Resting Peripheral Blood B Cells Presentation of Immune Complexes

Complement Opsonization Is Required for Presentation of Immune Complexes by Resting Peripheral Blood B Cells

This information is current as Susan A. Boackle, Margaret A. Morris, V. Michael Holers of September 25, 2021. and David R. Karp J Immunol 1998; 161:6537-6543; ; http://www.jimmunol.org/content/161/12/6537 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Complement Opsonization Is Required for Presentation of Immune Complexes by Resting Peripheral Blood B Cells1

Susan A. Boackle,* Margaret A. Morris,† V. Michael Holers,* and David R. Karp2†

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required Downloaded from exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events. The Journal of Immunology, 1998, 161: 6537–6543.

he development of a mature immune response requires complexes in the course of complement activation. Studies per-

the collaboration between B and T lymphocytes. Ag is formed in vivo have confirmed the critical role of CD21 in the http://www.jimmunol.org/ T internalized by B cells and processed into peptides that generation of a normal humoral immune response. Normal murine are presented to T cells in the context of specific MHC class II Ab responses to T-dependent Ags are inhibited by pretreatment molecules. Ag-specific T cells recognize these complexes via with mAbs to CD21 or soluble CD21 (4–6). Mice rendered CD21- TCR/CD3 with signals through these receptors resulting in the deficient using gene-targeting techniques demonstrate similar al- up-regulation of CD40 ligand on the T cell surface. Cross-linking terations (7, 8). B cell CD21 was found to be critical for these of B cell CD40 by CD40 ligand induces the expression of the effects based on studies in bone marrow chimeras, in which the costimulatory molecules CD80 and CD86 (1, 2), which bind CD28 immune defects were corrected by reconstitution with bone mar- on T cells. The T cells then secrete IL-2, which enhances B cell row from CR2ϩ/ϩ MHC-matched littermates. The role of B cell maturation and proliferation. An individual B cell is able to initiate CD21 was further confirmed in mice created by RAG-2-deficient by guest on September 25, 2021 these responses only if it presents adequate numbers of class II- blastocyst complementation (9). These animals specifically lack peptide complexes to specific T cells. Although nonspecific fluid- CD21 on their B cells, but have normal expression on their fol- phase endocytosis of Ag occurs poorly in resting B cells, Ag can licular dendritic cells (FDC). These studies demonstrated that the be internalized efficiently by binding to specific Ag receptors (3). targeting of complement-coated immune complexes to FDC is not Nonetheless, B cells are not believed to be critical for Ag presen- sufficient for the development of a normal humoral response in the tation because of the low frequency of Ag-specific B cells for any absence of complement receptors on B lymphocytes. Additional particular Ag. studies have supported an adjuvant-like role of complement in B Ag can be targeted to other B cell surface receptors, including cell responses. C3d linked to hen egg lysozyme (HEL)3 by recom- complement receptors such as CD21. CD21 is a 140-kDa glyco- binant DNA techniques was 1,000–10,000 times more immuno- protein located on the surface of mature B lymphocytes, as well as genic than HEL alone, inducing enhanced Ca2ϩ mobilization in on follicular dendritic cells, pharyngeal epithelial cells, thymo- vitro as well as augmenting primary and secondary Ab responses cytes, and some T cells. It binds the terminal products of C3 deg- in vivo (10). radation that have become covalently bound to Ag or immune CD21 may participate in the development of a normal immune response by several mechanisms. First, ligation of CD21 may result in various signals that are critical for normal B cell responses. *Departments of Medicine and Immunology, Division of Rheumatology, University Previous studies have shown that B cell proliferation and differ- of Colorado Health Sciences Center, Denver, CO 80262; and †Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas, TX entiation can be induced by cross-linking CD21 with polymeric 75235 C3d or certain anti-CD21 mAbs in the presence of T cell factors Received for publication June 23, 1998. Accepted for publication August 20, 1998. (11, 12) or phorbol esters (13). CD21 also plays a synergistic role The costs of publication of this article were defrayed in part by the payment of page in the activation of B cells induced by ligation of surface IgM, as charges. This article must therefore be hereby marked advertisement in accordance measured by calcium mobilization (14), proliferation (15), or in- with 18 U.S.C. Section 1734 solely to indicate this fact. duction of c-fos mRNA levels (16). Many of the signaling effects 1 This work was supported by National Institute of Health Grant U01-AI34621 induced by CD21 have been attributed to its physical association (D.R.K.), National Institute of Health Training Grant T32-AR07055 (S.A.B.), and National Institute of Health Grant R01-AI31105 (V.M.H.). Portions of this work have with CD19 and CD81 (TAPA-1) on the surface of B lymphocytes been previously been presented in abstract form at the Annual Meeting of the Amer- ican College of Rheumatology, Orlando, FL, October 1996. 2 Address correspondence and reprint requests to Dr. David R. Karp, University of 3 Abbreviations used in this paper: HEL, hen egg lysozyme; KLH, keyhole limpet Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235- hemocyanin; PE, phycoerythrin; CR, complement receptor; TAPA-1, target of anti- 8884. E-mail address: [email protected] proliferative antibodies-1.

