Reaction B Cell Complement Receptor 2 Transfer

B Cell Complement Receptor 2 Transfer Reaction Margaret A. Lindorfer, Hasmig B. Jinivizian, Patricia L. Foley, Adam D. Kennedy, Michael D. Solga and Ronald P. This information is current as Taylor of September 28, 2021. J Immunol 2003; 170:3671-3678; ; doi: 10.4049/jimmunol.170.7.3671 http://www.jimmunol.org/content/170/7/3671 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B Cell Complement Receptor 2 Transfer Reaction1

Margaret A. Lindorfer,* Hasmig B. Jinivizian,* Patricia L. Foley,† Adam D. Kennedy,* Michael D. Solga,* and Ronald P. Taylor2*

The B cell C receptor specific for C3dg (CR2) shares a number of features with the primate E C receptor (CR1). Previously, we have demonstrated, both in vitro and in animal models, that immune complexes (IC) bound to primate E CR1, either via C opsonization or by means of bispecific mAb complexes, can be transferred to acceptor macrophages in a process that also removes CR1 from the E. We have now extended this paradigm, the transfer reaction, to include B cell CR2. We used both flow cytometry and fluorescence microscopy to demonstrate that IC bound to Raji cell CR2, either via C opsonization or through the use of an anti-CR2 mAb, are transferred to acceptor THP-1 cells. This reaction, which appears to require Fc recognition of IgG bound to Raji cell CR2, also leads to transfer of CR2. Additional support for the B cell transfer reaction is provided in a prototype study in a monkey model in which IC bound to B cell CR2 are localized to the spleen. These findings may have important implications with respect to defining

the role of C in IC handling during the normal immune response. The Journal of Immunology, 2003, 170: 3671–3678. Downloaded from

omplement serves a major effector function in the adap- ability of IC bound to B cell CR2 to participate in a transfer reaction tive and innate immune systems as well as in facilitating analogous to that observed with E CR1. Our findings may have im- production of a robust immune response (1–4). Ags re- portant implications with respect to the role of C3dg-IC in the normal C 3 acted with Abs to form immune complexes (IC) are more immu- processing of Ags during the immune response. nogenic than free Ags, and opsonization of IC by C to generate http://www.jimmunol.org/ C3dg-labeled IC is critical in enhancing immunogenicity. In fact, interaction of opsonized IC with C receptor 2 (CR2), the C3dg Materials and Methods receptor on B cells and follicular dendritic cells (FDC), plays a key Human E and cell lines role in the prolongation and maturation of the immune response (5–11). Our laboratory has focused on the role of the primate E C The human monocytic THP-1 cell line (American Type Culture Collection (ATCC), Manassas, VA) was cultured in 10% FBS, in RPMI 1640 medium receptor 1 (CR1) in IC processing (12). We have demonstrated that (ATCC) supplemented with gentamicin (Life Technologies, Grand Island, IC bound to E CR1 via either C3b- or C-independent bispecific NY). THP-1 cells were treated with retinoic acid (Sigma-Aldrich, St. mAb constructs are transferred to acceptor phagocytes in a reac- Louis, MO) 3–4 days before use (28), and except for the experiment in Fig. 1, G and H, were dyed with the fluorescent dye PKH26 (Sigma-Aldrich), tion in which FcR recognition leads to removal of CR1 from E and by guest on September 28, 2021 according to the manufacturer’s directions. The B lymphoblastoid Raji cell uptake of the entire IC and CR1 by the acceptor cell (13, 14). line (ATCC) was cultured in the same medium supplemented with peni- B cell CR2 shares structural and functional features with E CR1 cillin and streptomycin (Life Technologies). Cultured cells were pelleted (15). The extracellular domains of both receptors are composed of from growth medium (200 ϫ g), resuspended in standard medium (SM, multiple copies of the short consensus repeat (SCR), a folding 10% FBS in RPMI 1640 medium (Life Technologies)), and held at room motif containing 60 aa, several of which are invariant. The ligand temperature until opsonized or dyed with PKH26. Whole blood from normal donors was anticoagulated with EDTA and stored in 50% Alsevers. for E CR1 is C3b; the ligand for CR2 is the downstream degradation product C3dg (9, 11, 15, 16). Under many conditions, CR1 and CR2 levels on B lymphocytes as well as CR1 on leukocytes and E are Antibodies reduced (17–21); in fact, soluble CR2 is found in the circulation in Anti-human CR2 IgG2a mAb HB135 (29) and bacteriophage ⌽X174 (30, normal individuals (22–24), and at increased levels in certain diseases 31) were labeled with Alexa (Al) 488, 594, or 633 (Molecular Probes, (23, 24). CR2 can be shed and/or reduced in copy number on B or T Eugene, OR), according to the manufacturer’s directions. Anti-⌽X174 cells, and one or more unidentified proteases may cut CR2 (22–24). mAbs 51.1, 7B7, and 7A4 have been previously described (30). Mouse Based on the intriguing similarities in the properties of CR1 and CR2 mAb 1H8 was produced by immunizing mice with C3b(i)-Sepharose (32); both in humans and in mouse models (25–27), we investigated the binding of this mAb to C3b(i)-Sepharose was blocked by purified C3d (Advanced Research Technology, San Diego, CA). Combinations of un- Ј labeled mAb HB135 (intact or its F(ab )2 fragments), Al488 mAb HB135, Al488 rabbit anti-mouse IgG (Molecular Probes), or unlabeled rabbit anti- *Department of Biochemistry and Molecular Genetics and †Center for Comparative Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908 mouse IgG (Southern Biotechnology, Birmingham, AL) were bound to Raji cells before transfer (Table I). Al633 goat anti-rabbit IgG (Molecular Received for publication August 21, 2002. Accepted for publication January 29, 2003. Probes), FITC anti-CD21 (clone BL13; Beckman Coulter Immunotech, The costs of publication of this article were defrayed in part by the payment of page Marseille, France), and allophycocyanin (APhCy) anti-CD21 (clone B-ly4; charges. This article must therefore be hereby marked advertisement in accordance BD PharMingen, San Diego, CA) were used to probe cells after transfer. with 18 U.S.C. Section 1734 solely to indicate this fact. The anti-CR2 mAbs HB135, BL13, and B-ly4 recognize distinct and non- 1 This work was supported by grants from EluSys Therapeutics (Pine Brook, NJ) (to overlapping epitopes on Raji cells, and on monkey B cells (only HB135 Ј M.A.L. and R.P.T.) and the National Institutes of Health (RO1-AR-43307) (to R.P.T.). and B-ly4 tested in monkeys; data not shown). The F(ab )2 fragment of 2 Address correspondence and reprint requests to Dr. Ronald P. Taylor, Box 800733, mAb HB135 was prepared with the ImmunoPure kit, according to the University of Virginia Health Sciences Center, Charlottesville, VA 22908-0733. E- manufacturer’s directions (Pierce, Rockford, IL). Anti-CR1 mAb 1B4 (33) mail address: [email protected] and anti-CR2 mAb FE8 (34) were used to block binding of C-opsonized IC 3 Abbreviations used in this paper: IC, immune complex; Al, Alexa; APhCy, allo- to E or Raji cells, respectively. FITC mAb F10-89-4 (mouse IgG2a; Se- phycocyanin; CR1/2, C receptor 1/2; FDC, follicular dendritic cell; MESF, molecules rotec, Raleigh, NC) and APhCy mAb H130 (mouse IgG1; Caltag, Burlin- of equivalent soluble fluorochrome; NHS, normal human serum; PF, paraformalde- game, CA), both specific for human CD45, were used to opsonize Raji cells hyde; SCR, short consensus repeat; SM, standard medium; TR, Texas Red. and probe for loss of CD45 after transfer, respectively.

