Band 3 protein clustering on human erythrocytes promotes binding of naturally occurring anti-band 3 and anti-spectrin antibodies

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Experimental Gerontology 35 (2000) 1025–1044 www.elsevier.nl/locate/expgero

Band 3 protein clustering on human erythrocytes promotes binding of naturally occurring anti-band 3 and anti-spectrin antibodies R. Hornig 1, H.U. Lutz* Institute of Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, CH 8092 Zurich, Switzerland Received 26 April 2000; accepted 25 May 2000

Abstract Recognition of senescent and oxidatively stressed human erythrocytes appeared to be initiated by band 3 clustering, followed by bivalent binding of naturally occurring anti-band 3 autoantibodies (anti-band 3 NAbs), and complement deposition. The number of RBC-associated anti-band 3 NAbs was, however, low compared to the total amount of IgG that bound in vitro to RBC containing band 3 oligomers. This implied the involvement of yet other types of NAb, among which we focussed on anti-spectrin NAbs, since eluates from RBC of thalassemic patients contained these NAbs. Binding of affinity-purified anti-band 3 and anti-spectrin NAbs was studied to RBC on which band 3 oligomers were generated by exoplasmic cross-linking. This pretreatment increased binding not only of 125 I-iodinated anti-band 3, but also of anti-spectrin NAbs by 7–10-fold at 0⬚C in the presence of nearly physiological IgG and HSA concentrations. Binding of anti-spectrin NAbs was not to spectrin as judged from surface-labeling of RBCs that were pretreated with cross-linker. Binding was dose and time dependent in both cases. Moreover, binding of anti-spectrin NAbs was not competed by high concentrations of anti-band 3 NAbs and anti-spectrin NAbs even stimulated binding of antiband 3 F(ab 0 )2 by 30%. This suggests that anti-spectrin NAbs bound to band 3 or a protein associated with band 3 by virtue of their inherent polyreactivity. 䉷 2000 Elsevier Science Inc. All rights reserved. Keywords: Naturally occurring antibodies; Red blood cell; Anti-spectrin; Anti-band 3; Band 3 oligomerization

Abbreviations: BS 3, bis(sulfosuccinimidyl)suberate; IOV, inside-out vesicles; DFP, di-isopropyl fluorophosphate; NAb, naturally occurring antibody; NEM, N-ethylmaleimide; PS, phosphatidylserine; sNHS-B, N-hydroxysuccinimidyl biotin * Corresponding author. Tel: ⫹41-1-632-3009; fax: ⫹41-1-632-1269. E-mail address: [email protected] (H.U. Lutz). 1 John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra ACT 2601, Australia. 0531-5565/00/$ - see front matter 䉷 2000 Elsevier Science Inc. All rights reserved. PII: S0531-556 5(00)00126-1

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1. Introduction Senescent as well as oxidatively stressed RBC are opsonized by preexisting antibodies (NAbs) and complement (Lutz et al., 1987, 1988) and engulfed by macrophages following their binding to Fc- and complement receptors (for review see Garratty, 1991). Recently it has been reported that even unopsonized damaged and oxidatively stressed human RBC are phagocytosed in vitro by subpopulations of animal macrophages through binding to scavanger receptors (Sambrano et al., 1994). Binding to macrophages was inhibited by ligands of scavenger receptor I and PS-containing liposomes (Pradhan et al., 1994). These data and the fact that senescent RBC are slightly enriched in PS in the outer monolayer (Connor et al., 1994) surmise physiological relevance, since PS exposure appears to be a phagocytosis trigger for apoptotic cells (for review see Bevers et al., 1998). In the case of senescent and oxidatively stressed RBC, it is questionable whether PS exposure could serve as a recognition signal, since RBC with an accelerated in vivo clearance from patients with several types of anemia did not expose additional PS (Boas et al., 1998). Thus, in vitro and in vivo recognition may occur by different routes. Physiologically relevant clearance mechanisms may require binding to two independent receptors (Lutz, 1995; Balasubramanian and Schroit, 1998) with the route being determined to a large extent by the ability of a ligand to bind to the target cell in the presence of other plasma constituents. Anti-band 3 NAbs did bind in the presence of plasma to oxidatively stressed RBC (Lutz et al., 1987) and stimulated C3b deposition via the classical pathway and through the amplification loop, because they preferentially formed C3b2 –IgG complexes that maintain this process (Lutz et al., 1993b). Their binding was to band 3 oligomers and was enhanced whether oligomerization of band 3 occurred by oxidative damage (Lutz et al., 1987), by binding of hemichromes to the cytoplasmic portion of band 3 (Low et al., 1985; Schlu¨ter and Drenckhahn, 1986) or by an altered band 3 modification as in dyserythropoietic anemia (Defranceschi et al., 1998). Oligomerization of band 3 resulted in increased binding of anti-band 3 NAb, since oligomers enable a bivalent binding of these antibodies which is required for them to overcome their low affinity (Lutz et al., 1987; Lutz et al., 1993a). The number of anti-band 3 NAb specifically associated with oxidatively stressed RBC was, however, low in 70% serum (ⱕ20 lgG per cell) (Lutz et al., 1987), as compared to the extent of opsonization achieved with induced anti-RBC antibodies. In contrast to this, binding of whole human IgG from plasma was almost two orders of magnitude higher, when band 3 was artificially oligomerized by Zn 2⫹ ions and the oligomers stabilized with a cross-linker (Turrini et al., 1991). Though the two sets of data cannot be compared directly, the evidence strongly suggests that band 3 oligomerization may not only enhance binding of anti-band 3 NAb, but also of other types of NAb, whose specificity was not investigated. It was known, however, albeit difficult to put into the right perspective that eluates from in vivo aged RBC of patients with b-thalassemia contained anti-spectrin NAbs (Wiener et al., 1986). It appears possible that anti-spectrin NAbs, which exist in human plasma (Lutz and Wipf, 1982), may assist in opsonizing RBC by binding to oligomers of negatively charged proteins, since the majority of these NAbs are polyreactive (Coutinho et al., 1995; Chen et al., 1998) and have an excess of cationic groups (Labrousse et al., 1994; Lutz et al., 1996). We therefore studied in detail the binding of affinity-purified anti-band 3 (Lutz et al., 1984) and anti-spectrin NAbs (Lutz

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and Wipf, 1982) to RBC in which band 3 oligomers were formed by the least perturbing method, an exoplasmic cross-linking of band 3 without addition of zinc ions.