(17–19). Signaling through CD19 has been reported to augment ological Reagent and Reference Repository (anti-H3; Braton Biotech, that of soluble IgM, with coligation of the two receptors lowering Rockville, MD). The murine mAbs to CD21 (HB5), CD3 (OKT3), CD14 the threshold for B cell activation by two orders of magnitude (20). (63D3), CD16 (B73.1), and CD11b (LM2/1.6.11) were obtained from the American Type Culture Collection (Manassas, VA). Phycoerythrin (PE)- Alternatively, CD21 may participate in the generation of a nor- labeled anti-CD19 (B43) was obtained from PharMingen (San Diego, CA). mal immune response by internalizing and directing C3-bound Ag Biotinylated anti-CD19 (B4) was obtained from Coulter (Miami, FL). into the class II processing pathway of B cells. This was first sug- FITC-labeled anti-CD69 was obtained from Becton Dickinson (Sunnyvale, gested by Lanzavecchia et al. (21) who demonstrated an aug- CA). FITC-labeled CD86 was obtained from The Binding Site (Birming- ham, U.K.). Rabbit polyclonal Ab to CD21, Ab73, was generated by im- mented anti-Ig-specific T cell response to transformed B cells munization with a soluble human CD21 produced by recombinant bacu- pulsed with anti-CD21 mAbs. Other investigators have utilized lovirus in insect cell culture. It recognizes short consensus repeats 1–2 of C3-linked ligands for CD21 to demonstrate this same phenome- CD21 including the C3d binding site (V.M.H., unpublished data). The non. In one report, tetanus toxoid was covalently cross-linked to mouse mAb to CD32, AT10 (26), was the kind gift of Dr. P. M. Morganelli C3b and C4b (22). In a second, immune complexes were generated (Veterans Administration Hospital, White River Junction, VT). The mouse mAb to CD35, 3D9, was the kind gift of Dr. Eric Brown (Washington by incubating normal human serum with keyhole limpet hemocy- University, St. Louis, MO) (27). anin (KLH) linked to a solid phase support (23). In both of these studies, transformed B cells pulsed with complement-containing Purification of peripheral blood B lymphocytes immune complexes elicited a greater response from Ag-specific T B cells were purified from peripheral blood by either negative or positive cells as compared with B cells pulsed with Ag or immune com- selection. For negative selection, PBMCs were depleted of monocytes and plexes lacking complement. We have recently extended these find- NK cells by treatment with 0.09 mg/ml leucine methyl ester for 45 min at