Table I. Raji cell transfer substrates, THP-1 cell treatments, and probes

Fig. Substrate THP-1 Treatment Probesa

2 C3dg/A1488-⌽X174 IC None APhCy CD21 3 A1488-HB135 None APhCy CD21 4 HB135/A1488-RAMS None APhCy CD21 A1633 GARB 5 FITC CD45 None APhCy CD45 5 A1488-HB135 None APhCy CD21 6 HB135 None APhCy CD21 A1488 RAMS Ј 6 F(ab )2-HB135 None APhCy CD21 A1488 RAMS 6 HB135/A1488-RAMS None APhCy CD21 6 HB135/A1488-RAMS ϩ huIgGb APhCy CD21 7 Naive None FITC CD21 7 HB135 ϩ⌽X174 IC, washedc FITC CD21

a Samples probed with mAbs specific for CD21 or CD45, A1633 goat anti-rabbit IgG (GARB), or A1488 rabbit anti-mouse IgG (RAMS) were blocked with mouse IgG, goat IgG, or rabbit IgG, respectively. b THP-1 ϩ huIgG: THP-1 cells pretreated with 2 mg/ml human IgG 5 min before mixing with Raji cells. Final human IgG: 1 mg/ml during transfer. c THP-1 ϩ⌽X174 IC: THP-1 cells incubated with preformed ⌽X174/anti-⌽X174 IC for 30 min at 37°C, washed three times with BSA/PBS, resuspended in RPMI 1640 medium. Downloaded from Cell opsonization and transfer reaction protocols with a BX40 fluorescence microscope (Olympus, Melville, NY), equipped with a Magnafire digital camera (Olympus), using FITC, FITC/Texas Red ⌽ To bind X174 to human E CR1 via immune adherence, washed E (10% (FITC/TR), or TR filters. hematocrit) were incubated in 50% normal human serum (NHS)/50% SM with Al488 ⌽X174 (15 ␮g/ml) and three anti-⌽X174 mAbs (2 ␮g/ml In vivo experiments each) for 7 min at 37°C. After three washes, E were resuspended in RPMI 1640 medium (Life Technologies) and combined at varying ratios (3:1 to All procedures were conducted following protocols approved by the Uni-