2. Materials and methods 2.1. Preparation of human RBCs Whole human blood (ORh ⫹) collected into CPD-adenine (Swiss Red Cross, Blood Donation Service, Zurich) was diluted with half a volume of PBS-G (10 mM NaKHPO4, 150 mM NaCl, 1 g/l d-glucose, pH 7.4). Proteases were inhibited by adding 0.8 mmol/l DFP to the diluting buffer. This suspension was passed over a cellulose column to remove white blood cells (Beutler et al., 1976; Lutz et al., 1992). The filtered blood was used immediately. RBCs were washed three times with 10 volumes of PBS-G. RBC numbers, hct and mcv were measured using a Sysmex F-800 microcellcounter (TOA Medical Electronics, Kobe, Japan). Where indicated, density fractionated RBC were isolated as given elsewhere (Lutz et al., 1992). 2.2. Isolation of RBC membranes and extraction of spectrin Washed RBCs were lysed with 30 volumes of cold hemolysis buffer (5 mM NaKHPO4, 1 mM EDTA, pH 7.4). Membranes were pelleted at 40 000g and washed twice in the same buffer. The first wash buffer was supplemented with 0.8 mmol/l DFP. RBC membranes were solubilized and alkylated by adding a final concentration of 1% SDS and 5.0 mmol/l NEM, diluted with hemolysis buffer to the initial volume of packed cells, and stored in aliquots at ⫺70⬚C. To study the accessibility of spectrin, peripheral membrane proteins were extracted from membranes (Bennett and Branton, 1977). 2.3. Chemical and enzymatic modifications of RBCs Washed RBCs were resuspended in PBS-G kept at the incubation temperature to a hematocrit of 5%. BS 3 or sNHS-B (Pierce, Rockford, IL) were dissolved in PBS-G and immediately added to give a final concentration of 1 to 5 mmol/l as indicated. The cell suspension was incubated at 0 or 37⬚C for 5 min and the reaction stopped by adding an equal volume of cold Tris-G (170 mM Tris, 125 mM HCl, 1 g/l d-glucose, pH 7.4) and pelleting the cells. The modified RBCs were washed once with a mixture of equal volumes of PBS-G and Tris-G and resuspended into PBS-G. The first supernatant was assayed spectrophotometrically at 405 nm for released hemoglobin, using RBCs lysed in water as a control for complete lysis. Less than 4 or 1.8% of cells treated with BS 3 at 37 or 0⬚C, respectively, were lysed. Enzymatic modification of RBCs. Treatment of RBCs by chymotrypsin (Sigma, St. Louis, MI) was performed essentially as described in Yu and Steck (1975). The digest was stopped by supplementing 50 mg/ml PMSF. Cells were pelleted by centrifugation, transferred into new tubes, washed three times with PBS-G and their membranes isolated by hypotonic lysis.

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2.4. Purification of human RBC membrane proteins Band 3 protein was purified from Triton X-100 extracts of RBC membranes, using anion exchange and affinity chromatography (Lukacovic et al., 1981). Prior to storage at ⫺70⬚C, the protein was extensively dialyzed against 20 mmol/l phosphate, containing 0.04% Triton X-100 (pH 7.4). Spectrin dimer was purified from peripheral protein extracts as described (Mariani et al., 1993). 2.5. Human IgG and albumin Whole human IgG (IgG) was outdated Sandoglobulin 䉸, which contained pooled human IgG from several thousands of blood donors. This material as well as 20% human serum albumin (HSA) were gifts from the Central Laboratory Blood Transfusion Service SRC, Bern. Both protein solutions were extensively dialyzed against appropriate buffers containing initially 50 mg/ml PMSF. Dialyzed IgG was then centrifuged for 10 min at 10 000 rpm. Heat-aggregated IgG was prepared by incubating 20 mg/ml IgG in PBS for 30 min at 63⬚C. The material was then centrifuged for 10 min at 40 000g to remove high molecular weight aggregates. 2.6. Affinity purification of naturally occurring antibodies Immobilized proteins. Heat-aggregated IgG was coupled to Affigel-10 (BioRad, Hercules, CA) using N-hydroxysuccinimide chemistry at a coupling ratio of 10 mg protein per ml of gel. Spectrin and band 3 protein were coupled to Affigel-15 at a ratio of 2–3 mg/ ml of gel in 20 mmol/l NaKHPO4 (pH 7.4). Buffers used for band 3 coupling also contained 0.04% Triton X-100 (Boehringer, Ingelheim, Germany). Following an overnight coupling in the cold with gentle rotation, unreacted esters were blocked with 100 mmol/l ethanolamine (pH 7.4). Washed and preeluted columns containing 4–10 ml of gel were connected in the following sequence for affinity purification of NAbs: heataggregated IgG, spectrin, band 3. IgG (70–100 g, at 20–30 mg/ml) was passed over these columns. A portion of the flowthrough (IgG ⫺ ˆ IgG depleted of these NAb) was dialyzed against PBS and frozen. The columns were washed overnight and bound protein eluted as described earlier for anti-spectrin (Lutz and Wipf, 1982) and anti-band 3 NAbs (Lutz et al., 1987, 1993a). The average yield per gram of IgG was 4–7 mg anti-band 3, 9–12 mg antispectrin NAb. The specificity of these NAbs was checked as given elsewhere (Lutz et al., 1993a). Concentrations of affinity-purified IgG NAbs were determined using a radioimmunoassay (Gee and Langone, 1981). 2.7. Preparation of F(ab 0 )2 fragments IgG or NAb were dialyzed against 25 mmol/l NaAc (pH 4.5). The material was then incubated overnight at 37⬚C in the same buffer with 10 mg pepsin (Boehringer, Mannheim) per mg IgG. After the digestion the mixture was supplemented with salt and buffer to reconstitute 20 mM Tris, 0.5% NaCl (pH 7.4). It was chromatographed over a Sephadex G-75 column (7 × 300 mm 2) in the same buffer to remove pepsin. Uncleaved IgG and Fcfragments were removed by batch adsorption to 0.1 volumes of protein G Sepharose at 4⬚C

