lymphocytes. antigen processing and presentation by ...

Natural antibody and complement-mediated antigen processing and presentation by B lymphocytes. This information is current as of November 2, 2016.

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J Immunol 1994; 152:1727-1737; ; http://www.jimmunol.org/content/152/4/1727

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 1994 by American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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B P Thornton, V Vetvicka and G D Ross

Natural Antibody and Complement-Mediated Antigen Processing and Presentation by B Lymphocytes’ Brian P. Thornton, V6clav VGtviEka, and Gordon

D. ROSS*

Department of Microbiology and Immunology, University of Louisville, Louisville, KY 40292

T

he original observations showing a role for C in the immune responsewere published 19 years ago by Pepys (1). Later,other laboratoriesreported that an inherited or acquired deficiency of C3 was associated with diminished responses to T-cell dependent Ags, including absent secondary responses and isotype switching (2-4). Patients with deficiencies of C3 and C4, aswell as the iC3b receptors CR3 and CR4, exhibited impaired responses to primary protein Ags such as KLH3 or bacteReceived for publication August 2, 1993. Accepted for publication November 25, 1993. The costs of publlcation of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indlcate this fact.

’

This work was supported by research grants from the American Cancer Society (IM-308C) and the Universlty of Louisville School of Medlclne.

’

Address correspondence and reprint requests to Dr. Gordon D. Ross, Department of Microbiology and Immunology, University of Louisville, Louisville, KY 40292.

’

Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; ATS, activated thiol Sepharose-4B; CRI, C receptor type 1, C3b/C4b/iC3b receptor, CD35; CR2, C receptor type 2, iC3b/C3dg/C3d receptor, CD21; CR3, C receptor type 3, iC3b receptor, CD1 1 b/CD18, Mac-1, M o l ; CR4, C receptor type 4, iC3b receptor, CDllc/CD18. p150,95; DTT, dithiothreitol; IC, immune complex; Slg, surface Ig; VBS, veronal-buffered saline; SPDP, n-succinimidyl 3-(2-pyridyldithio)propionate.MTT, (3-[4,5-dimethylthiazol-2-y]-2,5-diphe. nyl tetrazolium bromide; thiazolyl blue). Copyright 0 1994 by The American Association of lrnmunologsts

riophage +X- 174 (5-7). A later study of C-dependent enhancement of Ag recognition showed a probable role for IgM Ab specific for the immunizing Ag (8). Defining the role for C3 in the immune response has been difficult because nearly all lymphocyte subsets and immune accessorycells express C3 receptors (9). Major progress was made with mAbs to murine CR 1 and CR2 (10-12) and a soluble rCR2 chimeric moleculethat was bivalent in C3dbinding sites ( 1 3). Both primary and secondary responses to T cell-dependent and-independent Ags were suppressed by mAb that blocked the C3d-binding site shared by murine CRl and CR2but not by mAb that blocked only the C3b-binding site of murine CRl (10-12). The rCR2/ IgG chimera competed with B cells for soluble polymerized C3dg in vitro, and in mice it suppressed responses to T cell-dependent Ags in the same way as did anti-CR2 or cobra venom factor (13). The hypothesis for this investigation was that primary protein Ags become coated with iC3b/C3dg that promotes binding to B cell CR2. Because most soluble proteins do not activate the alternativepathway of C, activation of the classical pathway must occur via IC formation with IgM and/or IgG natural or polyreactive Abs reactive with the primary Ag ( 1 4). 0022-1 767/94/$02.00


Normal immune responses to primary protein Ags (those not seen previously by the immune system) have been shown to require C3 and the C3 receptor CR2 (CD21). This investigation tested the hypothesis that natural Abs to the primary protein Agkeyhole limpet hemocyanin (KLH) exist in normal serum and will form immune complexes (IC) that activate C and generate bound iC3b/C3dg, thereby promoting B cell CR2-dependent Ag processing. Both IgM and IgG anti-KLH were detectable in sera from 1 1 normal donors. IC generated with fresh serum bore iC3b and bound to CRI (CD35), CR2, and CR3 (CD11 b/CD18). Further treatment of IC with serum containing rCRl formedIC-bearingC3dg that bound only to CR2. When ICs were mixed with B lymphoblastoid cell clones, CR2-dependent processing of the KLH occurredthat was dependent on boundC3dg andCR2. Processing occurred regardless of whether the B cells bore KLH-specific surface Ig, although the efficiency of processing was greater with KLH-specific B cells. Both KLH-specific and nonspecific B cell clones presented K L H to KLH-specific T cells. The binding of KLH IC by normal B lymphocytes induced expression of the B7/BB1 Ag (CD80), the required co-stimulatory ligand for T cell CD28. Blocking experiments indicated that although bound C3 and CR2 were required to mediate IC binding to B cells, induction of CD80 expression required the secondary ligation of IC-associated IgG to B cell FcRll (CD32). These data support the hypothesis that responses to primary protein Ags involve IgG natural Abs and C3 that mediate Ag processing and presentation via B cell CR2 and FcRII. Journal of Immunology, 1994, 152: 1727.

1728

Materials and Methods

Pharmaceuticals, NewYork, NY); streptavidin-phycoerythrin (Tago, Inc., Burlingame, CA); D-biotin-N-hydroxy-succinimide ester (Boehringer Mannheim Biochemicals, Indianapolis, IN); cyclosporin A (Sandoz Research Institute, East Hanover, NJ); soluble rCRI was generously provided by Dr. Alfred Rudolph (T Cell Sciences, Cambridge, MA); human rIL-2 (Intergene, Purchase, NY); and Iodo-gen iodination reagent (Pierce, Rockford, IL).KLH was conjugated to FlTC in the same manner as Abs (26).