20:1) with THP-1 cells in RPMI 1640 medium. Aliquots of the mixtures versity of Virginia Animal Care and Use Committee. A cynomolgus mon- http://www.jimmunol.org/ were centrifuged at 200 ϫ g for 5 s and incubated at 37°C for varying key (6.2 kg) was anesthetized (ketamine, 10 mg/kg i.m.; atropine, 0.04 times. The transfer reaction for each aliquot was stopped by dilution into mg/kg s.c.), intubated, and maintained under anesthesia with isoflurane and ice-cold BSA-PBS, followed by centrifugation and resuspension of the cell 100% oxygen. Ab solutions (1 ml) were infused into the cephalic vein over pellet in 1% paraformaldehyde in PBS (1% PF-PBS). 1 min. Blood samples were drawn through an arterial catheter, anticoag- The details of Raji cell transfer experiments and fluorescent probing ulated with 10 mM EDTA, and washed twice with BSA-PBS and once with schemes are summarized in Table I. To bind ⌽X174 to Raji cell CR2 via 2 mg/ml mouse IgG in BSA-PBS, and the cell pellet was recovered in its C, Al488 ⌽X174 (10 ␮g/ml) and a mixture of two or three anti-⌽X174 original volume in 2 mg/ml mouse IgG in BSA-PBS. Samples were probed mAbs (2 ␮g/ml each mAb; comparable binding was obtained for both with a cocktail of PerCP anti-CD45 (BD PharMingen; CN 557513), PE cocktails) were incubated in 50% NHS/50% SM for 15 min at 37°Cto anti-CD20 (BD Biosciences; CN 347201), and either APhCy anti-CD21 deposit C3dg on the IC. An equal volume containing 5 ϫ 107 Raji cells/ml B-ly4 or Al633 goat anti-rabbit IgG. E were lysed with FACS lyse (BD by guest on September 28, 2021 in SM with 2 mg/ml goat IgG was then added to the serum-treated IC, and Biosciences), and remaining cells were washed twice with PBS and resus- the mixture was incubated for an additional 15 min at 37°C. After three pended in 1% PF-PBS. The B cell population was defined by a PerCP washes, the Raji cells were combined at varying ratios (1:3 to 1:1) with anti-CD45/PE anti-CD20 gate. The monkey was humanely euthanized 24 h THP-1 cells, and the transfer reaction was conducted as described for E. postinfusion, and samples of spleen and liver were snap frozen. After transfer, washed cells were resuspended, blocked, and probed (Table I), and then washed twice with BSA-PBS and resuspended in 1% PF-PBS. Results ⌽ We used similar conditions to examine direct binding of Al488 X174/ Binding and transfer of C-opsonized IC anti-⌽X174 IC to THP-1 cells in either medium, NHS, or NHS-EDTA. Alternatively, Raji cells (2.5 ϫ 107 cells) were ligated with anti-CR2 We have previously described binding of C-opsonized IC to pri- ␮ ␮ Ј mAb (12.5 g mAb HB135 or Al488 HB135, or 8 g F(ab )2 HB135) for mate E CR1 and their transfer to acceptor cells (13). We have also 30 min at 37°C, washed three times with BSA-PBS, and, for some experiments, incubated with 6.25 ␮g Al488 rabbit anti-mouse IgG for an addi- demonstrated that Ags can be bound to E in the complete absence tional 30 min at 37°C (see Table I). The Raji cells were washed three times of C by use of a bispecific complex, an anti-CR1 mAb chemically with BSA-PBS, resuspended at 1 ϫ 107 cells/ml in RPMI 1640 medium, cross-linked to an anti-Ag mAb. These E-bound IC are transferred and combined at varying ratios (1:3 to 1:1) with THP-1 cells. Control to acceptor cells in a concerted reaction in which CR1 is removed studies in which CD45 on Raji cells was targeted used a similar approach, from the E (12, 14, 30, 35). We first examined C-dependent bind- based on binding of FITC mAb F10-89-4. Preliminary binding tests with ⌽ ⌽ unlabeled anti-CD45 mAb F10-89-4 or anti-CD21 HB135, followed by ing of X174/anti- X174 IC to E CR1 and Raji cell CR2 under development with Al488 anti-mouse IgG revealed that Raji cells express various conditions (Table II). Binding of IC to either cell type levels of CD45 comparable to CD21, and that CD45 is also well expressed on THP-1 cells (data not shown). ⌽ ⌽ Flow cytometry Table II. Binding of X174/anti- X174 mAb IC to human E or Raji cells requires C Analyses were performed on a FACSCalibur (BD Biosciences, San Jose, CA). For in vitro studies, fluorescence intensity is reported in the figures as Bindingb to mean channel number, in which 256 channels correspond to one decade of fluorescence intensity. PKH26-dyed THP-1 cells were distinguished from Conditiona Human E Raji Cells Raji cells or E based on their FL2 fluorescence. To estimate the overall percentage loss of fluorophore in the in vitro and in vivo experiments (see Serum ϩϩ figure legends and footnotes to tables), mean fluorescence was converted to Medium ϪϪ molecules of equivalent soluble fluorochrome (MESF) based on calibration Heat-inactivated serum ϪϪ beads (Flow Cytometry Standards, San Juan, PR). Serum EDTA ϪϪ Serum; cells pretreated with ϪϪ Fluorescence microscopy cognate-blocking anti-CR mAbc

Frozen sections of monkey spleen and liver were ﬁxed in cold acetone, a See Materials and Methods. rinsed in PBS, probed with Al633 goat anti-rabbit IgG or Al633 goat anti- b Binding assessed by RIA and/or ﬂow cytometry. mouse IgG, in the presence or absence of competing IgG, and examined c Human E: mAb 1B4. Raji cells: mAb FE8. 10 ug/ml. The Journal of Immunology 3673