room temperature, and subsequently depleted of T cells by rosetting with Downloaded from ings by demonstrating that CD21 on transformed B cells mediates SRBCs. The remaining population was treated with 10 ␮g/ml OKT3, the presentation of Ag derived from complement-influenza-anti- 63D3, B73.1, and LM2/1.6.11, followed by labeling with goat anti-mouse influenza IgG immune complexes formed in vitro in a physiolog- magnetic beads (MACS; Miltenyi Biotec, Sunnyvale, CA), and selection ically relevant manner (24). The ability of CD21 to bind comple- for negative cells from a high-gradient magnetic separation column ment-coated Ags should enable a much larger number of B cells to (MACS; Miltenyi Biotec). For positive selection, PBMCs were stained with biotinylated mAb to CD19 (B4) and selected over a Cellprate LC participate in Ag presentation, since CD21 is expressed on all ma- avidin column (Cellpro, Bothell, WA). Cells were analyzed after purifica- ture B cells regardless of their specificity. tion by staining with PE-labeled anti-CD19 (B43; PharMingen, San Diego, http://www.jimmunol.org/ Fc receptors have also been demonstrated to play a role in aug- CA) and were determined to be 90–98% CD19-positive. The resting state menting B cell Ag presentation (25). They would likewise be ex- of these cells was confirmed by their lack of expression of CD69 and CD86. pected to extend the ability of the nonspecific B cell to participate in Ag presentation. However, previous data from this laboratory Preparation of immune complexes suggest that binding of immune complexes to B cells is dependent Influenza virus HK X-31 (recombinant between A/Aichi/2/68 and A/PR/ on the presence of complement in the complexes (24). The lack of 8/34) was grown in embryonated chicken eggs and live virus purified over immune complex binding in the absence of complement suggests a sucrose gradient. Virus was inactivated by UV irradiation. Immune com- that Fc receptor-mediated Ag presentation plays a less critical role plexes were generated by incubation of inactivated virus with RPMI 1640 in Ag presentation by nonspecific B cells. and 5% normal human serum from a single donor which contains high by guest on September 25, 2021 titers of influenza-specific IgG (24). Complexes were allowed to form at All of the studies to date have analyzed the function of CD21 in 37°C for 1 h. Serum samples that were inactivated by heat (56°C for 1 h) augmenting Ag presentation using transformed B cell lines, which or by incubation with methylamine (Sigma, St. Louis, MO) (100 mM, 37°C constitutively express costimulatory molecules thought to be im- for 1 h followed by exhaustive dialysis against PBS) were used to generate portant in Ag presentation. Resting B cells do not constitutively immune complexes lacking complement. In some experiments, synthetic immune complexes were generated by incubating biotinylated human IgG1 express these molecules. It is not known whether they function as (Sigma) with FITC-streptavidin (Life Technologies, Gaithersburg, MD) for APCs after binding complement-coated Ag. We wished to address 30 min at 4°C. Either normal human serum or methylamine-inactivated the question of whether targeting Ag to CD21 on resting B cells human serum was added to a final concentration of 5% and complement would result in the generation of sufficient peptide-MHC com- activation allowed to occur at 37°C for 1 h. plexes to elicit a T cell response. In addition, we wished to deter- Microcytofluorometry assays to analyze immune complex mine whether ligation of CD21 by complement-immune com- binding plexes would result in B cell activation and in the up-regulation of costimulatory molecules important in the generation of a normal T Immune complexes were bound to cells for 30 min at 37°C then washed twice with PBS. They were then analyzed by microcytofluorometry on a cell response. In the present study, we examined these questions FACScan (Becton Dickinson, Sunnyvale, CA). For blocking experiments, using resting B cells purified from the peripheral blood of human blocking Abs were incubated with cells for 30 min at 4°C before the ad- donors. We found that these cells are fully capable of presenting dition of immune complexes. Immune complex binding was detected using Ag derived from complement-influenza immune complexes to a goat anti-H3 serum followed by FITC-sheep anti-goat IgG in the case of the influenza-containing complexes, and directly for the IgG/FITC-strepta- panel of influenza-specific T cell clones. This process is comple- vidin complexes. In experiments involving PBMC populations, B cells ment-dependent and likely occurs via CD21. However, CD21 li- were labeled with PE-labeled CD19, and histograms were generated by gation by complement-immune complexes does not result in B cell gating on the CD19-positive cells. activation and the induction of well-characterized costimulatory Generation of T cell clones molecules in the absence of T cell help. These data support the hypothesis that CD21 participates in the generation of a normal A panel of influenza-specific human T cell clones was generated from a immune response by its ability to direct Ag into the class II Ag donor who possesses the MHC class II haplotype: DRB1*0101/ DRB1*0401, DQB1*0501/DQB1*0301 (24). T cells were characterized processing pathway, in a process that occurs independently of B for MHC class II restriction and Ag specificity by testing their IL-2 re- cell activation. sponse to influenza virus or peptide presented by B cell lines and PBMC of known haplotypes (S.A.B., unpublished data). MHC class II restrictions Materials and Methods were confirmed by blocking T cell responses with mAbs to class II mol- Antibodies ecules. Characterization of TCR was performed as described (28). CL19 is restricted by HLA-DR1 and -DR4/Dw4, CL23 and CL58 by HLA-DR1, Polyclonal goat Abs to influenza hemagglutinin (A/Aichi/2/68) were ob- CL35 by HLA-DQ1, and CL62 by HLA-DRw53. CL19 and CL58 both tained from the National Institute of Allergy and Infectious Diseases Bi- recognize the immunodominant peptide of hemagglutinin, HA307-319. The Journal of Immunology 6539