mAbs and polyclonal Abs The MN-41 hybridoma secreting anti-CR3a-chain(CD1Ib)-specific mAb was obtained from Drs. Allison Eddy and Alfred Michael of the University of Minnesota, Minneapolis. HB-5 anti-CR2,OKT-3 (antiCD3). and OKT-4 (anti-CD4) hybridoma cells were obtained from the American Type Culture Collection (Rockville, MD). Each mAb was isolated from ascites fluid by sequential steps of caprylic acid and ammonium sulfate precipitation followed by Mono-Q FPLC (Pharmacia) anion exchange chromatography (27). Purified carrier-free O W - 7 anti-CR2 mAb was provided by Dr. Gideon Goldstein of Ortho Pharmaceuticals (Raratan, NJ). Rabbit anti-C3c and C3d were generated as previously described (28), then mixed together and used for the preparation of F(ab'), anti-C3 (26). Rabbit anti-KLH serum was generated by immunization with purified KLH and a portion of the IgG fraction was coupled to FITC (26). Rabbit anti-CR1 serum was generated by immunization with rCRl and then F(ab'), fragments of IgG were generated (29). Rabbit anti-CR2 serum generated by immunization with rCR2 was kindly provided by Dr. Mike Holers, Washington University, St. Louis, MO. The IgG fraction of this antiserum was isolated by ammonium sulfate precipitation followed by a DEAE-Sephacel anion exchange chromatography (26). Both this polyclonal anti-CR2 and OKB7 anti-CR2 mAb were shown to be capable of stimulating Raji cell proliferation in serum-free medium (30). Ascites fluid containing rat anti-C3c mAb (clone 4) was provided by Professor Peter J. Lachmann, Cambridge University, UK. Sheep anti-IgM-FITC was purchased from Sigma. B4 RDl mAb (antiCD19-phycoerythrin) and mouse myeloma IgG-phycoerythrin were purchased from Coulter Cytometry Division (Miami Lakes, FL). The IgG fraction of anti-CD80 mAb was provided by Dr. Edward Clark of the University of Washington, Seattle, and conjugated to biotin (31). F(ab) fragments of anti-FcRII mAb AT10 were generously provided by Dr. Martin Glennie of Tenovus Research Laboratory, Southampton, UK.

ELISA for natural Ab to KLH Immulon micro-ELISA plates (Dynatech, Alexandria, VA) were coated with 50 p g h l of KLH in carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. After washing (PBSRween 20/3% BSA) and blocking the plates with 0.2% gelatin for 30 min at room temperature, 100 pl of serial twofold dilutions of serum with Tween 20 were added to the wells and incubated for 2 h at room temperature. Alkaline phosphatase-conjugated goat anti-human polyvalent Ig (a, p , A chain specific; 1:700 dilution; Sigma) or alkaline phosphatase-conjugated goat anti-human Ig ( p chain specific; 1: 12,000 dilution or y chain specific; 1:1O,ooO dilution; Sigma) were added and incubated for 2 hat room temperature, followed by color development with p-nitro-phenol phosphate carbonate-bicarbonate buffer, pH 9.6. Theplates were evaluated by measuring the absorbance at 410 nm with a Dynatech MR-600 microplate reader. Serum from a patient with congenital agammaglobulinemia (IgM < 20 mg/dl, IgG < 40 mg/dl, IgA < 6.7 mg/dl) was provided by Dr. Karen Cost (University of Louisville) as a negative control. Normal serum Ab levels measured in parallel were: IgM 63 to 277 mg/dl, IgG 723 to 1685 mg/dl, and IgA 69 to 382 mg/dl.

Reagents Reagents were obtained from the following sources: FCS (HyClone Laboratories, Logan, UT); culture medium AIM V (GIBCO, Grand Island, NY); RPMI 1640 medium (Whittaker, Walkersville, MD); KLH (Pacific Biomarine, Venice, CA); gelatin (Bio-Rad,Richmond,CA); avidinalkaline-phosphatase, Ficoll, MTT, mitomycin C, L-cysteine, and Sigma 104 p-nitrophenyl phosphate substrate tablets (Sigma Chemical Co., St. Louis, MO); DEAE-Sephacel, SPDP, Dextran T500, Sephadex (3-25, Sepharose 6B, Superose 6, and ATS (Pharmacia LKB Biotechnology, Piscataway, NJ); DTT (Kodak, Rochester, NY); Hypaque M (Winthrop

Size fractionation of serum IgM natural Abs A 3-ml sample of serum from a normal donor with relatively high IgM anti-KLH activity was concentrated to 1 ml in a dialysis bag by dehydration with solid sucrose. Half of the concentrated serum was loaded onto a 1.6 X 50 cm column of Superose 6 connected to a Waters HPLC system (Millipore, Milford, MA). Fractions of 0.3 ml were collected and analyzed for total protein by measuring absorbance at 280 nm and for anti-KLH IgM by ELISA as described above.


B cells play a significant role inAg processing and MHC class 11-mediated presentation of Ag peptides to Th cells (15-19). Although the initial assumption was that Ag uptake by B cells required the presence of Ag-specific SIg, later studies showed that Ag linkage to non-SIg membrane molecules (e.g., class I molecules or transferrin receptors) was also effective in allowing subsequent Ag processing and presentation to Ag-specific T cells (18). As with Ag nonspecific macrophages and dendritic cells, the specificity of responses involving Ag processing by nonspecific B cells should occur with subsequent presentation toAgspecific Th cells. CR2 appears to be an appropriate target molecule to promote nonspecific Ag uptake by B cells because CR2 is expressed on the majority of mature B cells (20-22). Some evidence for C3-enhanced Ag processing by B cells was reported by Arvieux et al. (23) who studied the presentation of tetanus toxoid Ag-C3b or -C4b complexes by B lymphoblastoid cells to a tetanus toxoidspecific Th cell line. In another report, pneumococcal polysaccharide-C3d complexes were shown to be bound to B cells via CR2, and the presence of bound C3d was shown to enhance in vitro responses (24). In a third investigation (25),influenza virus complexed with C3 was shown to be processed by EBV-transformed B cells via CR1 and CR2, resulting in presentation of influenza virus Ags to normal peripheral blood T cells. This investigation tested the hypothesis that low levels of natural Ab form IC with primary Ag, resulting in C3 deposition onto the Ag, uptake of the Agby B cells via CR2, processing of the Ag, and subsequent presentation of the Ag to Ag-specific Th cells. Natural Abs to KLH were demonstrated in the sera from all 1 1 donors examined, and KLH mixed with normal serum formed IgM/IgG-KLH-C3 IC that were taken up and processed by B lymphoblastoid cells lacking KLH-specific surface Ig. The B cell lines that had taken up KLH in this nonspecific manner stimulated KLH-specific, MHC class 11-compatible Th cells to proliferate, indicating that Ag presentation could occur in this way. This approach differs from that used by Arvieux et al. (23) inthat processing and presentation were demonstrated with IC generated with a primary protein Ag, natural Ab, andphysiologically bound C3dg, rather than with a secondary Ag bearing chemically linked C3b. In addition, Ag-specific B and T cell clones were derived from unimmunized donors.