FIGURE 1. Transfer of C-opsonized IC, bound to E CR1 by immune adherence, to THP-1 cells. A and B, After 60 min, 76% of the IC were removed from E incubated with THP-1 cells. Symbols: F, IC-opsonized E and THP-1 cells; E, IC- opsonized E only; ‚, naive E; Ⅺ, THP-1 cells only. Representative of three similar experiments. C–F, Fluorescent images of PKH26-dyed THP-1 cells before (C) and after (D–F) a 30-min incubation with green fluorescent IC bound to E, as in A and B. G and H, An undyed THP-1 cell is located near several E-containing bound green fluorescent IC at time 0 (G). Asso- ciation of the Al488 ⌽X174 with THP-1 cells is evident after 60 min (H). required NHS, and if C activity was abrogated by heat inactivation THP-1 cell observed with the FITC/TR filter before incubation or addition of EDTA, no binding was detected. If E or Raji cells with E. After 30 min of transfer, a red acceptor THP-1 cell is Downloaded from were pretreated with mAbs specific for the ligand binding site on identified in Fig. 1E, and Fig. 1, D and F, displays the green flu- CR1 or CR2, respectively, IC binding was eliminated. Experi- orescent IC (containing Al488 ⌽X174 initially bound to E) asso- ments with mAb 1H8, specific for C3dg, confirmed that this C ciated with this cell. Fig. 1G shows green fluorescent IC bound to fragment was associated with Raji cells with bound C-opsonized E before transfer to undyed THP-1 cells; Fig. 1H illustrates that IC. Treatment of these cells with mAb FE8 led to release of Ͼ80% after a 60-min incubation, most of the green fluorescent IC are

of both the bound IC and C3dg (data not shown). indeed associated with THP-1 cells. http://www.jimmunol.org/ For purposes of comparison, we investigated transfer of C-op- Similar experiments were performed with Raji cells as donor sonized IC from E to THP-1 cells. ⌽X174 was labeled with Al488 cells. When the Al488 ⌽X174/anti-⌽X174 IC was incubated in to allow direct detection of IC, and THP-1 cells were labeled with NHS for 30 min at 37°C, stable binding of Al488 ⌽X174 to Raji PKH26 to distinguish them from donor cells. C-opsonized IC cells was detectable by flow cytometry (Fig. 2A, E). However, in bound to E CR1 are stable in medium for 60 min (Fig. 1A, E). the presence of THP-1 cells, the IC were rapidly transferred (Fig. However, when combined with THP-1 cells, E-bound IC were 2, A and B; F). We probed the cell mixtures for CR2 and found, rapidly released and transferred to THP-1 cells (Fig. 1, A and as previously observed by others (24), that Raji cells display a slow B, F). rate of spontaneous loss of CR2 when incubated alone (Fig. 2C, E This transfer reaction was also followed by fluorescence micros- and ‚). Coincubation with THP-1 cells greatly increased the rate by guest on September 28, 2021 copy. Fig. 1C shows the red fluorescence from a PKH26-dyed of loss of CR2 from Raji cells containing bound IC, but not from

FIGURE 2. Simultaneous transfer of C-opsonized IC, bound to CR2 by immune adherence, and CR2 from Raji cells to THP-1 cells. A–D, After 30 min, Ͼ90% of the IC and 38% of CR2 were removed from Raji cells incubated with THP-1 cells, compared with 13% loss of IC and 14% loss of CR2 from Raji cells incubated alone. Symbols: F, IC- opsonized Raji and THP-1 cells; E, IC-opsonized Raji cells only; Œ, naive Raji and THP-1 cells; ‚, naive Raji cells only; Ⅺ, THP-1 cells only. Repre- sentative of four similar experiments. E, Samples from an experiment similar to that described in A–D were examined by fluorescence microscopy. Top panels, A red fluorescent PKH26-dyed THP-1 cell and a Raji cell with IC-bound green fluorescent Al488 ⌽X174, at time zero, viewed under dim white light with the TR filter (WL/TR), or with the FITC/ TR, TR, and FITC filters. Lower panels, A THP-1 cell and two Raji cells after 30 min of transfer. The THP-1 cell (identified by red fluorescence with TR) also exhibited green/yellow fluorescence due to transfer of Al488 ⌽X174 (FITC/TR and FITC). 3674 CR2 TRANSFER REACTION

FIGURE 5. Binding of a CD45-specific IgG2a mAb to Raji cells, followed by incubation with THP-1 cells, does not promote loss of CD45. Results for the positive control for transfer, based on opsonization with IgG2a anti-CD21, are also shown. The residual fraction of fluorescence of the opsonizing mAb (FL1, before transfer) or probing mAb (FL4, after transfer) was calculated based on mean fluorescence intensity after con- Ϯ FIGURE 3. Simultaneous transfer of an anti-CR2 mAb and CR2 from version to MESF. Average SD, three independent experiments. Raji cells to THP-1 cells. After 30 min, 73% of mAb HB135 and 74% of