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overnight. The supernatant was concentrated by ultrafiltration and passed over a Sephadex G-200 column yielding the F(ab 0 )2 fragments having a purity of over 85%. The concentrations of F(ab 0 )2 are expressed in IgG equivalents. 2.8. Radioiodination of proteins and RBCs Purified NAbs and IgG ⫺ were 125I-iodinated (1 mCi per 50–100 mg protein) by using chloramine T as an oxidant (Lutz et al., 1993b). Recovered protein amounted to about 50%, which resulted in specific activities ranging from 15 to 30 × 10 6 cpm/mg. Radioiodination of recombinant protein G and avidin (Sigma, St. Louis, MI) was performed following the same protocol, except that Sephadex G25 columns were used. The labeled proteins were stored in aliquots at ⫺20⬚C. Upon thawing they were dialyzed overnight against PBS containing HSA or gelatin. Surface iodination of RBCs. BS 3-treated RBC (200 ml) were surface-iodinated in a final volume of 1 ml with 200 mCi of 125I-iodide for 60 min at 25⬚C in the presence of 22 mmol/ l a/b-d-glucose, 1.32 U/ml lactoperoxidase (Sigma, St. Louis, MI) and 1 mg/ml glucose oxidase (Boehringer, Mannheim) (Reichstein and Blostein, 1975). The reaction was stopped by a two-fold dilution and pelleting of the cells, following by three washes with PBS-G in a new tube in the cold. 2.9. Binding of

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I-iodinated NAbs to RBCs

Washed, BS 3-treated and untreated RBC were resuspended to a constant cell number ranging from 2.0 to 3.0 × 10 9 ml ⫺1 in PBS-G. RBC suspensions (25 ml) were added to an equal volume of 125I-iodinated NAb in PBS-G (1 × 10 6 cpm) containing IgG as indicated. The labeled NAb was supplemented with HSA before dialysis against PBS to yield a final concentration of 25 mg/ml. In some experiments, complement-inactivated human serum was included. Where indicated unlabeled NAb was added to achieve the given concentration. Specific activities were calculated from the radioactivity of added NAbs and the total NAb concentration in the assay. When labeled F(ab 0 )2 of NAb was used, its concentration was adjusted with the appropriate NAb to that of the NAb. RBCs and labeled NAb were incubated for the given times at 0 or 37⬚C. The NAb binding reaction was stopped on ice by five-fold dilution with PBS-G, containing a final concentration of 12 mmol/l EDTA and centrifugation (3 min) of an aliquot through a mixture of 80% dibutyl phtalate and 20% diisononyl phtalate (Ross et al., 1985). The tips were cut from the tubes frozen in dry ice and the radioactivity bound to the pelleted cells was determined by gamma counting. 2.10. Radioimmune assays Binding of 125I-iodinated NAbs to covalently immobilized spectrin dimer or band 3 protein was carried out as described previously (Lutz et al., 1993a). Briefly, proteins were covalently immobilized on Chemobond plates (Lutz et al., 1990). Binding of anti-spectrin, anti-band 3 NAbs and that of IgG depleted of the two NAbs was studied at 100 ng/ml of the labeled immunoglobulins, diluted to a specific activity of 11 × 10 6 cpm/mg in 50 mg/ ml HAS. Samples were supplemented with the given concentration of whole human IgG in a buffer containing 1% gelatin, 0.04% Triton X-100 and 50 mg/ml HSA. Unspecific

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binding was determined by subtracting the binding recorded for IgG depleted of the two NAbs (IgG ⫺). It amounted to less than 3% for anti-spectrin to spectrin, 30% for anti-band 3 to band 3 and up to 76% for anti-spectrin to band 3. 2.11. Electrophoresis and immunoblotting Samples in 1% SDS were mixed with sample buffer containing 40 mmol/l dithiothreitol as reducing agent (Laemmle et al., 1987), unless indicated otherwise. After boiling for 3 min at 100⬚C reduced sulfhydryl groups were blocked with 50 mmol/l NEM. Samples were immediately loaded onto SDS-PAGE (Fasler et al., 1988). Gels were run for about 45 min at 50 mA in a standard apparatus (BioRad, Hercules, CA). In some experiments a high resolution SDS-PAGE was used with a Tris–Tricine buffer (Scha¨gger and von Jagow, 1987). Gels were stained with Coomassie blue or the polypeptides blotted for 3 h onto Immobilon P, polyvinyl difluoride-membranes (Millipore, Bedford, MA) at a constant current of 140 mA. Blots were incubated overnight as indicated with either 125I-iodinated NAb or 125I-iodinated F(ab 0 )2 fragments of NAbs or whole IgG in the same buffer containing sodium azide and 2–10 mg/ml IgG, 0.04% Triton X-100 and optionally 0.5–1% gelatin (Sigma, St. Louis, MI). 2.12. Densitometric quantification of polypeptides in stained polyacrylamide gels and on immunoblots Coomassie blue R-250 stained gels were scanned (Molecular Dynamics, Sunnyvale, CA). The amount of protein per band was calculated from the signal, which a known amount of an appropriate protein gave in the same scan. RBC membrane proteins of known concentration, run on inner lanes of the same gel, were used as standard, assuming that 30% of total membrane protein concentration was band 3 protein. Immunoblots were exposed overnight and digitized on a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). In some experiments a gel slice containing a known amount of radioactivity was quantified and the Phosphorimager data converted into cpm. The background signal was determined and subtracted from the reading. Figures showing stained gels or immunoblots were visualized using linear gray tone scales. 3. Results 3.1. Band 3 protein dimer formation by BS 3 is temperature controlled Human RBC were treated for 5 min with the non-penetrating, bivalent cross-linker BS 3 at 0 and 37⬚C. The BS 3 cross-linked species are not cleaved by reduction and appeared as band 3 dimers on immunoblots from RBC membrane proteins, separated on SDS PAGE (Fig. 1). Substantial cross-linking to the dimer evidently required incubation at 37⬚C. No band 3 oligomers larger than the dimer were visible on immunoblots from membranes of RBC treated with 5 mmol/l BS 3 for 5 min. To assess the amount of cross-linked band 3 dimer, binding of anti-band 3 NAb was studied to blotted band 3. Binding of 125I-labeled anti-band 3 NAb and its F(ab 0 )2 to blotted band 3 and band 3 dimer linearly increased with