COMPLEMENT-DEPENDENT ANTIGEN PROCESSING

1729

Journal of Inimunology

Preparation of IgM/lgG-KlH-iC3b/C3dg IC

ELlSA of IC for bound IgM, IgG, C3, and KLH A sandwich ELISA was used to analyze the composition of KLH-IC. Micro-ELISA plates were coated with 50 pg/ml of rabbit anti-KLH IgG in carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. After washing (with PBS/Tween 20/3%BSA) and blocking the plates with 0.2% gelatin for 30 min at room temperature. 200 pl/well of a 1: 15 dilution ofIC generated from heat-inactivated serum or fresh serum (in PBS) was added to the wells and incubated for 2 h at room temperature. The presence of complex-bound IgM, IgG, C3, or KLH was detected by addition of 200 ~ l / w e l l of either alkaline-phosphatase-conjugated goat antihuman IgM (p-chain specific, 1 : 12,000). alkaline-phosphatase-conjugated goat anti-human IgG (y-chain specific, I : 10,000), biotinylated clone 4 anti-human-C3c mAb, or biotinylated rabbit anti-KLH IgG. The assay was developed by incubation with 200 pl/well of avidin-alkalinephosphatase (1:8,000) in those wells containing biotinylated Ab, followed by 200 pl to all wells ofp-nitro-phenol phosphate substrate diluted in carbonate-bicarbonate buffer. pH 9.6. The plates were evaluated by measuring the absorbance at 410 nm as above.

Cell lines The B lymphoblastoid lines Raji and Daudi, the T lymphoblastoid line 8402. and the monkey line B9S-8 were obtained from American Type Culture Collection. Cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, and antibiotics.

Peripheral blood neutrophils and lymphocytes Blood from healthy volunteers was drawn into sodium citrate anticoagulant. After sedimentation of erythrocytes with dextran, mononuclear cellsand neutrophils were separated by centrifugation on a two-step (density = 1.08 and 1.105)density gradient of FicoH-Hypaque (32). Cells were washed five times in RPMI 1640 medium and maintained in an ice bath until used.

K l H-specific, EBV-transformed B lymphoblastoid cell clones The method described by Lanzavecchia was used (33). Briefly, IO’ mononuclear cells isolated from a donor with relatively high titer of anti-KLH natural Ab were incubated in I O ml of RPMI 1640 medium supplemented with 10% FCS, antibiotics, 30% spent culture supematant from the B95-8 cell line (as a source of EBV), and 600 ng/ml of cyclosporin A. After 16 h incubation at 3 7 T , the cells were washed three times with warm RPMI 1640 medium, suspended at S X 104/mlin RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, antibiotics, and cyclosporin A, and distributed into 96-well flat-bottom culture plates at 200 pl/well. After 13 days cultivation, the supernatants were screened for anti-KLH Ab secretion by ELISA. Positive wells were cloned by limiting dilution and screened again for anti-KLH Ab production. Clones of cells demonstrating anti-KLH secretion were subsequently tested for KLHspecific SIg by tests for membrane surface staining with KLH-FITC and flow cytometry, using murine myeloma IgG-FITC and non-anti-KLHsecreting B cell lines as negative controls.

Bulk cultures of Th cells enriched for K l H specificity The method described by Gosselin et al. (34) was used. Briefly, 2 X IO’ mononuclear cells isolated from a donor with high anti-KLH natural Ab titer were incubated in AIM V medium supplemented with 30 pg/ml of KLH for 3 days. Nonadherent cells, isolated by washing the culture flasks three times with RPMI 1640 medium, were cultured for an additional 14 days in AIM V medium supplemented with 30 pg/ml of KLH, 30 U/ml of rIL-2. and 2S% autologous serum. After removal of dead cells by centrifugation through a cushion of Ficoll-Hypaque (density = 1.08 g/ml), the remaining population of viable cells consisted of 292% CD4+ Th cells.

Assay of IC binding to complement-receptors ICs were analyzed for binding to lymphoid cells or neutrophils by immunofluorescence staining with rabbit anti-KLH-FITC and flow cytometry. Cells ( 5 X 105 in 0.1 mlof RPMI 1640 in12 X 75 mm plastic tubes) were incubated with KLH-IC (or uncomplexed KLH as a negative control) for 30min at 37°C and then washed by centrifugation for 10 min at 400 X g through a 3-ml cushion of12% BSA in PBS with 10 mM sodium azide (12% BSA/F’BS/azide). The cells were stained by incubation with rabbit anti-KLH-FITC for 30 min at 4°C. followed by centrifugation through a cushion of 12% BSA/F’BS/azide. To analyze the specificity of IC binding to CRI, CR2, and CR3, cells were treated with

Ag processing The modified method of Chain et al. (35) was used. Briefly, S X IO6 APC in 3 ml of RPMI 1640 were incubated for 1 h at 37°C with ‘ZsI-labeled KLH IC (iodinated with Iodo-gen as recommended by Pierce) at a concentration of 50 pg/ml and a sp. act. of 2.5 FCi/Fg protein. After incubation, the cells were washed extensively at 4°C (until there were less than 1% counts in the supernatant) and then incubated an additional 1 to 4 h at 37°C. The supernatants were collected and analyzed for TCAsoluble radioactivity after precipitation of insoluble radioactivity with cold 10% TCA.


Soluble ICs were generated with fresh human serum and the KLH A& linked to a solid phase support on which the IC could be washed free from uncomplexed serum proteins and later eluted for analysis. Free sulfydryl groups were introduced into KLH using SPDP by reaction of 5 ml ofKLH (10 mgiml in PBS) with 8.33 p1 of a 12.5 mg/ml stock solution of SPDP in 1 0 0 % ethanol for 30 min at room temperature. Unreacted SPDP was removed by passage of the KLH through a 3 X 12 cm column of Sephadex G-25 equilibrated with 0.1 M sodium acetate/O.l M NaCI, pH 4.5, and then concentrated by ultrafiltration to 5 ml with a YM-30 membrane (AmiconCorp., Lexington, MA).KLH-SPDP was reduced by addition of SO mM DTT. and excess DTT was removed after 20 min at room temperature with the same Sephadex G-25 column equilibrated with PBS/I mM EDTA. After concentrating to 5 mlwith a YM-30 membrane, the reduced KLH-SPDP was added to 60 ml of ATS that had been equilibrated with PBS/I mM EDTA and aspirated to a nearly dry cake on a Buchner funnel with sintered glass filter. The gel slurry was placed in a SO-ml conical centrifuge tube and mixed on a rocking platform overnight at 4°C. The KLH-ATS sluny was poured onto a Buchner funnel with sintered glass filter and washed with several column volumes of VBS containing 0.5 mM magnesium chloride and 0.15 mM calcium chloride (VBS”). After aspiration to nearly a dry cake, 1 mlof KLH-ATS was mixed with 14 mlof fresh human serum in a 15-ml conical tube and placed on a rocking platform at37°C for 2 h. Unbound serum proteins were eluted by pouring the KLH-ATS into a 0.5 X 14 cm column and washing at 4°C with 500 mlof 5 X concentrated VBS” stock solution or until the OD,,, was ~ 0 . 0 0 5 . The soluble Ig-KLHIC3b ICs were eluted from the ATS matrix with IS mM L-cysteine in PBS, pH 7.0, dialyzed against PBS overnight at 4°C. and concentrated to 5 mlwith a YM-30 membrane. For conversion of complex-bound iC3b into C3dg. the same procedure was first used to generate fixed iC3b and then the ATS-bound ICs were incubated a second time with serum supplemented with soluble rCR 1 to serve as a factor I cofactor. For this purpose, serum from the same donor was dialyzed overnight at 4°C against half-ionic strength VBS2+containing 2.5% dextrose and 10 mM sodium azide. Thirty micrograms per ml of rCRl was added to the dialyzed serum and then the serum was mixed with the ATS-KLH-Ig-iC3b on a rocking platform for 2 h at37°C. After washing away unbound serum proteins as before. the IgM/IgG-KLH-C3dg ICs were eluted with cysteine as described above.