CR2 were removed from Raji cells incubated with THP-1 cells, compared Downloaded from with 24% loss of mAb HB135 and 16% loss of CR2 from Raji cells in- from Raji cells to THP-1 cells; the intrinsic and extrinsic probes cubated alone. Symbols: as in Fig. 2, A–D. Representative of four similar demonstrated similar kinetics of transfer to THP-1 cells. To more experiments. closely simulate a B cell-bound IC in the absence of C, Raji cells previously incubated with unlabeled mAb HB135 were further reacted with Al488 rabbit anti-mouse IgG (Fig. 4). The intrinsic naive Raji cells (Fig. 2C, F compared with Œ). Both the intrinsi- Al488 label (rabbit anti-mouse IgG; Fig. 4, C and D) was trans- cally labeled Al488 IC (Fig. 2B) and CR2, as indicated by the ferred at a rate similar to that observed for the transfer of mAb http://www.jimmunol.org/ extrinsic anti-CR2 probe (Fig. 2D), were transferred to THP-1 HB135 (Fig. 3, A and B). After transfer, cell mixtures were probed cells. In contrast to the behavior of the intrinsic Al488 IC label, the with Al633 goat anti-rabbit IgG (Fig. 4, A and B) or APhCy anti- extrinsic signal rose rapidly and then slowly declined to 70% of its CR2 (Fig. 4, E and F). As seen for IC prepared with C (Fig. 2, C peak value (Fig. 2D). Thus, both IC and CR2 were removed from and D), the extrinsic probes showed an initial rapid rise in the the donor cell and transferred to the acceptor cell with very similar THP-1-associated signal, followed by a decrease over the next 20 kinetics. The subsequent decrease in magnitude of extrinsic signal min. When Al488 HB135 and unlabeled rabbit anti-mouse IgG attributable to CR2 on THP-1 cells may reﬂect either internaliza- were used to opsonize Raji cells, similar kinetics of transfer of the

tion by or release from the THP-1 cells. intrinsic and extrinsic labels was observed (data not shown). by guest on September 28, 2021 We also followed transfer of bound IC from Raji cells to THP-1 In view of the similarities in structure and function of CR1 and cells by fluorescence microscopy. The top panels of Fig. 2E show CR2, we thought it likely that Raji cell CR2 should be processed a red fluorescent PKH26-dyed THP-1 cell and a Raji cell opso- in the transfer reaction in a similar fashion to the processing of E nized with green fluorescent IC immediately after mixing. The CR1. In contrast, it would seem reasonable that structurally unre- bottom panels illustrate transfer of green IC from Raji cells to lated cell surface proteins would not be transferred after engage- THP-1 cells after 30 min. ment by specific mAbs and exposure to THP-1 cells. To address this question, it is necessary to use isotype-matched mAbs to allow Binding and transfer of C-independent anti-CR2 mAb-based for testing in the paradigms illustrated in Fig. 3. Based on these complexes considerations, we have examined the fate of CD45 (36) on Raji To extend this work to a C-independent paradigm, we opsonized cells when it is bound by IgG2a mAb F10-89-4 and then incubated Raji cells by ligation with the anti-CR2 mAb HB135 (29). As with THP-1 cells (Fig. 5). Although small amounts of the anti- shown in Fig. 3, both CR2 and the anti-CR2 mAb were transferred CD45 mAb are lost from the Raji cells during the experiment,

FIGURE 4. Simultaneous transfer of an anti-CR2 mAb-based IC and CR2 from Raji cells to THP-1 cells. After 30 min, 79% of the IC and 67% of CR2 were removed from the Raji cells, compared with 8% loss of IC and 23% loss of CR2 from Raji cells incubated alone. The Al633 goat anti-rabbit IgG probe also reported 75% loss of IC for the transfer sample. Symbols: as in Fig. 2, A–D. Rep- resentative of three similar experiments. The Journal of Immunology 3675 development with a noncompeting second mAb specific for CD45 Table III. Binding of A1488 ⌽X174/anti-⌽X174 mAb IC to THP-1 indicates no loss of CD45 from the Raji cells under conditions that cells does not require C do lead to removal of CD21, when it is bound by IgG2a mAb HB135. Moreover, this targeted loss of CD21 during the transfer Mean Fluorescence Intensity, Channel No.a reaction does not lead to loss of CD45 (data not shown). A1488 ⌽X174 To determine whether the Fc portion of IgG was necessary for Condition IC A1488 ⌽X174 None the transfer reaction, we opsonized Raji cells with the F(abЈ) frag- 2 Serum 337 Ϯ 20 76 Ϯ 2ND ment of mAb HB135 and compared its transfer with that of cells Medium 538 Ϯ 997Ϯ 261Ϯ 1 reacted with intact mAb HB135 (Fig. 6, F vs Œ). As demonstrated Serum/EDTA 94 Ϯ 889Ϯ 3ND previously (Fig. 3), intact mAb HB135 and associated CR2 were a Average Ϯ SD, n ϭ 2, representative of two similar experiments. simultaneously transferred from the Raji cell to the acceptor cell. Ј However, for cells opsonized with the F(ab )2 fragment of mAb HB135, neither the mAb fragment nor CR2 was removed from the were incubated alone, with naive THP-1 cells, or with THP-1 cells donor cell or taken up by THP-1 cells. Thus, the Fc portion of the containing bound IC. The results (Fig. 7) indicate that the modest substrate appears to be necessary for transfer. To further examine loss of CR2 from naive Raji cells alone (छ) is not increased by the possible role of FcR in the transfer reaction, we preincubated incubation with THP-1 cells, with or without bound IC (‚, E). THP-1 cells with human IgG. Human IgG in the reaction mixture However, as illustrated previously above, CR2 is removed and blocked transfer of an anti-CR2 mAb/rabbit anti-mouse IgG IC transferred from the Raji cells when IgG2a mAb HB135 is first from Raji cells (Fig. 6). Loss of both Al488 rabbit anti-mouse IgG bound to the cells, and they are then reacted with THP-1 cells; Downloaded from Ⅺ and CR2 from the Raji cells was reduced (Fig. 6, A and C, ), and binding of IC to the THP-1 cells substantially inhibits this reaction, the rate of loss was similar to the rates observed in the absence of presumably because of FcR blockade. In a similar experiment, Raji THP-1 cells (data not shown). Moreover, in the presence of human cells were opsonized with Al488 HB135. Pretreatment of the THP-1 IgG, THP-1 cells did not take up either Al488 rabbit anti-mouse cells with IC reduced the loss of Al488 HB135 on the Raji cells from Ⅺ IgG or CR2 (Fig. 6, B and D, ). 62 Ϯ 2% to 35 Ϯ 1%, relative to naive THP-1 cells. Finally, transfer In view of the apparent role of FcR in the transfer reaction, it is of NHS-opsonized, Al488-labeled IC from Raji cells to THP-1 cells http://www.jimmunol.org/ possible that binding of IgG, contained in IC, to FcR on the THP-1 is also inhibited when IC are prebound to the THP-1 cells. The flu- cells might activate these cells and lead to nonspecific proteolysis orescent signal of Raji cells opsonized with Al488 ⌽X174/anti- of Raji cell CR2. We examined the conditions that optimize bind- ⌽X174 IC in NHS decreased from 540 Ϯ 4to292Ϯ 8 (channel ⌽ ⌽ ing of X174/anti- X174 IC to THP-1 cells. Robust binding, pre- number) when naive THP-1 cells were acceptors, vs 540 Ϯ 4to sumably mediated by FcR, occurs when IC are added to THP-1 408 Ϯ 3 when THP-1 cells were pretreated with IC, corresponding to cells, but binding is reduced in serum, and is eliminated in serum- a reduction in transfer from 90 to 70%, respectively. EDTA (Table III). In analogy to the findings in Fig. 6, we suggest that human IgG in serum and serum-EDTA blocks IC binding by Primate study by guest on September 28, 2021 competing for binding sites on FcR. The lower, but discernible In vitro calibration studies with whole blood from four cynomol- level of binding obtained in serum is most likely caused by C gus monkeys established that the anti-human CR2 mAb HB135 activation, followed by C3b-mediated immune adherence of the (but not an isotype control) recognizes CR2 on monkey B cells; in opsonized IC to CR1 (37). Therefore, in subsequent experiments, THP-1 cells were pretreated with preformed IC in the absence of serum. To test for nonspecific proteolysis of CR2, naive Raji cells