CL62 recognizes a peptide contained within the influenza nucleoprotein. Identification of the peptide Ags for the other clones has not been completed. Ag presentation assays Highly purified peripheral blood B cells or B cell lines were incubated with complement-influenza immune complexes for4hat37°C, washed to remove free immune complexes, then plated with T cells in a 96-well round bottom plate at stimulator:responder ratios from 2:1 to 5:1. T cell clones were washed twice to remove excess IL-2 before plating. In some experiments, peripheral blood B cells were incubated for an additional 20 h before addition of T cells. As shown below, this additional incubation period was found to be critical for the presentation of immune complexes by resting B cells. To test whether Ag processing occurred before contact with T cells, the resting peripheral blood B cells were first incubated with immune complexes for 4 h, washed, then incubated for 20 h at 37°C. The cells were then fixed with 0.75% paraformaldehyde in serum-free medium for 5 min at 37° and then for 1–2 h at 4°C. Ten percent FCS in PBS was added to quench the fixative, and the cells were washed and plated with T cells. In all cases, T cell supernatants were harvested after 24 h at 37° and tested for IL-2 by CTLL-2 bioassay as described (29). Downloaded from Microcytofluorometry assay to analyze B cell activation and up-regulation of costimulatory molecules

Peripheral blood B lymphocytes were incubated with immune complexes FIGURE 1. Binding of immune complexes to peripheral blood B cells for 4 h, washed, and plated in 96-well round bottom plates at 20,000 cells/ occurs in a complement-dependent manner. Influenza-containing immune well in RPMI 1640 containing 10% heat inactivated human serum (HIHS). complexes formed in either normal human serum (thick line) or heat-in- After 20 h, T cell clones were added at 20,000 cells/well. Cells were har- activated human serum (thin line) were incubated with highly purified pe- vested after 24 h, stained with PE-labeled anti-CD19 and FITC-labeled http://www.jimmunol.org/ anti-CD69 or CD86, and analyzed by dual color microcytofluorometry. ripheral blood B cells. Binding of immune complexes was detected by Histograms were generated by gating on the CD19-positive cells. staining with anti-H3 Ab, which is specific for influenza hemagglutinin. Peripheral blood B cells bound high levels of complement-containing im- Results mune complexes and did not bind immune complexes that lacked complement. The background fluorescence of cells not incubated with immune Complement-influenza immune complexes bind to peripheral complexes is indicated by the dotted line. blood B cells in a complement- and CR-dependent manner We have previously demonstrated that immune complexes formed under physiological conditions bind to CD21-transfected fibro- by guest on September 25, 2021 blasts and to transformed B cells in a complement- and CD21- plement receptors was seen, whereas no binding to the B cell Fc dependent fashion (24). The following experiments were per- receptor could be demonstrated in mixtures prepared in serum in formed to confirm that binding of these complexes to peripheral which complement was inactivated. Again, identical data were ob- blood B cells occurs in a similar manner. Immune complexes were tained regardless of the type of immune complex used. formed from either acquired anti-influenza IgG and purified virus, or biotinylated IgG and streptavidin. The complexes were exposed to normal or