blocking Abs to each complement receptor type before addition of IC or ICs were incubated with rabbit antikc3 F(ab’),before incubation with the cells. To assess binding of IC to B and T cells, two-color analysis was performed with FITC labeling of IC in combination with phycoerythrinlabeled anti-CD19 to identify B cells or anti-CD3-phycoerythrin to identify T cells. Flow cytometry data was obtained with a Coulter Profile I1 (Coulter) and stored in the list mode. The presence of KLH on cells was assessed later by analyzing the stored list mode data with the EPICS Elite Flow Cytometry Workstation software (Coulter). Histograms generated with the Elite software were exported as metafiles to Freelance Graphics for Windows (Lotus Developments Corp., Cambridge, MA) to combine several histograms into one graph.


1730 n 9c

0.5

1:lOO

01:200

1:300

L

0.15

0.3

d 4-

c

Q

d

0.1

0

0

0.05

0.1

n

"

DH BPT MW GDR Ern

CO

JH

RHL RFB Zhi

Men LO

nm

B cell clones were incubated with mitomycin C (25 pg/ml) for60 min in the dark to block their ability to proliferate. After six washes in RPMI 1640 medium with 5 % FCS, the mitomycin C-treated B cells (5 X IO4) were mixed with an equal number of KLH specificity-enriched CD4' T cells in 0.2 ml of RPMI 1640 medium supplemented with glutamine, 10% FCS, antibiotics,and various concentrations ofKLH IC. After 3 days incubation, T cell proliferation was measured using the MTT assay (36).

JH

RHL RFB Zhl

Men LO

Serum Donors FIGURE 2. ELISA of sera from normal donors for the presence of IgG natural Abs to KLH. Serum from a patient with agammaglobulinemia (DH) served as a negative control. Three dilutions of each serum sample (1 :I 00, 1 :200, 1 :300) were tested. The buffer control was 50.08 and wassubtracted fromall serum values. Individual sera were tested 2 1 0 times with consistent results; however, the data shown represents one characteristic experiment when all sera were tested at one time.

280 O.D. at

O.D. at 410 nm

7

0.45

6 -5-

(O.D. at 280 nm) "~,"_;_.1_1_.._1_~

0.3

4-

Activity

3-

lnduction of the CD80 Ag

2-

Samples of 5 X lo5 PBMC in 0.1 ml of RPMI 1640 were incubated with 100 pg/ml of the different types of IC, 0.1 ml rabbit anti-KLH/KLH IC (150 pg/ml KLH and 100 pg/ml rabbit anti-KLH incubated for 30 min at 4"C), or 500 Fg/ml of rabbit IgG anti-CR2 forup to 4 h at 37°C. After centrifugation through a cushion of 12% BSAiPBSiazide, cells were stained with anti-IgM-FITC and anti-CD80-biotin for 30 min at 4°C. The cells were washed by centrifugation through a cushion of 12% BSA/PBS/azide and incubated for an additional 30 min at 4°C with streptavidin-phycoerythrin followed by removal of unbound stain with 12% BSARBSiazide as above. Finally, the cells were resuspended in 200 pl of 1% BSA/PBS/azide and analyzed by two-color flow cytometry for SIgM+ B cells that were induced to express phycoerythrin-labeled CD80 Ag.

1-

Results

CO

0

Md 0.05

1

11 41 31 21

51 91 81 71 61

108 118 128 138

Fraction Number

FIGURE 3. Superose 6 molecular sieve chromatography of normal serum IgM natural Abs to KLH. Serum from donor RHL who had a relatively high titer of IgM anti-KLH Ab was fractionated and column fractions were tested for total protein (OD at 280 nm) and IgM anti-KLH activity by ELISA. Two peaks of IgM anti-KLH activity were detected, one eluting with an apparent m.w. of 900 kDa presumably represents pentameric IgM, andthe second elutingwith an apparent m.w. of 190 kDa presumably represents monomeric IgM.

Natural Abs to KLH in normal human serum

Natural IgM and IgG Abs to KLH were detected in serum from 11 normal donors by a sensitive ELISA (Figs. 1 and 2 ) . Serum from a patient with congenital agammaglobulinemia (DH) was used as a negative control. There was little correlation between serum IgM and IgG anti-KLH levels in individual donors. For example,one donor (RHL) consistently had a higher IgM anti-KLH level than the other donors but also had the lowest level of IgG anti-

KLH, and conversely, donor GDR had the highest level of IgG anti-KLH but a relatively low level of IgM anti-KLH. At least some of the IgM anti-KLH may represent SIgM shed from the surface of B cells because size fractionation of serum from donor RHL showed that a portion of the IgM anti-KLH was monomeric (Fig. 3).


FIGURE 1. ELISA of sera from normal donors for the presence of IgM natural Abs to KLH. Serum from a patient with agammaglobulinemia (DH) served as a negative control. Three dilutions of each serum sample (1 :loo, 1 :200, 1 :300) were tested. The buffer control was 50.06 and wassubtracted fromall serum values. Individual sera were tested 2 1 0 times with consistent results; however, the data shown represents one characteristic experiment when all sera were tested at one time.