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Fig. 1. Cross-linking of RBC membrane proteins by BS 3. RBC were treated for 5 min with 5 mmol/l BS 3 at: (A) 0; or (B) 37⬚C as described. Membranes from treated and untreated (U) cells were prepared. Reduced and alkylated membrane proteins were separated on SDS-PAGE (5% acrylamide). Gels were either stained with Coomassie blue (CB) or corresponding blots (blot) incubated with 10 6 cpm/ml of 125I-iodinated anti-band 3 NAb (1 mg/ml) in a buffer containing 0.04% Triton X-100, 25 mg/ml HSA and 10 mg/ml IgG.

the band 3 concentration (not shown). Their binding to the BS 3-generated dimer was 1.5– 2.1 times higher than to randomly immobilized band 3 monomers on blots (Table 1). Thus, anti-band 3 NAb binding appeared to be favored by the geometry of the band 3 molecules ˚ spacer arm of BS 3. This preference for the organized dimer as connected by the 11.4 A compared to randomly immobilized band 3 was found at both NAb concentrations tested (0.1 and 1 mg/ml). An average preference of 1.8-fold was used to correct NAb binding to band 3 dimer in order to quantify the extent of band 3 cross-linking from immunoblots as shown in Fig. 1. At 37⬚C, 37 ^ 4.5% of total band 3 protein was cross-linked after treatment with 5 mmol/l BS 3, whereas at 0⬚C, only 12 ^ 2.2% migrated in the dimer Table 1 Binding of anti-band 3 NAb and its F(ab 0 )2 fragment to band 3 monomer and dimer on blots (Membranes of RBCs treated with BS 3 (2.5 mmol/l) at 37⬚C and of untreated control cells were isolated and their proteins separated in reduced and alkylated form on SDS-PAGE (5% acrylamide). The amount of membrane protein added was varied from 0.1 to 15 mg to determine anti-band 3 binding as a function of protein concentration. Band 3 monomer and dimer were quantified from Coomassie blue stained SDS-PAGE. Proteins from gels run in parallel were blotted. The transfer yield from SDS-PAGE to blot was the same for the monomer and the BS 3-induced dimer of band 3. Immunoblots were incubated with 10 6 cpm/ml 125I-iodinated anti-band 3 NAb or 125I-iodinated F(ab 0 )2 fragments, supplemented with unlabeled anti-band 3 NAb to the given concentrations in the presence of 10 mg/ml IgG in buffer containing 0.04% Triton X-100. Bound radioactivity was quantified. Anti-band 3 binding to the BS 3induced dimer was corrected for the binding of anti-band 3 to the band 3 dimer region of untreated RBC, by subtracting the corresponding protein and binding values recorded for untreated RBC. Bound antibody was plotted for monomer and dimer against the protein concentration, yielding linear fits with R values ⬎0.93. The ratios of the slopes of these linear fits were calculated for each experimental condition and are given (binding to band 3 dimer/binding to band 3 monomer). Antibody concentration (mg/ml)

0.1 1.0

Binding to band 3 dimer/binding to band 3 monomer Anti-band 3 NAb

Anti-band 3 F(ab 0 )2

1.5 2.1

1.9 n.d.

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Fig. 2. Extent of band 3 cross-linking after a BS 3-treatment. RBCs were treated for 5 min with 5 mmol/l BS 3 as outlined in Fig. 1 at the given temperatures. A sample treated with BS 3 in the presence of 70 mmol/l Tris at 0⬚C (Tris, BS 3) and a control without BS 3 (Tris) was run in parallel. Reduced and alkylated membrane proteins from these RBCs were separated on SDS-PAGE and blotted. Blots were incubated with labeled anti-band 3 as in Fig. 1 and bound NAb quantified. Mean values from three experiments are given ⫹ 1SD. The amount of band 3 crosslinked to dimers was similar, when 2.5 mmol/l BS 3 was used at 37⬚C, but slightly decreased, when only 1 mmol/l BS 3 was used (not shown). Data are corrected for a higher affinity of anti-band 3 NAb for the band 3 dimer (see Table 1).

band (Fig. 2). In the presence of Tris, which competed with band 3 for the cross-linker, band 3 dimer was 5.6% and controls with untreated RBC never contained more than 2.8%. The low extent of band 3 cross-linking by BS 3 at 0⬚C (Figs. 1 and 2) was not due to a lack of chemical reactivity of N-hydroxysuccinimide esters, since a monovalent, biotinylated homologue, sNHS-B, was incorporated to similar extents with the same preference for band 3 at 0 and 37⬚C (Table 2). Its incorporation reached at 0⬚C, 72% of that at 37⬚C. BS 3 with the same functional group should have yielded close to 26% band 3 cross-links (ˆ72% of the extent of cross-linking at 37⬚C), if cross-linkable band 3 dimers had been available at 0⬚C. In contrast to this, BS 3 cross-linked only 12 ^ 2.2% of band 3 at 0⬚C. Hence, band 3 cross-linking was temperature dependent. Increased temperature alone was, however, not sufficient to generate stable oligomers, since an incubation of RBC at 37⬚C increased the content of unreducible band 3 dimers by 3.8% as compared to 24.8% in the presence of BS 3 (Table 2). Labeling of RBCs with sNHS-B demonstrated that the main target of N-hydroxysuccinimide esters was band 3 protein, since an exoplasmic digest with chymotrypsin produced labeled fragments typical for band 3 (Fig. 3), and since little biotin was incorporated into other proteins. Glycophorin A was not labeled, since no significant label remained above 55 kDa following digestion. In conclusion, BS3 specifically generated covalently linked band 3 dimers upon incubation at 37⬚C and did not react with glycophorins or cytoskeletal proteins. 3.2. Anti-band 3 NAb binding was dependent on band 3 cross-linking on intact RBC Binding of 125I-iodinated anti-band 3 NAbs at 0⬚C was high to RBC treated with BS 3 at

