important pressed on the surface of immature B cells (Campana in controlling the strength of signaling from the antigen et al., 1985; Dorken et al., 1986; Loken et al., 1987; receptor. In speculating on the physiologically relevent Andersson et al., 1996; Erickson et al., 1996). Because ectodomain interactions that might regulate intracellular SHP-1 negatively regulates signaling in immature bone domain function, three types must be considered: (1) marrow B cells (Cyster and Goodnow, 1995a), the iden- interactions between molecules on the same cell; (2) tity of the membrane docking molecule for SHP-1 in interactions between molecules on different cells; and immature B cells has been unclear. By using the CD22 (3) interactions with soluble antigen complexes and knockout as a staining control, it has now been shown other molecules. compellingly that CD22 is expressed on the surface of (1) Tuning the Antigen Receptor by Interactions both pre-B and immature B cells (Nitschke et al., 1997). between Ectodomains on the Surface CD22 may therefore act as an important membrane of the Same Cell docking molecule for SHP-1 in immature as well as ma- The proximity of CD22, CD45, and other regulatory mole- ture B cells. In the transition from immature to mature B cules with the antigen receptor is likely to be critical in cells there is an approximately 5-fold increase in surface regulating antigen receptor signaling. The use of extra- CD22 expression. Whether this increase equates to an cellular domains to mediate such associations might increased extent of negative regulation of antigen recep- provide a means of regulating the extent of the associa- tor signaling in mature B cells compared to immature B tion, depending on cellular context. CD22 contains a cells (O’Keefe etal., 1996) remains to be established. The sialic acid ␣2–6Gal binding lectin in its two amino-termi- concomitant increase in surface expression of positive nal domains (Stamenkovic et al., 1991; Sgroi et al., 1993; regulatory molecules CD45, CD19, and CD21 (Tedder Engel et al., 1995; Law et al., 1995; van der Merwe et et al., 1997) suggests that this interpretation may be too al., 1996), and it may be capable of binding to IgM mole- simplistic. Furthermore, to the extent that it has been cules on the cell through ␣2,6-sialylated sugars expressed tested, immature B cells do not show greater activation on IgM (Hanasaki et al., 1995). Competition for binding of intracellular signaling pathways in response to anti- to other ␣2,6-sialylated proteins could then alter this gens than mature B cells (Yellen et al., 1991; Cyster association (e.g., when the cell is in a particular microen- and Goodnow, 1995a). An alternative explanation for the vironment) and in turn affect antigen receptor signaling. increased expression of these regulatory molecules on As B cells become activated, they increase expression the cell surface as B cells mature is that their extracellu- of ␤-galactoside ␣2,6-sialyltransferase (␣2,6-ST) (Er- lar domains serve important functions in the interactions ikstein et al., 1992). Sialylation of CD22 itself can block Immunity 514

Figure 3. Possible Roles for the CD22 Ecto- domain (A) CD22 recognizes the sialylated carbohydrate side chains of membrane IgM on resting B cells and negatively regulates antigen receptor signaling. Late after activation, the B cell increases ␣2,6-ST expression, and this may cause theCD22 lectin domain to become blocked by its own sialic acid groups, reduc- ing its binding to IgM and permitting en- hanced BCR signaling. (B) In a nonlymphoid microenvironment, the level of ␣2,6-sialylated carbohydrate may be low, favoring CD22–IgM association and negative regulation. When the B cell enters a lymphoid microenvironment that is rich in ␣2,6-linked sialic acid, CD22 may be drawn away from IgM, augmenting signaling. (C) BCR binding of an antigen–serum IgM complex or serum IgM itself (in the case of an autoreactive B cell) may recruit CD22 into an association with membrane IgM and inhibit signaling. Complement C3d fixation on the IgM complex might be expected to over- ride such inhibition by recruiting CD21/CD19.