Ag presentation

)R Ern

DH

Serum Donors

Journal of Immunology

1731

IgG

c3c

E E 0

1

G c Q

d

0.5

O K 8 7 antl-CR2

0 n

Fresh serum Serum heated

1

+ Rajl cells 7.8% K L H +

1

56"C, 30 min

Serum used for KLH immune complexes

Log Fluorescence Intensity

Binding of KLH IC to cells with C3 receptors

FIGURE 5 . Flowcytometry analysis of KLH IC binding to Raji B lymphoblastoid cells. After incubation ofIgM/lgGKLH-C3 IC (30 &ml) with Raji cells for 30 min at 37"C, the cells were washed and the proportion of cells with bound IC was determined by staining with rabbit anti-KLH-FITC. The staining obtained with uncomplexed KLH (Raji cells plus KLH) was used as the negative control to assess nonspecific staining. Both the IC generated with fresh serum (IgM/lgGKLH-iC3b) and the IC prepared with serum containing rCR1 (IgM/lgG-KLH-C3dg) bound to Raji cells. Incubation of IgM/ IgG-KLH-iC3b IC with F(ab'), antiLC3 for 30 min at 4°C before incubation with Raji cells reduced IC binding from 84.3 to 1.5% (98.2% inhibition).Similarly, the prior incubation of Raji cells with OKB7 but not HB-5 anti-CR2 mAb (20 pg/mI) prevented the Raji cells from later binding IC. OKB7 inhibited IC binding by 90.7%,, whereas HB-5 produced little inhibition when compared with the untreated Raji cell control. Results from one characteristic experiment are presented.

In the absence of erythrocyte C R l , the bound C3 present on KLH ICgeneratedwith whole serumshould be primarily in the form of iC3b (37), and thus should promote binding of the IC to CR1, CR2, CR3, and CR4 (27, 38). Figure 5 shows that binding of IgMAgC-KLH-iC3b IC to Raji cells that express only CR2 and lack other types of C3 receptors (28) was dependent on both bound C3 and CR2, because binding was inhibited by either F(ab'), antiX3 or OKB7 anti-CR2 but not by HB-5 anti-CR2, which does not block the C3d-binding site of CR2 (39). The KLH IC continuedto bind to Rajicells via CR2 after complexbound iC3b was converted to C3dg by a second treatment of the ATS-bound IC with serum containing rCR 1. The Daudi B cell line that expresses a very low density of CR2 and the T cell line 8402 that lacks detectable C3receptors did not bind IC (data not shown). Analysis of IgMAgG-KLH-iC3bbinding to CD19' B cells among PBMC demonstrated attachment to the ma-

jority of B cells and inhibition of binding by either antiCRl or anti-CR2 (Fig. 6). Nearly complete inhibition of B cell binding of the iC3b-bearing IC was obtained with a mixture of anti-CR1 and anti-CR2 (Fig. 6). The 10.1% of B cells that bound the iC3b-IC in the presence of anti-CR1 and anti-CR2 may represent the small number of CD5' B cells in PBMC that are known to express CR3 and CR4 (40, 41). Although T cells have been reported to express a low surface density of CR2 (42-45), no IC binding was detectable by direct analysis of Ig-KLH-C3dg binding to CD3' peripheral blood T cells (data not shown). Tests for immune complex attachment to neutrophils demonstrated that iC3b-ICboundprimarily to CRI and CR3 and that C3dg-IC did not bind to neutrophils (Fig. 7). Absence of binding of the C3dg-IC toneutrophils indicated that the IC

Generation of KLH IC with normal serum

KLH was disulfide linked to ATS in such as way that KLH IC could be generated on the Sepharosematrix with whole serum and later washed free of serum proteins before elution with L-cysteine. An ELISA was used to analyze the composition of IC generated in this manner. Serum from donorRHL was used aseitherfreshserum to generate TgM/IgG-KLH-C3 IC or serum heatinactivatedat 56°C for 30 min to generate IgM/IgG-KLH IC lacking bound C3. Figure4 shows thatICgenerated from eitherheatinactivated or normal serum contained both IgM and IgG but that only the IC generated with fresh serum contained significant amounts of C3.


FIGURE 4. ELISA of KLH IC generated with normal serum for the presence of IgM, IgG, and C3. IC generated from fresh serum and serum heated to 56°C for 30 min contained comparable levels of IgM and IgG anti-KLH Abs. The IC generated from serum heated to 56°C for 30 min bore only very small amounts of fixed C3.

1732

COMPLEMENT-DEPENDENT ANTIGEN PROCESSING PMN +

PBMC +

I

+ PMN + igMllgG-KLH-iC3b 37.3% KLH

danti-CR1 Rab

+

MN-41 anti-CR3 + PMN +

S I

il i,~ OKB7 antl-CR2 + PBMC +

~

Log Fluorescence Intensity

FIGURE 6. Two-color flow cytometry analysis of IC binding to CD19' normal B cells. PBMCs were incubated simultaneously with KLH IC and anti-CD19-phycoerythrin mAb. After staining of the bound KLH IC with rabbit anti-KLH-FITC, the level of FlTC staining on CD19-phycoerythrin' cells was determined. PBMCs incubated with uncomplexed KLH (PBMC + KLH) served as a negative control for nonspecific staining by the anti-KLH-FITC. Treatmentof PBMC with 10 pghl of either rabbit F(ab'), anti-CR1 or OKB7 anti-CR2 mAb partially inhibited IC binding to CD19' B cells. The residual IC staining obtained after blockade of both CR1 and CR2 may be due to the CD5' B cell subset that is known to express CR3 and CR4.

contained insufficient amounts of IgG to mediate attachment solely via Fc receptors. IC prepared with heat-inactivated serum also failed to bind to B cells or neutrophils (data not shown). Processing of KLH IC by B lymphoblastoid cells

EBV-transformed B cell lines were generated with the PBMC from two donors who had a relatively high level of serum anti-KLH and whose lymphocytes gave a proliferative response to KLH (not shown). From each of the B cell lines, clones of B cells were isolated that either expressed or lacked KLH-specific SIg, as determined by their ability to bind KLH-FITC. By immunofluorescence flow cytometry analysis, the majority of cells from both KLH-specificandnonspecific B cell clones expressed CR1 and CR2 and lacked detectable CR3 and CR4 (not shown). Each type of B cell clone was examined for its ability to process KLH, asdetermined by the conversion of TCA-insoluble '251-KLH protein into TCA-soluble 1251KLH peptides (Fig. 8). The IgM/IgG-KLH (Ig-KLH) IC were processed only by the KLH-specific B cell clones andnotby the nonspecific B cell clones, demonstrating that with such IC lacking C3, processing was solely SIg

Log Fluorescence Intensity FIGURE 7.