FIGURE 6. Transfer does not occur if the substrate lacks an Fc region, or FIGURE 7. Binding of IC to THP-1 cells does not promote loss of CR2 if FcR on the THP-1 cell are blocked. After 30 min, 77% of mAb HB135 and from naive (Na) Raji cells, but does inhibit loss of CR2 from Raji cells op- 72% of CR2 were removed from Raji cells ligated with mAb HB135, com- sonized with HB135. The overlap of the छ, E, and ‚ indicates that naive Raji pared with 27% loss of mAb and 11% loss of CR2 from Raji cells with HB135 cells alone as well as naive Raji cells incubated with either naive THP-1 cells Ј F(ab )2. Representative of two similar experiments. After 30 min, 53% of the or IC-containing THP-1 cells had the same signal at 30 min. However, after 30 IC and 61% of CR2 were removed from Raji cells incubated with THP-1 cells, min, 74% of CR2 was removed from the Raji cells opsonized with mAb while only 14% of the IC and 17% of CR2 were removed from Raji cells HB135 and incubated with THP-1 cells; 47% was lost from the HB135-op- incubated with THP-1 cells in the presence of 1 mg/ml human IgG. sonized Raji cells when IC were prebound to the THP-1 cells. 3676 CR2 TRANSFER REACTION

FIGURE 8. In vivo experiment: IC bound to monkey B cells are removed in concert with loss of CR2. Al488 mAb HB135 was infused i.v., and 1 h later rabbit anti-mouse IgG was infused. Infusions are denoted by arrows on the x-axis. Blood samples were processed and analyzed, as described in Materials and Meth- ods. A and B, The percentage of Al488 HB135 and percentage of APhCy CD21-positive cells, of the doubly positive PE CD20/PerCP CD45 population, are plotted. C and D, The MESF values for these populations are plotted. In vitro calibrations in which equivalent amounts of Al488 mAb HB135 were added to anticoagulated whole blood (E) gave virtually identical binding to B cells. Downloaded from

addition, Al633 rabbit anti-mouse IgG only bound to B cells after A and B); however, the majority of the B cell-bound mAb HB135 pretreatment with mAb HB135. We also found that typically demonstrable at 60 min had been removed from the cells (Fig. 8C), ϳ