heat- or methylamine-treated serum then were incubated with PBMCs, and binding to CD19-positive cells was analyzed by microcytofluorometry. Binding was complement-dependent, as it did not occur with immune complexes formed in serum in which complement had been inactivated (Fig. 1). Identical results were obtained whether the complexes were formed between influenza virus and acquired Abs, or between biotinylated IgG and streptavidin (data not shown). Additional experiments were performed to identify the specific receptors involved in the binding of immune complexes to peripheral blood B cells. These cells express moderately high levels of the B cell C3 receptors, CD21 and CD35, in addition to the B cell Fc␥RII, CD32, and little, if any, complement receptor 3 (CD11b/ CD18; CR3) (data not shown). Blocking the binding domains of either CD35 (CR1) or CD21 alone partially inhibited binding, whereas blocking both CD21 and CD35 simultaneously com- pletely abrogated immune complex binding (Fig. 2). There was no effect on immune complex binding when the cells were pre-incu- FIGURE 2. Immune complexes bind to CD21 and CD35 on peripheral bated with a blocking mAb to CR3 or CD32, nor could anti-CD32 blood B cells. Peripheral blood B cells were treated with medium alone (A) or with blocking Abs to CR1 (3D9) (B), CR2 (Ab73) (C), CR3 (LM2/ enhance the inhibition of complex binding seen with anti-CD21 1.6.11) (D), CD32 (AT10) (E), or CR1 plus CR2 (F) before incubation alone (data not shown). These data suggest that fluid-phase acti- with complement-containing immune complexes (thick line). Fluorescence vation of complement by soluble immune complexes generates of cells treated with isotype-matched species-specific control Abs before ligands for both CD35 (C3b and C4b) and CD21 (presumably the addition of immune complexes is indicated by the thin line. B cells iC3b). One particle may have either or both types of ligand. Bind- were labeled with PE-labeled anti-CD19 and histograms were generated by ing of complement-containing immune complexes to both com- gating on CD19-positive cells. 6540 B CELL ANTIGEN PRESENTATION REQUIRES COMPLEMENT OPSONIZATION

FIGURE 4. Immune complex-pulsed B cells process Ag before contact FIGURE 3. T cell clones respond to complement-containing immune with T cells. Resting peripheral blood B cells were incubated with medium ␮ complexes presented by peripheral blood B cells after prolonged incuba- alone, 1 M HA307-319 peptide, or immune complexes prepared in the tion. Resting B cells purified from peripheral blood were pulsed with im- presence of complement for 4 h, washed, incubated an additional 20 h, then mune complexes containing normal human serum (filled columns), meth- fixed. Cells were plated with the HA307-319-specific T cell clone, CL58, ylamine-treated human serum (gray columns), or medium alone (open as well as the nucleoprotein-specific T cell clone, CL62. The responses of columns) for 4 h, washed, then incubated in medium for 20 h before in- CL58 to live (open bar) and fixed B cells (filled bar) and the responses of Downloaded from cubation with T cells at a stimulator:responder ratio of 1:2. Supernatants CL62 to live (shaded bar) and fixed B cells (hatched bar) were measured were analyzed for IL-2 content. Background incorporation of thymidine by by analyzing IL-2 content in 24-h supernatants. CTLL-2 in this experiment was ϳ5000 cpm. Data are representative of three separate experiments. than direct processing and presentation by B cells. The T cell