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Table 2 Incorporation of sNHS-B and BS 3 into RBCs (RBCs were labeled for 5 min with sNHS-B (5 mmol/l) instead of BS 3 at 0 or 37⬚C, but otherwise as in Fig. 1. Membrane proteins were separated in reduced and alkylated form on SDS-PAGE and blots incubated with 10 5 cpm/ml of 125I-labeled avidin. Bound avidin was quantified.) Temperature (⬚C)

0 37

Total a (%)

72 100

sNHS-B incorporated into band 3 in % of Total c

Monomer ⫹ dimer d

83 83

2.3 6.1

BS 3 incorporated in band 3 dimer in % of monomer ⫹ dimer b

12.2 37

a The total incorporation of sNHS-B into RBC membranes at 0⬚C is given in percent of that incorporated at 37⬚C. b The amount of band 3 dimer generated with BS 3 at 0 and 37⬚C is given in percent of that in monomer ⫹ dimer (data from Fig. 2). c The extent by which sNHS-B incorporated into band 3 (monomer ⫹ dimer) is given in percent of the total amount incorporated at either condition in all bands (including proteins at the buffer front). d The amount of sNHS-B bound to band 3 dimer is given in percent of that which was bound to band 3 monomer ⫹ dimer at either condition.

Fig. 3. Reactivity of N-hydroxysuccinimide esters with RBC membrane proteins. RBCs were labeled for 5 min with 5 mmol/l sNHS-B at 37⬚C and treated with chymotrypsin (Chy) or left untreated (no). Membranes were isolated and membrane proteins separated on Tris–Tricine SDS-PAGE (10.5% acrylamide). The blotted proteins were incubated with 10 5 cpm/ml of 125I-labeled avidin. The positions of the known chymotryptic fragments of band 3 protein (55, 38, 21 kDa) and of glycophorin A (PAS 1) are marked.

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Fig. 4. Anti-band 3 NAb binding to BS 3-treated RBCs at 0⬚C. BS 3-treated RBCs were incubated at 0⬚C for the given times with 125I-labeled anti-band 3 NAb or IgG ⫺ in the presence of 5 mg/ml IgG and 25 mg/ml HSA as described. Radioactivity bound to pelleted RBCs was determined and binding of lgG ⫺ subtracted. The mean of bound NAb ^ 1 SD from three independent experiments is given for binding of 125l-labeled anti-band 3 NAb at 1.5 mg/ml to RBCs treated with BS 3 at 37⬚ (W) or to untreated RBCs (X). Analogous results are shown for RBC treated with BS 3 at 37⬚C and incubated at 0⬚C with labeled anti-band 3 NAb at 2.5 mg/ml (A). These data are from two independent experiments with error bars indicating the deviation of the two measurements. NAb binding of RBCs treated with BS 3 at 0⬚C (B) was low, but significantly higher than to untreated RBCs in four out of four anti-band 3 NAb preparations.

37⬚C and marginal to control cells and RBC treated with BS 3 at 0⬚C (Fig. 4). Thus, binding was dependent on the presence of band 3 cross-links which were primarily formed when RBC were treated with BS 3 at 37⬚C (Figs. 1 and 2). BS 3 applied at 37⬚C caused a 10-fold increase in anti-band 3 NAb binding at 1.5 mg/ml by 90 min and a 20-fold increase at 2.5 mg/ml, respectively. Hence, binding of anti-band 3 NAbs was concentration dependent at nearly physiological concentrations of IgG and HSA. Binding was specific for anti-band 3 NAb because: (i) it was measured in the presence of a 10 4-fold excess of other IgG; and (ii) the binding data were corrected for unspecific binding by subtracting values obtained from BS 3-treated RBC incubated with 125I-iodinated IgG that was depleted of anti-band 3 and anti-spectrin NAbs. Its binding was about six times lower than that of anti-band 3 and anti-spectrin NAbs together, but did not increase with time (not shown). Surprisingly, anti-band 3 NAb binding to RBCs containing band 3 cross-links did not increase by raising the temperature during binding (Fig. 5, 37⬚C; Fig. 4, 0⬚C). In fact, antiband 3 NAb binding was up to 70% less by 30 min at 37⬚C at comparable anti-band 3 concentrations (Fig. 5). On the other hand, anti-band 3 NAb binding to RBCs increased significantly, when binding was measured at 37⬚C on cells pretreated at 0⬚C with BS 3, while binding to untreated RBC did not increase (Fig. 5). This effect was found in both of two experiments. 3.3. Anti-spectrin NAb binding to RBC was similarly dependent on band 3 cross-links Binding of anti-spectrin NAbs was studied by using the same stringent conditions as for anti-band 3 NAb. Unexpectedly, anti-spectrin NAb binding to RBCs at 0⬚C increased up to

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Fig. 5. Anti-band 3 NAb binding to BS 3-treated RBCs at 37⬚C. RBCs were treated for 5 min with BS 3 at 37⬚C (A), at 0⬚C (B), at 0⬚C in the presence of Tris (V) or in the absence of BS 3 (X). RBCs were incubated for the given times with 125l-iodinated anti-band 3 NAb or IgG ⫺ (3 mg/ml) at 37⬚C in the presence of 5 mg/ml IgG and 25 mg/ ml HSA. The mean of bound NAb ^ 1 SD from four experiments is shown. Standard deviations are not given for binding to RBC treated with BS 3 at 0⬚C (B), since these values varied, but were below binding to cells treated with BS 3 at 37⬚C and higher than binding to untreated RBCs in all four experiments.

seven-fold for RBCs pretreated with BS 3 at 37⬚C and up to three-fold for those pretreated at 0⬚C (Fig. 6A). Likewise, binding of anti-spectrin NAb to BS 3-treated RBC was lower, when determined at 37⬚C instead of 0⬚C (Fig. 6B). Furthermore, anti-spectrin NAb binding increased with prolonged incubation times at 37⬚C for RBCs that were treated with BS 3 at 0⬚C, while binding to untreated cells remained unchanged (Fig. 6B). Thus, binding