Flow cytometry analysis of IC binding to isolated neutrophils (peripheral mononuclear cells; PMN). Bound ICs were detected as described in the legends to Figures 5 and 6, with uncomplexed KLH again used as a negative control. Treatment of PMN with 10 &nl of either rabbit F(ab'), anti-CR1 Ab, MN-41 anti-CR3 mAb, or a combination of these Abs for 30 min at 4°C inhibited the subsequent binding of IgM/lgC-KLH-iC3b IC by 57, 62, and 82%, respectively,compared with the untreated control PMN. As expected, the unactivated PMN did not bind C3dg-bearing IC. The results obtained in one characteristic experiment are shown.

dependent and that there were insufficient amounts of IgG on the IC to generate FcRII-dependent processing. On the other hand, the IC bearing bound iC3b or bound C3dg that bound to CR1 and CR2 or only CR2, respectively, were processed by B cell clones with or without KLH-specific SIg. The efficiency of processing was greater with KLHspecific B cells than with nonspecific B cells, and bound iC3b was more efficient than was bound C3dg, probably because bound iC3b mediated binding of IC via both CR1 and CR2. Presentation of KLH after B cell complement receptor-dependent Ag processing

PBMC cultures that were greatly enriched for Th cells specific for KLH were generated from the same two donors from whom the B cell clones had been generated to assure TCR/MHC class I1 compatibility inAg presentation. Tests for stimulation of these T cell cultures showed a requirement for both KLH-and MHC-compatible monocyte (data not shown) or B cell APC (Fig. 9). IgM/IgGKLH IC lacking bound C3 were only presented efficiently


. ."

IgMligG-KLH-C3d

1733

journal of Immunology

~~

-

-p 2

None

-

B cell wecificitv andI KLH-specific KLH-specific tE&cific v) 12yooo IgKLH-iC3b IgKLH-C3dg Ig-KLH Q) 10,000 + 0 Non-specific Non-specific Non-specific Q ) m 8yooo - Ig-KLH-iC3b Ig-KLH-C3dg Ig-KLH -

O

0 1.5 IC v)

6,000

=n

[7 Ig-KLH-C3dg

L

-

w Q

1

n

a 5 4,000 U Q I-

Ig-KLH

E-

Ig-KLH-iC3b

0 0.5

2,000

n -

1

3

2

-

n 4

KLH-specific B cells

Non-specific B cells

by the KLH-specific B cell clones, whereas both the iC3band C3dg-IC were able to stimulate a KLH-specific T cell proliferative response when presented by either KLH-specific or nonspecific B cells. Complement receptor- and FcRII-dependent stimulation of B cell CDBO expression

B cellAg presentation requiresinduction of the B cell membrane co-stimulatory molecule CD80,which provides a second signal for T cell activation by binding to CD28 or CTLA-4 (46-48). Effective presentation with B lymphoblastoid cells (Fig. 9) was expected because transformed B cells express CD80 constitutively (49). It was important to show that Ig-KLH-C3 IC could induce normal B lymphocytes to express CD80, because the absenceof CD80 onB cells presenting Ag to T cells is thought to result in T cell anergy (50, 51). PBMC were incubated with KLH IC for 3 h and monitored for induction of B cell expression of CD80 by two-color flow cytometry (Fig. 10). IC bearing iC3b or C3dg induced CD80 expression on SIgM+ B lymphocytes within 1 h. In addition, a somewhat lower level of CD80 expression was induced with the KLH IC prepared with heat-inactivated serum (IgM/IgG-KLH), suggesting that CD80 expression might result from the IgG on these ICs binding to the B cell Fc receptor FcRII (CD32). This was confirmed by demonstrating that ICs prepared with rabbit IgG anti-KLH(RabIgG-KLH)and bearing much larger amounts of IgG than the normal human serum-derived IC, was very effective in inducing CD80 ex-

FIGURE 9. B cell presentation of KLH to KLH-specificT cells. Mitomycin C-treated B cell clones were used that expressed eitherKLH-specific SlgM or nonspecificSlgM in combination with Th cell cultures enriched for KLH specificity and expressing TCR that were compatible with the B cell MHC class I I molecules. T cell proliferation that was indicative of presentation was measured with an MTT assay after 3 days of cultivation. The MTT dye is cleaved by active mitochondria producing a product that absorbs light at 51 0 nm, thus allowing measurement of the OD,,, to be used as an indication of growing cells. The amount of proliferation observed with no added KLH Ag (None) or unopsonized pure K L H (data not shown) were approximately equivalent. The results given represent the mean values of three experiments, and the error bars represent ? 1 SD.

pression in the absence of bound C3. On the other hand, rabbit IgGanti-CR2failed to induce CD80 expression (Fig. IO), despite theparallel finding that this particular Ab preparation was capable of stimulating Raji cell CR2-mediated proliferation in serum-free media (not shown). The induction of B cell CD80 expression by both the iC3b- and C3dg-bearing IC was blocked by either F(ab'), anti-C3 or F(ab) anti-FcRII,showingarequirement for both bound C3 and IgG on the IC and C3 receptors and FcRII on the B cells (Fig. 11). However, the induction of CD80 by the rabbit IgG-bearing IC, in combination with the failure of rabbit anti-CR2 to induce CD80, suggests that FcRII is solely responsible for inductionof CD80, and that bound C3 and C3 receptors function by promoting the uptake of IC that bear insufficient amounts of IgG natural Ab to be bound independently by FcRII.Thus, bound C3dg and CR2 play an important synergistic role in the induction of CD80 by IgG and FcRII.

Discussion This investigation tested the hypothesis that the immune response to a primary protein Ag involves the fixation of C3 onto the Ag via natural Ab and the formation of IC capable of activating the classical pathway of complement. Such bound C3 was shown to promote Ag uptake by B


HOURS FIGURE 8. Processing of KLH IC by cloned B lymphoblastoid cells. EBV-transformed B cell clones were used that expressed either KLH-specific SlgM or SlgM that was nonspecific for KLH. ICs labeled with l Z 5 l (2.5 pCi/pg and 50 pg/ml) were incubated with 5 X 10' cloned B cells at 37"C, and the supernatants were analyzed at hourly intervals for the conversion of TCA-precipitable protein into TCA-solublepeptides. The results given represent the mean values obtained from three experiments. Significant processing was observed at 3 and 4 h, and error bars shown at these time point represent '.I SD.

1734


Rab IgG-KLH

IgMllgG-KLH-iC3b

IgM/lgG-KLH-C3dg

IgM/lgG-KLH

-

+

L

I

-4-

Rab anti-CRP

+

= I " I m

1-

c E 9 201

3

2

1

Hours FIGURE 10. Inductionofnormal

B cell expression of CD80 Ag byKLH IC. Two-color flow cytometry analysis of IC induction of the CD80 Ag onfreshly isolated PBMC. PBMCs were stained with anti-IgM-FITC. The IgM' cells were gated and analyzed for the induction of CD80Ag by staining with biotinylatedanti-CD80Aband strepavidin-phycoerythrin. Biotinylated murine myeloma IgG and strepavidinphycoerythrin wasused as a negative control. The results from a typical experiment are expressed as the percentage of SlgM' cells that are CD80'.