clones were shown to be negative for CR1, -2, and -3, and thus http://www.jimmunol.org/ Peripheral blood B cells present Ag from complement- would not be expected to bind intact immune complexes (data not containing immune complexes in a CЈ-dependent manner shown). Furthermore, B cells from HLA-unmatched donors that We and others have shown that CD21 can participate in the pre- were pulsed with complement-containing immune complexes were sentation of Ag derived from complement-containing immune unable to transfer Ag to the T cell clones (data not shown). These complexes by transformed B cells. These cells constitutively ex- data suggest that resting B cells are directly involved in the pre- press the costimulatory molecules CD80 and CD86 and are similar sentation of Ag that has bound to the cell surface via CD21. to cells that have been triggered by B cell receptor cross-linking Peripheral blood B cells are not activated by ligation of CD21 (30). We wished to determine whether resting peripheral blood B cells have a similar capacity for Ag presentation via CD21. Al- To further analyze the events that occur in the B cell subsequent to by guest on September 25, 2021 though resting B cells are not thought to be good APCs, ligation of immune complex binding, the induction of the early activation CD21 may augment this ability by enhancing uptake or processing marker CD69 and the costimulatory molecule CD86 was mea- of immune complexes or by providing signals which improve the sured. Resting B cells were purified from peripheral blood. The capacity of the cells to present Ag. resting state of these cells was confirmed by their lack of expres- A panel of influenza-specific T cell clones was used to analyze sion of CD69 and CD86 at time 0 (data not shown). The cells were the ability of resting B cells to present Ag derived from comple- pulsed with immune complexes for 4 h and then allowed to incu- ment-coated immune complexes. Resting B cells purified from the bate at 37°C for an additional 20–44 h. Cells were harvested and peripheral blood of HLA-matched donors were pulsed with com- stained with FITC-labeled anti-CD69 or anti-CD86. There was lit- plement-influenza immune complexes for 4 h, then incubated for tle or no evidence of B cell activation after a 48-h exposure to an additional 20 h before addition of the T cells. The T cell re- immune complexes as measured by CD69 expression, or of co- sponse was analyzed by measurement of IL-2 in the cell superna- stimulatory molecule up-regulation as measured by CD86 expres- tants after a 24-h incubation. Significant T cell activation was seen sion (Fig. 5). In addition, there was no change in the levels of the only in response to B cells that were pulsed with immune com- adhesion molecules CD11a/CD18 (LFA-1) or CD58 (LFA-3) or in plexes containing complement, and not to B cells pulsed with im- the costimulatory molecule CD80 on the B cells (data not shown). mune complexes prepared in the absence of complement (Fig. 3). Similar results were found with cells harvested 20 h after being Fixed B cells were used to determine whether Ag uptake and pro- pulsed with immune complexes. These findings suggest that CD21 cessing occurred during the period when the B cells were cultured ligation by immune complexes does not result in activation of the alone, or only after coculture with the T cell clones (Fig. 4). When B cell as measured by traditional markers. the B cells were fixed before addition of the T cell clones, their ability to activate the T cells was reduced but not ablated. Fixed B T cell signals are required for B cell activation and induction of cells that had been pulsed with HA307-319 peptide also had re- costimulatory molecules duced ability to activate the HA307-319-specific T cell clone, Additional experiments were performed to determine the condi- CL58, suggesting that fixation itself decreases the effectiveness of tions required for the induction of activation markers and costimu- B cells to act as APCs. The fixation did not cause the B cells to latory molecules in this system. Peripheral blood B cells were become nonspecific stimulators of the T cell clones as CL62 did pulsed with complement-influenza immune complexes for4hand not produce IL-2 in response to the peptide-pulsed B cells. allowed to incubate an additional 20 h before the addition of HLA- All five of the T cell clones demonstrated an augmented re- matched influenza-specific T cell clones to the culture. Cells were sponse to Ag derived from complement-influenza immune com- harvested after 24 h, stained with anti-CD19 and either anti-CD69 plexes. It is unlikely that this is due to transfer of immune com- or anti-CD86, and analyzed by dual color microcytofluorometry. plexes or processed peptide to T cells for autopresentation rather Some cells were pulsed with HA307-319 peptide as a positive The Journal of Immunology 6541

tion expressed CD69 and none expressed CD86. These data suggest that T cell factors are critical in the full activation of resting peripheral blood B cells, and that these T cell responses are elicited by the recognition of Ag derived from complement-containing immune complexes in the absence of B cell activation.