Fig. 6. Anti-spectrin NAb binding to BS 3-treated RBC at: (A) 0; and (B) 37⬚C. RBCs were treated for 5 min with BS 3 at 37⬚C (A), at 0⬚C in the absence (B) or in the presence of Tris (V) or used untreated as a control (X). RBCs were then added to 10 6 cpm of 125I-iodinated anti-spectrin NAb or IgG ⫺ to give a final concentration of 3 mg/ml in the presence of 5 mg/ml of IgG and 25 mg/ml HSA. These suspensions were kept for the given times at: (A) 0; or (B) 37⬚C. The mean of bound NAb ^ 1 SD from three independent experiments are given in A, in which three different anti-spectrin NAb preparations were used. Results shown in B are from a single experiment in which all four types of cell modification were analyzed simultaneously.

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Fig. 7. Proteins accessible to surface 125I-iodination after a BS 3-treatment. RBCs were treated in parallel for 5 min with 0, 1, 2.5 or 5 mmol/l BS 3 at 37⬚C and surface- 125I-iodinated subsequently by using lactoperoxidase. Membranes were prepared and extracted with low salt buffer to remove peripheral proteins. Peripheral protein extracts (SE) and the remaining inside-out vesicles (IOV) from equal amounts of membranes were separated on SDS-PAGE (5% acrylamide). (A) Coomassie blue-stained SDS-PAGE; and (B) autoradiograph of the same gel.

of anti-spectrin NAbs to RBCs increased with the extent of band 3 dimers generated by BS 3 in a similar way as that of anti-band 3 NAb. Binding of both NAbs to RBC containing band 3 cross-links further increased by raising the total IgG concentration from 5 to 15 mg/ml, where it reached even at 37⬚C 100 and 50 ng/10 10 RBC, respectively (not shown). The binding of anti-spectrin NAb to RBCs was not due to their binding to exposed spectrin, as this cytoskeletal protein was not accessible to lactoperoxidase-catalyzed surface 125I-iodination of BS 3-treated RBCs (Fig. 7). The two spectrin bands in peripheral membrane protein extracts were not labeled. Furthermore, IOV, which retained most of the labeled band 3 dimers, showed no label in the residual, tightly membrane associated spectrin (Mariani et al., 1993). Thus, binding of 125I-iodinated anti-spectrin NAbs to RBCs was to surface loops of integral membrane proteins rather than to exposed spectrin. 3.4. Anti-spectrin modulated anti-band 3 binding to BS 3-treated RBC Binding of F(ab 0 )2 fragments from both anti-spectrin and anti-band 3 NAbs to BS3-treated RBC reached about 50% of that of intact NAbs (Fig. 8A and B), implying the existence of Fcand Fab-mediated interactions. To further analyze potential NAb–NAb and/or NAb–RBC interactions, binding of either NAb was studied in the presence of increasing concentration of the other NAb. Binding of anti-spectrin F(ab 0 )2 was not altered by high concentrations of antiband 3 NAb, whether anti-spectrin concentrations were low or high (Fig. 8B and C). On the other hand, binding of anti-band 3 F(ab 0 )2 to BS3-treated RBCs increased up to 30% with increasing concentrations of anti-spectrin NAb (Fig. 8A). The same results were found at a 200-fold higher concentration of anti-band 3 F(ab 0 )2 (Fig. 8C). Thus, anti-spectrin NAb stimulated binding of anti-band 3 NAb to BS3-treated RBC without having to interact with their Fc portions. These findings show that the two NAbs did not compete for the same

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Fig. 8. Binding of anti-spectrin NAb enhances that of anti-band 3 NAb. Density-fractionated RBC (fraction 3) were treated for 5 min with 2.5 mmol/l BS 3 at 37⬚C. These RBCs were incubated with 10 6 cpm of labeled NAb or their F(ab 0 )2 fragments at 2 mg/ml, if not otherwise indicated, and increasing concentrations of the other, unlabeled NAb in a buffer containing 5 mg/ml IgG and 25 mg/ml HSA. Bound radioactivity was determined after incubation for 90 min at 0⬚C. The amount of bound NAb or their F(ab 0 )2 fragments was calculated from bound label, using the specific radioactivity and the total protein concentration of the NAb or F(ab 0 )2 in their labeled and unlabeled form. Bound antibody is given in ng and bound F(ab 0 )2 in ng equivalent to intact antibody. (A), (B) Binding of 125I-iodinated NAbs (A, n ˆ 1) and their F(ab 0 )2 (B, n ˆ 2) in the presence of increasing concentrations of the other NAb is shown for anti-band 3 in (A) and for anti-spectrin in (B). (C) Binding of 125I-iodinated F(ab 0 )2 of anti-band 3 (X, n ˆ 2) or of anti-spectrin (O, n ˆ 1) at 20 mg/ml of labeled F(ab 0 )2 in the presence of the given concentrations of the other unlabeled NAb.

binding sites on BS3-treated RBC. The ability of anti-spectrin NAbs to slightly enhance binding of anti-band 3 NAbs suggests that anti-spectrin NAb patched additional band 3 molecules to which F(ab 0 )2 fragments of anti-band 3 NAb could bind. These findings further imply that binding of anti-spectrin NAbs to RBC containing band 3 cross-links could be due to their polyreactivity rather than contaminating anti-band 3 NAbs. 3.5. The polyreactivity of anti-spectrin NAbs Most IgM NAbs and many IgG NAbs are polyreactive (Ichiyoshi and Casali, 1994).

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Fig. 9. Binding of 125I-iodinated F(ab 0 )2 of anti-spectrin NAbs and of whole human IgG to RBC membrane proteins. F(ab 0 )2 were prepared from whole human IgG (IgG) and anti-spectrin NAbs (anti-S) and 125I-iodinated to similar extents. Their binding was studied to blots from an SDS-PAGE run with reduced and alkylated RBC membranes proteins (7 mg). Blots were incubated with labeled F(ab 0 )2 in buffer containing 0.04% Triton X-100 and 0.5% gelatin (no addition) or in 66% complement-inactivated serum (⫹66% serum) that was dialyzed against the buffer. F(ab 0 )2 fragments of anti-spectrin NAbs and IgG were added in comparable concentrations, equivalent to 70–80 ng/ml of IgG.