70% of monkey B cells (identified as CD20 and CD45 positive) coincident with the loss of the majority of the CD21 epitope (Fig. http://www.jimmunol.org/ were CR2 positive (see below). We performed a study on a mon- 8D). In addition, the rabbit IgG bound to the B cells at 60 min was key that had a low titer of circulating anti-mouse IgG, due to an i.v. also cleared (final MESF ϭ 445). Both spleen and liver sections infusion of mouse IgG 1 year earlier. Al488 HB135 was infused were examined for localized Al488 mAb HB135 and rabbit IgG, i.v. as a bolus at a dose of 50 ␮g/kg. The Al488 HB135 bound and both proteins were clearly demonstrable and colocalized in the rapidly (within 2 min) to circulating B cells (Fig. 8, A and C), and spleen (Fig. 9), but were not detectable in the liver (data not a fraction of cell-bound mAb was cleared within 30 min. ELISA shown). We performed a similar experiment (but only out to 3 h) measurements confirmed a low level of anti-mouse IgG in the on another cynomolgus monkey, and we observed the same pat- preinfusion samples, which was eliminated 30 min after infusion terns of B cell binding, sequestration, and clearance of bound li- of Al488 mAb HB135 (data not shown). gands and CR2 (data not shown). by guest on September 28, 2021 Rabbit anti-mouse IgG (100 ␮g/kg) was infused at the 60-min mark (Fig. 8, second arrow), and it immediately bound to the Discussion mouse mAb already on the B cells, as demonstrated by an increase Comparison of E CR1 and B cell CR2 in the transfer reaction in the Al633 goat anti-rabbit signal (MESF ϭ 1520, 2 min after The experiments reported in this work provide evidence that ex- infusion; background 450 before infusion). Shortly after this sec- tends the E CR1 transfer reaction to B cell CR2. We find that ond infusion, a fraction of the CR2-positive B cells (defined by C3dg-opsonized IC bound to Raji cell CR2 are transferred to ac- either Al488 HB135 or the APhCy anti-CD21 probe) was tempo- ceptor macrophages in a reaction that is quite similar to the transfer rarily removed from the bloodstream (Fig. 8, A and B), and the reaction for C3b-opsonized IC bound to E (Figs. 1 and 2). These remaining CR2-positive B cells underwent a rapid loss of both findings indicate that the kinetics of the two transfer processes are bound Al488 mAb HB135 as well as the CD21 epitope (Fig. 8, C comparable. These results, along with our previous studies (12– and D). At the 24-h point, the fraction of CR2-positive B cells 14), indicate that in each case the transfer reaction follows a con- found in the bloodstream returned to the preinfusion values (Fig. 8, certed process in which the C3 fragment-opsonized IC and donor cell C receptor are removed at the same rate. Moreover, CR2 removed from Raji cells is taken up by acceptor THP-1 cells, which again suggests that CR2 and its IC ligand are transferred together. In analogy to the E model in which substrates are bound to E via CR1-specific mAbs, we investigated B cell CR2 opsonization with an anti-CR2 mAb (Figs. 3–7). In this model, the anti-CR2 mAb HB135 is used as a surrogate for C3dg-opsonized IC; that is, the mAb IgG is noncovalently associated with the B cell CR2 in place of the C3dg-Ag-IgG IC. Robust and stable binding of both these substrates to Raji cell CR2 is achieved after a brief incubation. Although the kinetics and affinity of binding of these substrates to B cell CR2 may be different, we suggest that the model allows FIGURE 9. Fluorescence immunochemistry of frozen spleen sections. analysis of the transfer reaction in the absence of C. A–D, Sections were probed with Al633 rabbit anti-mouse IgG in the absence or presence of mouse IgG. The presence of the mouse IgG eliminated Substrates constructed with the anti-CR2 mAb HB135 are also the red fluorescence signal due to Al633 rabbit anti-mouse IgG (B vs D), transferred to THP-1 cells from Raji cells, and the kinetics of the but the green fluorescence due to the infused Al488 HB135 was not af- transfer process as well as the concerted properties of the reaction fected (A vs C). E–H,AsinA–D, with Al633 goat anti-rabbit IgG in the are quite similar to those observed in the experiments with C op- absence or presence of rabbit IgG. sonization. That is, mAb HB135 by itself, or in combination with The Journal of Immunology 3677 rabbit anti-mouse IgG, is taken up by THP-1 cells in a process that CR2 were all removed from the B cells in a concerted reaction. leads to loss of CR2 from Raji cells along with its appearance on Apparently, the reaction did not go to completion because lower, THP-1 cells. The fact that the signal on the THP-1 cells due to but measurable levels of B cell-bound Al488 mAb were demon- extrinsic probes (e.g., APhCy anti-CR2 or Al633 anti-mouse IgG) strable on the presumably released B cells at 24 h. The fact that decreases at longer transfer times suggests that a fraction of sub- both infused Al488 mAb HB135 and rabbit IgG could be found strates taken up by the THP-1 cells is internalized after transfer. colocalized in the spleen is consistent with this hypothesis (Fig. 9). The signal on the THP-1 cells due to intrinsic IC labels reaches a We found no evidence for localization of fluorescent material to maximum and does not decrease, indicating that the results cannot the liver, suggesting that with respect to organ localization, han- be explained by release of transferred ligands at later times. dling of IC bound to B cell CR2 may be somewhat different from The E transfer reaction requires Fc recognition of bound IC (12, handling of IC bound to E CR1. 13, 35), and studies of the process with Raji cells suggest a similar requirement (Fig. 6). Although mAb HB135 is a suitable ligand for Ј Possible extension to other acceptor cells transfer, its F(ab )2 fragments are not transferred; moreover, when THP-1 cells are incubated with human IgG (to block FcR), transfer In view of the importance of C, and in particular CR2 and its of mAb HB135 is abrogated. It is important to note that under ligand, C3dg, in the immune response (1–11, 39), the in vitro and these two conditions, in which the ligand bound to Raji cells is not in vivo demonstration of a robust and rapid transfer reaction and taken up by THP-1 cells, CR2 also remains on the Raji cells. That the localization of CR2-associated IC to the spleen in the monkey is, when the Fc component is not present or the IC substrate cannot model may reflect an important and natural process. Studies by effectively engage the FcR on the acceptor cell (Fig. 6), CR2 trans- several groups, initiated Ͼ30 years ago, revealed that IC infused Downloaded from fer is eliminated. Use of prebound IC to engage FcR on the THP-1 into animals are either rapidly phagocytosed by macrophages in cells also partially inhibited transfer of substrates and loss of CR2 the spleen and/or liver, or taken up by resident, but not Ag-specific, from Raji cells (Fig. 7). Finally, we find no evidence that IC bind- B cells in the spleen and then later transferred to FDC (40–46). ing to the THP-1 cells can activate these cells to promote direct Although the mechanism for transfer to FDC was never clearly loss of CR2 when these cells are incubated with naive Raji cells elucidated, it requires an intact C system and it is believed to play