Discussion These experiments demonstrate that resting polyclonal B cells are capable of participating in Ag presentation. By binding complement-containing immune complexes to the B cell complement receptors Ag can be targeted to all peripheral blood B cells. Al- though both CD21 and CD35 participate in binding of immune complexes to the B cell, CD21 likely plays the major role in immune complex internalization. Polymeric C3d, the specific ligand for CD21, can be internalized (31), whereas CD35 has not been shown to undergo endocytosis. Furthermore, Raji B cells, which express CD21 in the absence of CD35, are capable of presenting immune complex-derived Ag to T cell clones (24). Since CD35 is Downloaded from a cofactor for the conversion of C3b to iC3b and iC3b to C3d, immune complex bound to this receptor on the surface of the resting B cell may contain C3 products at an intermediate stage in their degradation to C3d. There was no measurable binding of the im- FIGURE 5. CD69 and CD86 expression on B cells pulsed with com- mune complexes to the B cell Fc receptor, CD32, in the presence plement-containing immune complexes. Resting peripheral blood B cells of human serum. Ligation of certain isoforms of this receptor has http://www.jimmunol.org/ were incubated with media alone (A and C) or with immune complexes prepared in the presence of complement (B and D). Cells were harvested been shown to dampen early events in B cell activation initiated by after 24 h, stained with FITC-labeled mouse mAbs to CD69 (A and B)or cross-linking of surface Ig (32). Whether there is an in vivo role for CD86 (C and D) (thick lines) and analyzed by flow cytometry. Control CD32 in the later activation events described here has not been staining with isotype matched mAb is indicated by the thin lines. determined. The physiological immune complexes formed in these experiments contain acquired anti-influenza IgG. While these data sup- control for T cell activation and its reciprocal effects on B cell port the role of complement opsonization in B cell Ag presentation activation. The B cells that had been pulsed with either immune following an established humoral immune response, it is likely that complexes or HA307-319 peptide before incubation with T cells other immune complexes can be presented in this manner. Natural by guest on September 25, 2021 expressed high levels of CD69 and moderate levels of CD86 on Abs directed against a variety of foreign particles are present con- their surface (Fig. 6). This was in contrast to those cells that had stitutively and can activate the classical pathway. Furthermore, the been incubated in medium alone, in that few cells in this popula- alternative pathway of complement activation can be initiated by many microbial pathogens in the absence of specific Ab. We have shown that influenza is opsonized by complement and binds equally well to B cells regardless of whether the complexes are formed by the alternative or the classical pathway of complement activation (data not shown). Since complement-opsonized complexes can be formed in the absence of high titer-specific Ig, CD21-mediated Ag presentation may also play a role in the responses to Ags for which a humoral immune response has not yet been mounted. Resting peripheral blood B lymphocytes were fully capable of presenting complement-bound Ag to a panel of class II-restricted Ag-specific T cell clones. Interestingly, a longer incubation period before exposure to T cells was required for Ag presentation in resting B cells as compared with transformed B cells. Critical costimulatory molecules expressed constitutively on transformed B cells may need to be up-regulated on resting B cells before they can function as competent APCs. This is an unlikely explanation in FIGURE 6. Effect of T cells on the expression of CD69 and CD86 by this system as the up-regulation of classical costimulatory mole- immune complex-pulsed peripheral blood B cells. Peripheral blood B cells cules (CD80/86 and LFA-1/LFA-3) was not induced by incubation were incubated with medium alone (A and D), with 1 ␮M HA307-319 with complement-containing complexes, nor do the T cell clones peptide (B and E), or immune complexes prepared with normal human used in these experiments require these molecules for their acti- serum (C and F) for 4 h and then washed and incubated with medium vation (data not shown). Alternatively, the ability of resting B cells alone. After 24 h, responder T cells from a HA307-319-specific clone (CL19) were added. After an additional 24-h incubation, cells were har- to internalize and process Ag may be less efficient than in trans- vested and stained with a directly labeled PE-CD19 and either a directly formed B cells. The B cells clearly had processed Ag and targeted labeled FITC anti-CD69 (A–C) or anti-CD86 (D–F) Ab. Histograms were specific peptide to their class II molecules in the absence of T cell generated by gating on CD19-positive cells. Specific staining is indicated help, since fixation of the cells after exposure to immune com- by the thick lines; isotype-matched controls by the thin lines. plexes did not ablate their ability to activate the T cells. 6542 B CELL ANTIGEN PRESENTATION REQUIRES COMPLEMENT OPSONIZATION