the ligand-binding domain (Braesch-Andersen and Sta- expression of ␣2,6-ST by B cells along with the high menkovic, 1994), and this alteration could be a means expression of heavily glycosylated CD45 makes this in- by which the cell regulates the extent of sIg–CD22 asso- teraction very probable. The physical dimensions of ciation (Figure 3A). CD45 and CD22 are likely to be similar, which might Different splice variants of CD22 that lack either im- further favor an interaction. The functional signifance of munoglobulin domains 3 or 3 and 4 have been described such an interaction is unexplored. (Stamenkovic and Seed, 1990; Wilson et al., 1991; Engel (2) Tuning the Antigen Receptor by Ectodomain- et al., 1995), raising the possiblity that alternate splicing Mediated Interactions between Molecules of the extracellular domain might also be used by the on Adjacent Cells cell as a means of altering association with sIg. Such a The best-characterized antigen receptor regulatory mechanism has previously been suggested to regulate molecules in this class are CD4 and CD8 on T cells. interactions between CD45 and the CD4–TCR complex Their recruitment into the TCR–major histocompatibility (Leitenberg et al., 1996). As T cells differentiate in re- (MHC)–peptide complex plays a positive role by bringing sponse to antigen receptor signals they express variants p56lck into the signaling complex (Weiss and Littman, of CD45 with fewer amino-terminal exons. An increased 1994). Less well-characterized examples include the association of these isoforms with CD4 appears to occur regulation of TCR signaling by interactions between B7 and has been suggested to improve the strength or molecules on presenting cells and CD28 or CTLA4 on quality of antigen receptor signaling (Leitenberg et al., T cells, and the inhibition of NK cell cytotoxicity by inter- 1996). Another interaction that may take place on the actions between MHC molecules on target cells and surface of B cells and that may be of functional impor- killer inhibitory receptorson NK cells. The possibility that tance occurs between CD22 and CD45. CD45 binding CD22 and CD45 interact between cells during antigen to CD22 has been well documented, and the strong presentation by B cells to T cells has been suggested Review 515

(Stamenkovic et al., 1991; Aruffo etal., 1992). CD22 is not antigen that take place during an immune response and expressed by other antigen-presenting cells, however, so amplify or down-regulate the response as appro- indicating either that these cells express other mole- priate. cules that serve the same purpose, or that the interaction The observation that a major ␣2,6-sialylated ligand plays a role that is particular to B cell–T cell interactions. for CD22 present in serum is IgM raises the possibility The latter possibility deserves further consideration that CD22 might also participate in this type of interac- since many studies suggest that T cells are poorly acti- tion (Hanasaki et al., 1995). CD22 might act as a form vated following an encounter with antigen presented by of IgM FcR, being recruited to the antigen receptor when B cells; perhaps CD22-mediated cross-linking of CD45 the cell binds IgM–antigen complexes. Such an interplay inactivates the positive regulatory function of the phos- could be especially important in blocking the response phatase on the T cell. A general dilemma with implicating of self-reactive B cells that recognize the idiotype of a a role for CD22 and CD45 in T cell–B cell interactions secreted IgM molecule (Figure 3C). Measuring the rela- has been the large dimensions of these proteins (van tive affinity of CD22 for different glycoproteins, and es- der Merwe and Barclay, 1994). The dimensions of CD4 tablishing whether the protein backbone of sialylated and CD8 are similar to the TCR and MHC and can there- ligands contributes to binding affinity should help to fore be accommodated near these complexes at cell– define which of the many possible CD22 ectodomain cell interfaces (van der Merwe and Barclay, 1994). interactions are of physiological importance. Whether CD45 and CD22 could be accommodated at In summary, CD22 plays an important role as a nega- such an interface is unknown. tive regulator of antigen receptor signaling in B lym- A possibility that has already been discussed is that phocytes, exemplified by the exaggerated signaling, the ectodomains of these large molecules function increased cell death, and reduced response to T-inde- more in communicating to the cell its microenvironmen- pendent antigens of CD22-deficient cells. CD22 is also tal context rather than participating directly in close cel- important in limiting the size of the B-1 cell compartment lular associations. Lymphocytes continually recirculate and in controlling production of IgM and IgA. CD22 around the body, moving through nonlymphoid and stands as a striking example of a transmembrane mole- lymphoid tissues. A B lymphocyte might encounter anti- cule with an extracellular ligand-binding domain of un- gen outside a lymphoid microenvironment, but it may known function and an intracellular domain that plays be advantageous to delay full activation of the cell until an integral role in controlling the quality or quantity of it enters an appropriate lymphoid microenvironment. Al- signals from the antigen receptor. As the interactions though relative expression of ␣2,6-sialylated carbohy- between the cytoplasmic domains of CD22 and related drates has not been widely characterized, it has been molecules with intracellular signalingmolecules become suggested that a lower level of such glycans in non- better defined, the major challenge for the future will be lymphoid tissues may promote CD22–sIg associations to understand the nature of the extracellular cues that that dampen signaling, whereas in lymphoid tissue ␣2,6- regulate cytoplasmic domain function, intracellular sig- sialylated glycans on other cells may draw CD22 away naling, and cell fate. from the sIg association and enhance signaling (Figure 3B; Doody et al., 1995). Within lymphoid tissues, ligands Acknowledgments for CD22 are differentially expressed, being highest within B cell zones (Sjoberg et al., 1994). Perhaps when J. G. C. is a Pew scholar and C. C. 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