Such NAbs bind with an affinity lower than that for the antigen to a number of even unrelated proteins (Labrousse et al., 1994). Anti-spectrin NAbs belong to this category of NAbs, since their affinity for spectrin increased with their cationic charge and their binding was inhibited by increasing salt concentrations (Lutz et al., 1984). Their polyreactivity was low, but measurable even at physiological concentrations of HSA and IgG (Fig. 9). This figure shows binding of 125I-iodinated F(ab 0 )2 fragments of anti-spectrin NAbs to RBC membrane proteins in the absence or the presence of 66% serum. Binding to spectrin was lowered by the presence of 66% serum, while that to band 3 and band 4.2 remained. Serum had a similar effect on the binding of F(ab 0 )2 of whole IgG to spectrin (Fig. 9, IgG). On the other hand, 66% serum did not significantly inhibit binding to band 3 for either type of F(ab 0 )2. This result demonstrates that polyreactivity of anti-spectrin NAbs is real, but low. It was similarly low to native proteins as established by a radioimmune assay, where binding to spectrin was 185 pg/well and to band 3 only 1.5 pg/well at physiological IgG concentrations. The binding of anti-spectrin to band 3 was 8% of that of anti-band 3 NAbs. Hence, it is possible that polyreactivity of anti-spectrin NAbs was involved in mediating binding to RBC containing band 3 cross-links.

4. Discussion 4.1. Band 3 oligomerization Sixty percent of band 3 is diffusible within the membrane at 37⬚C (Nigg and Cherry, 1980), while only 37% of band 3 protein was cross-linked to dimers within 5 min by the bivalent cross-linker BS 3 at 37⬚C (Fig. 2). This yield is in accordance with the findings that

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protein diffusion is not completely unrestricted, with some band 3 being confined to a membrane domain during a relatively long time, either by obstacle clusters (Saxton, 1992) or by interactions with cytoskeletal elements (Liu and Derick, 1992). Even within the diffusible band 3 fraction 80% of the molecules may transiently interact with the cytoskeleton, and only the remainder seems to be completely unrestricted in its motion (Tomishige et al., 1998). Based on the comparison between band 3 oligomerization and chemical reactivity of N-hydroxysuccinimides at 0⬚C, effective cross-linking of band 3 protein required elevated temperatures. This finding implies that cross-linking was (a) interdimeric, between two preexisting band 3 dimers and involved lateral and rotational mobility. Alternatively, it implies that cross-linking was (b) intradimeric and occurred between associated band 3 monomers, but involved rotational mobility and/or conformational changes. The data clearly show that NAbs did not bind to preexisting functional dimers (Tanner, 1997) or tetramers (Hanspal et al., 1998) as such. Based on results from Jennings and Nicknish (1985) and Salhany et al. (1990) intradimeric cross-links are formed first, while incubations with BS 3 for up to an hour generated a small amount of band 3 tetramer. Irrespective of whether the rapidly generated BS 3 cross-linked dimers were intra- or interdimeric, the temperature-dependent cross-linking generated band 3 molecules that were sufficiently altered in their topology to allow NAb binding. Theoretically, N-hydroxysuccinimidyl-groups react unspecifically with amino-groups in exposed protein loops. Nevertheless, N-hydroxysuccinimidyl incorporated quite specifically into band 3, as was found previously (Jennings and Nicknish, 1985). The presence of sNHS-B in both chymotryptic, transmembrane fragments of band 3 suggests that at least two sites located in the 55 and the 38 kDa fragment of band 3 reacted with BS 3 with a preference for the site in the 55 kDa fragment. Despite N-hydroxysuccinimidyl groups bound to two sites within the exoplasmic portion of band 3, we found almost exclusively band 3 dimers with BS 3. This could imply that a single amino group per band 3 was reactive with BS 3, because of a structural or an imposed heterogeneity among band 3 molecules. This is quite probable, since the two negative charges on the BS 3 molecule may convey to this molecule properties similar to those found in the well known inhibitor, 4,4 0 diisothiocyano-stilbene-2,2 0 disulfonate, which interacts predominantly with K539 (Kietz et al., 1991). Alternatively, the reactivity of band 3 with BS 3 may differ for preexisting, functional band 3 dimers containing either polylactosaminyl residues or short oligosaccharides (Landolt-Marticorena et al., 1998). 4.2. Band 3 cross-linking increased binding of anti-band 3 and of anti-spectrin NAbs We show that changes in topology of band 3 protein are sufficient to expose binding sites for anti-band 3 NAbs (Fig. 4) as well as for anti-spectrin NAbs (Fig. 6A). Binding of both anti-band 3 and anti-spectrin NAbs was at 0⬚C up to 10 times higher to RBC containing BS 3-induced band 3 dimers. Thus, mild cross-linking of band 3 alone rather than oxidative, intracellular damage affecting lipids and other proteins was sufficient to induce band 3 oligomers in a manner preserving the architecture of the RBC membrane. Binding of affinity-purified anti-band 3 and anti-spectrin NAbs (which therefore represented all NAbs of the particular specificity that are encoded by the human germline) was Fab mediated to at least 50%. Anti-band 3 and anti-spectrin NAb binding was specific,