- 0

natural Ab promoted the fixation of C3 onto IC with normal serum. KLH IC bearing C3dg bound to B cells via CR2, regardless of the antigenic specificity of the B cells, and this promoted the processing of the KLH into small peptides that were presented successfully to KLH-specific Th cells. The resultsalsosuggestedthat the secondary ligation of B cell FcRIIby IgG natural Abs containedin IC may be requiredtostimulate B cellexpression of the CD80 co-stimulatorymolecule that must bind to T cell CD28 or CTLA-4 to prevent T cell anergy. This mechanism of Ag uptake by B cells may improve the immune response at low Ag dosages by allowing processing and presentation to be conducted by any CR2-bearing B cell, regardless of its antigenic specificity. As with Ag nonspecific macrophages and dendritic cells, thespecificity of the immune response would be retained if nonspecific B cells only presented Ag to specific Th cells, and if differentiation of the presenting B cells into Ig-secreting cells only occurred when specific SIg was present on the B cells. To generate bound C3 on a primary protein Ag, natural Ab reactive with that Ag must be present to form IC and activate the classical pathway of complement, because soluble proteins do not usually activate the alternative pathway of complement. Both IgM and IgG Abs tothe primary protein Ag KLH were detected in the sera of all 11 donors tested. These Abs formed IC and generated bound C3 on the KLH that promoted Ag attachment to C3 receptors. Some IgM anti-KLH was monomeric and thus may represent shed B cell Slg. This could be confirmed in future investigations by COOH-tern1inal sequence analysis of pchains that woulddistinguish the membrane form of IgM. If this were the case, then the repertoire of serum IgM natural Abs would be expected to be the same as that of allavailable B cells,andthus sufficient to havethis mechanism be operative for any Ag. Evidence that natural Abs have such a wide range of specificities has been reported (14). Alternatively, some primary Ags may be recognized by the polyreactiveAbsproduced byCD5' B cells that individuallyhave a very broad range of reactivity with several Ags (14, 52). In either case, Abs would be available in low concentrations that were reactive with any possible Ag and thus able to mediate IC formation, activation of the classical pathway, and Ag uptake via CR2. Generation of IC with an Ag that was disulfide bonded to a gel matrix allowed IC formation with whole serum, becauseuncomplexedserumproteinscould be washed away from the IC before their elution by mild reduction with cysteine. Analysis of the soluble IC both by ELISA and by their ability to bind to specific receptors showed the presence of IgM, IgG, and iC3b or C3dg. It is likely that IC attachment to CR1 was due to iC3b rather than to C3b, because bound C3b has a half-life in serum of -90 s on surfaces that do not activate the alternative pathway of C. Such IC bearing bound iC3b might be ingested by phagocytic cells via CR1 or CR3 and CR4, but if such IC were to remain in the blood and escape phagocytosis, then the

pBM Rab IgG-KLH

+ ATlO Fab

anti-FcRII

+ ATlO Fab anti-FcRii + Rab F(ab'hanti-CB IgM/lgG-KLH-C3dg

+ AT10 Fab

anti-FcRiI

+ Rab F(abh anti-C3 IgM/lgG-KLH

+ AT10 Fab

=

antl-FcRII I

0

10

I

I

20

I

I

30

I

I

40

Percent SlgM' CD80'

FIGURE 11. Two-color flow cytornetry analysis of the inhibition of IC induction of the CD8O Ag on freshly isolated PBMC. B cells were evaluated for dual expression of SlgM and CD80, as described in Figure 10. Pretreatment of PBMC with 20 p d m l of either AT1 0 F(ab) anti-FcRII or rabbit F(ab'), antLC3 for 30 min at 4°C followed by incubation with different IC for 60 rnin at37°C inhibited B cellinduction of CD80 compared with the untreated control for each IC. The of three characteristic experiments are results fromone shown.

cells via CR2, followed by Ag processing and presentation of Ag peptides to MHC-compatible, Ag-specific Th cells. Both IgM and IgG natural Abs to the primary protein Ag KLH were detected in all normal sera examined, and this

Journalof Immunology

linked to the tetanus toxoid Ag rather than being bound physiologically via natural Abs and the classical pathway. Such chemically linked C3b is frequently resistent to conversion into bound iC3b and C3dg and this may explain why this study found a greater role for CRl than CR2, despite a higher density of CR2 than CRl on the B cell line APC. Nevertheless, the findings were similar to those of this investigation. Although Ag presentation was demonstrated with EBVtransformed B lymphoblastoid cell clones, there is some question as to whether successful presentation would also occur with normal B lymphocytes. With normal B cells, Ag presentation requires the activation of several co-stimulatory molecules that bind to specific ligands on Th cells (48). One of the co-stimulatorymoleculesthat must be induced on B cells is CD80, which is a ligand for either CD28 or CTLA-4 on Th cells (46, 47, 60, 61). If Ag processing does not stimulate the expression of CD80 on B cells, then it is thought that Ag presentation may result in T cell anergy rather than an immune response (50, 5 1, 62). Although Ag presentation with T cell activation did occur with nonspecific Bcells,the EBV-transformed B cells used for presentation are known to express CD80 constitutively (49, 63). It was thus important that KLH IC induced the expression of CD8O on peripheral blood B lymphocytes. Induction of CD80 required bound C3 and C3 receptors, as well as IgG natural Ab and FcRII. However, C3 receptors mediated only IC attachment, and the secondary ligation of FcRII by IgG in IC was required to stimulate the expression of CD80. A polyclonal anti-CR2 that was able to stimulateB cell proliferation failed to stimulate the expression of CD80, whereas IC prepared with purified rabbit IgG anti-KLH did stimulate CD80 expression. Moreover, F(ab) anti-FcRII blocked the expression of CD80 stimulated by either C3-bearing IC or the rabbit IgG-bearing IC. Although these studies suggest a role for B cell FcRII in Ag presentation by B cells, it is also known that large amounts of IgG Ab directed to an immunizing Ag can suppress the immune response (64). Perhaps there are threshold levels of FcRII stimulation that induce CD80 without suppressingBcell activation. By comparison, there are thought to be levels of CD19 ligation that either stimulate or suppress B cell activation (65). These findings support the hypothesis that the immune response to a primary protein Ag may involve IgM and IgG natural Ab, the formation of IC that promote activation of the C classical pathway, fixation of C3dg onto the Ag, and processing of theAg by CR2-bearingBcells. Nonspecific B cells that process the Ag via CR2 present the Ag to specific Th cells in much the same way as Ag nonspecific macrophages and dendritic cells take up and present Ag. A major question for future investigation is whether nonspecific B cellsthat present Ag are stimulated to proliferate and secrete nonspecific Ig or whether nonspecific B cell processing of an Ag can in some way enhance the uptake of that Ag by specific B cells.