(Fig. 7). a key role in the immune response. Our in vitro studies demon- http://www.jimmunol.org/ As in the E transfer reaction (12), it is likely, but not definitively strate that transfer can occur between donor Raji cells and acceptor proven, that the transfer reaction is initiated by recognition of Raji macrophages, in a reaction most likely mediated by FcR. The iden- cell-bound IC by FcR on the macrophage, allowing close juxta- tity of other cells that may remove IC and associated B cell CR2 position of the cells. This binding step is followed by proteolysis remains to be defined, but FDC, which have high levels of Fc␥RII of CR2, thus allowing uptake of released IC and associated CR2 by as well as CR2, are likely candidates (4, 6, 7). As stated by Hum- THP-1 cells. The identity of the protease(s), presumably associated phrey (44): “Why and how B lymphocytes with CR1 and CR2 with the THP-1 cells, which may cleave E CR1 or B cell CR2, receptors should transfer immune complexes containing comple- remains to be defined. Whether the transfer reaction and cleavage ment to FDC, which are even more rich in CR1 and CR2 receptors, and release of cell surface proteins are most specific and/or unique remains a mystery.” We suggest that close association of B cells, by guest on September 28, 2021 to C receptors or perhaps include other proteins that express the containing C3dg-opsonized IC, with FDC can be mediated by both SCR (38) remains to be determined. However, ligation of an un- Fc␥RII and CR2 on the FDC. This association may then allow related cell surface protein, CD45, by an IgG2a mAb does not lead transfer of the IC to the FDC for future presentation of intact Ag to loss of this protein in the transfer reaction (Fig. 5). Raji and to specific B cells. THP-1 cells both express high levels of CD45, and the modest loss It has been reported by several groups that B cell-associated of anti-CD45 mAb from the Raji cells may be due to simple re- HIV, apparently bound to B cell CR2 as C3dg-labeled IC, is par- equilibration, but could not have been mediated by the transfer ticularly infectious for T cells (19, 21, 47). Levels of B cell-asso- reaction, as CD45 was not lost from the Raji cells. ciated CR2 are reduced in AIDS (19–21), and T cells appear to have the resident surface protease that can facilitate constitutive Implications of the monkey model proteolysis of T cell-associated CR2 (24). Thus, it is reasonable Our studies of the B cell CR2 transfer reaction in the monkey that because of GP120-CD4 interaction, juxtaposition of T cells model (Figs. 8 and 9) display some similarities and two notable with C3dg-opsonized HIV IC on B cells can promote a transfer differences compared with the primate E CR1 reaction: temporary reaction leading to B cell CR2 cleavage and HIV uptake by and B cell sequestration, and spleen localization. The results clearly productive infection of the T cells. Although B cell CR2 is reduced indicate that Al488 mAb HB135 binds to monkey B cells in vivo. in AIDS, a recent report suggests that FDC CR2 is in fact stable in The early events seen in the first 30 min of the experiment are most this disease (47). Studies on another class of SCR-containing pro- likely caused by the low level of circulating anti-mouse IgG teins, the selectins, have demonstrated rigorous requirements for present in this monkey’s circulation. However, when the rabbit proteolytic release of these proteins from leukocytes (38). Thus, anti-mouse IgG was infused at 60 min, thus forming IC in situ on we speculate that because of one extra SCR (48), FDC CR2 is not B cell CR2, a large fraction of the monkey B cells containing the as easily proteolyzed as B cell CR2. Therefore, FDC CR2 may bound IC was temporarily removed from the circulation. By 24 h, provide a stable site for localization and retention of intact Ag. the percentage of CR2-positive B cells in the circulation had re- The studies of mouse CR2 initiated by Kinoshita and colleagues turned to the original value, but the cells had far lower levels of (27, 49, 50) have demonstrated that infusion of an anti-CR2 mAb CR2, and, in addition, the amount of B cell-bound Al488 mAb in the mouse leads to loss of splenic B cell CR2. Although the HB135 and rabbit IgG had decreased considerably. The fact that mechanism that leads to this CR2 loss has not been clarified, we CR2 levels were so low at 24 h indicates that it is unlikely that B suggest it reflects the B cell transfer reaction in the mouse. Our in cells found in the circulation at that time were newly synthesized vitro studies, as well as our findings in the primate model, provide B cells. Rather, we suggest that while the B cells were sequestered substantial evidence supporting the basic tenets of the B cell trans- (presumably in the spleen; see below), the transfer reaction oc- fer reaction. Future investigations, based on the use of anti-CR2 curred, and that Al488 mAb HB135, rabbit anti-mouse IgG, and mAbs in Fc␥R- and C-deficient mice (51), should lead to a more 3678 CR2 TRANSFER REACTION detailed understanding of the role of C in enhancing the immune 25. Boackle, S. A., J. M. Brown, and V. M. Holers. 2000. Role of complement response to Ags in IC. receptors type 1 (CR1/CD21) in murine SLE. 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