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since the concentrations of anti-band 3 and anti-spectrin NAbs used in these assays were in the order of 1–3 mg/ml, which is small compared to the total IgG concentration included (5–10 mg/ml), but yet nominally 10 times higher than physiological. The effective NAb concentrations were, however, lower, since they were determined as IgG concentrations in eluates from affinity columns that do not reach a 10 5-fold degree of purification. Binding of both NAbs to BS 3-treated RBC was lower when probed at 37 than at 0⬚C (Figs. 4–6). This unexpected result was confirmed in many experiments and is explicable. Band 3 oligomers, which were generated by BS 3, were covalently stabilized only in form of dimers, since we could not detect covalently linked tetramers. Thus, these oligomers remained diffusible at 37⬚C and dissociated, whereby NAb binding was disfavored. These results are in agreement with data from Turrini et al. (1991), who did not find a significantly enhanced binding of whole human IgG to RBC at 37⬚C when RBC were treated with BS 3 alone. Taken together, these results show that binding of anti-band 3 NAbs at quasi-physiological conditions required band 3 oligomerization rather than a chemical modification to a neoantigen (Kay et al., 1983). 4.3. Anti-spectrin NAb binding may be due to their polyreactivity A BS 3 treatment of RBCs not only enhanced binding of anti-band 3 NAbs at 0⬚C, it also stimulated binding of anti-spectrin NAbs or their F(ab 0 )2. The enhanced binding of antispectrin NAbs to intact RBCs appeared puzzling, although it is in accordance with in vivo data showing anti-spectrin NAb association with RBCs from patients with hemoglobinopathies (Wiener et al., 1986) and with RBC from phenylhydrazine-treated animals (Schlu¨ter and Drenckhahn, 1986). The enhanced binding of anti -spectrin NAbs was not due to spectrin exposure, since surface- 125I-iodination of BS 3-treated RBC did not reveal labeled spectrin (Fig. 7). Anti-spectrin NAbs displayed similar binding kinetics to BS 3-treated RBC as anti-band 3 NAbs, suggesting that the target of anti-spectrin NAbs was linked to band 3 oligomers or was band 3 protein. These results are compatible with the idea that anti-spectrin NAbs bound to surface loops of oligomerized band 3 protein or associated proteins. Anti-spectrin NAb binding was not due to anti-band 3 NAb contaminating anti-spectrin preparations, since binding of F(ab 0 )2 of anti-spectrin NAbs was not competed by high concentrations of anti-band 3 NAbs. Anti-spectrin NAbs bound to independent sites, since they even stimulated binding of F(ab 0 )2 of anti-band 3 NAbs and thus recruited or stabilized additional band 3 oligomers to which anti-band 3 NAbs bound. Recruitment of an additional 30% of band 3 dimers calls for either a high enough affinity to patch antigens or binding of antispectrin NAbs to a band 3 associated protein, like glycophorin (Lutz, 1987), through which additional band 3 molecules became oligomerized. At present we do not understand how anti-spectrin NAbs interacted with BS 3-treated RBC, except that their binding required band 3 oligomers. It is possible, but not established that their binding was due to their polyreactivity. The role of polyreactivity among NAbs is poorly understood and polyreactive NAbs have so far been described primarily for cationic NAbs (Ichiyoshi and Casali, 1994; Lacroix-Desmazes et al., 1995). Anti-spectrin NAbs are also cationic (Lutz et al., 1996) and have striking similarities with anti-DNA NAbs and autoaggressive anti-DNA antibodies, of which the latter also bound to intact

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kidney cells in glomerular nephritis patients (Naparstek et al., 1990). In analogy to anti-spectrin NAbs, anti-DNA Abs may directly bind to surface consitutents on kidney cells, since a DNAse-treatment of the target cells did not affect, but a protease treatment affected their binding (Raz et al., 1993) or was inhibited by synthetic peptides of a negatively charged protein (Gaynor et al., 1997). Irrespective of the binding mechanism, anti-spectrin NAb bound to RBC with band 3 oligomers and thereby opsonized these cells at quasi-physiological conditions. The concerted action of the two types of NAb may be a necessity to reach a high extent of opsonization and may provide a means to secure clearance in case of a lack of a particular NAb or a particular protein. This and the potential involvement of yet other polyreactive NAbs, which have not been investigated, may be the reason why band 3 knock outs (Peters et al., 1996) and band 3-deficient cattle (Inaba et al., 1996) remained capable of clearing RBCs. A very recent report describes that elevated anti-spectrin antibodies accelerated clearance of infused senescent RBC from a hypertransfused animal (Graldi et al., 1999). While these authors think of an exposure of spectrin on senescent RBC, their findings do not contradict our interpretation. Future work will have to focus on the target of polyreactive anti-spectrin NAbs. Acknowledgements We thank Dr P.J. Spa¨th from the Central Laboratory for Blood Transfusion Services, SRC, Bern for providing outdated Sandoglobulin 䉸 and highly purified human serum albumin. We further acknowledge the skillful technical help by Mena Nater and Pia Stammler with the immunoblots. This work was supported by funds to H.U.L. from the Swiss National Science Foundation 3200-045844 and in particular by the Swiss Federal Institute of Technology through a grant entitled “Tissue homeostasis”. References Balasubramanian, K., Schroit, A.J., 1998. Characterization of phosphatidylserine-dependent beta(2)-glycoprotein I macrophage interactions—implications for apoptotic cell clearance by phagocytes. J. Biol. Chem. 273, 29 272–29 277. Bennett, V., Branton, D., 1977. Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. J. Biol. Chem. 252, 2753–2763. Beutler, E., West, C., Blume, K.-G., 1976. The removal of leukocytes and platelets from whole blood. J. Lab. Clin. Med. 88, 328–333. Bevers, E.M., Comfurius, P., Dekkers, D.W.C., Harmsma, M., Zwaal, R.F.A., 1998. Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance. Biol. Chem. 379, 973–986. Boas, F.E., Forman, L., Beutler, E., 1998. Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia. Proc. Natl. Acad. Sci. USA 95, 3077–3081. Chen, Z.J., Wheeler, C.J., Shi, W., Wu, A.J., Yarboro, C.H., Gallgher, M., Notkins, A.L., 1998. Polyreactive antigen-binding B cells are the predominant cell type in the newborn B cell repertoire. Eur. J. Immunol. 28, 989–994. Connor, J., Pak, C.C., Schroit, A.J., 1994. Exposure of phosphatidylserine in the outer leaflet of human red blood cells-relationship to cell density, cell age, and clearance by mononuclear cells. J. Biol. Chem. 269, 2399–2404. Coutinho, A., Kazatchkine, M.D., Avrameas, S., 1995. Natural autoantibodies. Curr. Opin. Immunol. 7, 812–818.

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