iC3b would be converted into C3dg after interaction with erythrocyte CRl and factor I (37). C3dg mediates IC binding primarily to CR2 because phagocytic cell CR3 does not bind C3dg unless it is activated by agents such as PMA or P-glucan (53). CR2 are expressed in highest density on follicular dendritic cells (54) and B cells, and much less CR2 is expressed by T cells (22,45). The ICgenerated with normal serum bound to the higher densitiesof CR2 expressed by normal B cells and theB cell line Raji but apparently did not contain enough bound iC3b or C3dg to bind to the lower densities of CR2 expressed by either T cellsor the B cell line Daudi. IC formed with heat-inactivated serum bore insufficient amounts of IgG natural Ab to mediate IC attachment to FcR. However, the amount of IgG within IC was sufficient to stimulate B cell FcRII when IC attachment was mediated via bound C3 and C3 receptors. This mechanism by which large amounts of bound C3 assist in B cell recognition of small amounts of IgG is reminiscent of neutrophil and monocyte phagocytosis in which small amounts of opsonizing IgG are recognized as a consequence of particle attachment to the phagocyte surface via C3 receptors (55, 56). After the ligation of IC to CRland/or CR2, theKLH Ag was processed into TCA-soluble peptides and presented to MHC-compatible, KLH-specific Th cells. Processing and presentation occurred regardlessof whether the B lymphoblastoid cells expressed KLH-specific SIg. In anormal immune response this would mean that CR2 could function in Ag trapping and presentation with all B cells and not just that small subsetof B cells bearing SIg specific for the primary Ag. This might be important with very small Ag dosages, and indeed, a role for C in the immune response has been more readily demonstrated with small Ag dosages (3, 5 , 57). Nevertheless, both processing and presentation were more efficient with B cells bearing KLH-specific SIg, and thus,these findings do not excludethe possibility that CR2 could also function in lowering the threshold of Ag dose required to stimulate Ag-specific B cells through its membrane interaction with CD19 (58). CR 1 may function cooperatively with CR2 and FcRII in Agprocessing and presentation when bound iC3b is present on IC. Although C3dg-bearing IC did not bind to CR1 or CR3, the current experiments did not allow distinction of whether CR2 and/or FcRII was responsible for Ag endocytosis and processing. It has been suggested that the membrane complexes of CR1 and CR2 present on B cells may facilitate the uptake of IC bearing C3b or iC3b (59), and that the factor I cofactor activity of CR1 in these complexes may be used along with B cell-derived factor I (30) to convertIC-associatedC3b/iC3bintoC3dg that would stimulate B cells via CR2/CD19 membrane complexes (58). Arvieux et al. (23) have also reported C3-mediated Ag uptake and presentation via B cell C3 receptors. However, a tetanus toxoid secondary Ag was used rather than a primary Ag such as KLH, and C3b or C4b were chemically

1735

1736

Acknowledgments We are grateful for the generous donation of mAbs from Drs. Allison Eddy, Alfred Michael, Gideon Goldstein, Peter Lachmann, Edward Clark. and Martin Glennie. We thank Dr. Mike Holers for the donation of rabbit anti-CR2 serum, Dr. Alfred Rudolph and TCell Sciences for the donation of K R l , and Dr. Karen Cost for the donation of serum from a patient with congenital agammaglobulinemia. We also thank Dr. Rafael Fernandez-Botran of the University of Louisville for help in generating KLH specificity-enriched Th cell cultures. Finally, we acknowledge the excellent technical assistance of Ms. Lynda O’Rear and Ms. Jana VEtviEkovb.

References production in vivo: effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J. Exp. Med. 140:126. 2. Klaus, G. G. B.,and J. H. Humphrey. 1977. The generation of memory cells. I. The role of C3 in the generation of B memory cells. Immunology 33:SI. 3. Bottger, E. C., S. Metzger, D. Bitter-Suermann, G. Stevenson, S. Kleindienst, and R. Burger. 1986. Impaired humoral immune response in complement C3-deficient guinea pigs: absence of secondary antibody response. Eur. J. Immunol. 16.1231. 4. O’Neil, K. M., H. D. Ochs, S. R. Heller, L. C. Cork, J. M. Moms, and J. A. Winkelstein. 1988. Role of C3 in humoral immunity: defective antibody production in C3-deficient dogs. J. Immunol. 140: 1939. 5 . Ochs, H. D., R. J . Wedgwood, S. R. Heller, and P. G. Beatty. 1986. Complement, membrane glycoproteins, and complement receptors: their role in regulation of the immune response. Clin. Immunol. Immunopathol. 40:94. 6. Wedgwood, R. J. and H. D. Ochs. 1985. Assessment of the humoral immune response in immunodeficiencies. In Recent Advances in Primary Immunodeficiency Diseases, Serono Symposia, Vol. 28. F. Aiuti, F. Rosen, and M. D. Cooper, eds. Raven Press, New York, p. 227. 7. Ochs, H. D., S. Nonoyama, Q. Zhu, M. Famngton, and R. J. Wedgwood. 1993. Regulation of antibody responses: the role of complement and adhesion molecules. Clin. Immunol. Immunopathol. 67: s33. 8. Heyman, B.,L. Pilstrom, and M. J . Shulman. 1988. Complement activation is required for IgM-mediated enhancement of the antibody response. J. Exp. Med. 167:1999. 9. Ross,G. D. 1989. Complement and complement receptors. Curr. Opin. Immunol. 2:50. 10. Heyman, B., E. J . Wiersma, and T. Kinoshita. 1990. In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody. J. Exp. Med. 172:665. 11. Thyphronitis, G., T. Kinoshita, K. Inoue, J.E. Schweinle, G. C. Tsokos, E. S. Metcalf, F. D. Finkelman, and J. E. Balow. 1991. Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo. J. Immunol. 147:224. 12. Wiersma, E. J., T. Kinoshita, and B. Heyman. 1991. Inhibition of immunological memory and T-independent humoral responses by monoclonal antibodies specific for murine complement receptors. Eur. J, Immunol. 21:2501. 13. Hebell, T., J. M. Ahearn, and D. T. Fearon. 1991. Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254:102. 14. Casali, P., and A.L. Notkins. 1989. CD5’ B lymphocytes, polyreactive antibodies and the human B-cell repertoire. Immunol. Today 10.364. 15. Chesnut, R. W., S. M. Colon, and H. M. Grey. 1982. Requirements for the processing of antigens by antigen-presenting B cells.I. Functional comparison of B cell tumors and macrophages. J. Immunol. 129t2382.

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