Our studies demonstrate that polyclonal B cells participate in Ag presentation in vitro; a recent study using a novel mAb specific for peptide-class II complexes suggests that this process also occurs in vivo (33). After i.v. injection of high titers of soluble HEL, nearly all B cells in lymphoid tissues were found to have HEL 46-61-I-Ak complexes on their surface using a mAb specific for this complex. The mechanism by which these B cells load class II molecules with peptide was not determined, nor was it clarified whether lower amounts of injected Ag resulted in display of peptide by the majority of B cells. Nonetheless, these studies suggested that polyclonal B cells are a major player in the in vivo presentation of Ag, and it is possible that one way in which they participate in this process is by uptake of Ag-C3d complexes by CD21. The recruitment of T cell help by Ag-nonspecific B cells could result in the polyclonal expansion of B cells and the production of nonspecific Abs. To maintain selectivity of B cell responses, the ability of the B cell to respond to T cell help may be dependent upon coligation of surface Ig and CD21. This is supported by a recent report by Thornton, et al. (34), in which the incubation of Downloaded from PBMC with complement-coated KLH immune complexes resulted in the synthesis of Abs specific for KLH, rather than a polyclonal Ab response. This finding suggests that although CD21 can serve as a vehicle for internalizing Ag, it will not transduce signals which result in the independent production of Ab in the absence of signals through sIg. http://www.jimmunol.org/ The ability of B cells to present Ag in different circumstances is modeled in Fig. 7. Resting B cells of diverse antigenic specificities can take up Ag by CD21. Since they do not express costimulatory molecules, as demonstrated by our studies, they are not involved in the initial activation of naive T cells (top row). However, they may be involved in the continued activation of T cells that have been previously primed by dendritic cells or activated monocytes (bot- FIGURE 7. Model of CD21- or IgM-mediated Ag presentation by B tom row). Previously activated T cells are believed to have less cells. Resting polyclonal B cells can take up Ag in immune complexes by stringent requirements for costimulation. These T cells are initially either CD21 (top and bottom rows) or by surface IgM alone or in conjunc- by guest on September 25, 2021 activated in the periarteriolar lymphoid sheath (PALS) region of tion with CD21 (second and third rows). Ag presentation mediated by the spleen or the parafollicular cortex of lymph nodes and subse- CD21 alone does not activate the B cell. Activation of cognate T cells quently move into the marginal zone where they come in contact depends on their requirement for costimulation. Uptake of Ag involving with B cells of multiple specificities. Ag-specific B cells located in IgM on Ag-specific B cells can cause activation, making them able to stimulate naive as well as memory T cells. this region which have bound Ag by IgM alone or in conjunction with CD21 (second and third rows) can be activated and induced to up-regulate costimulatory molecules. These cells can participate that it may play a role in the breakdown of tolerance thought to be in cognate B-T interactions with memory, and perhaps, naive T important in the pathogenesis of this autoimmune disease. cells. However, B cells specific for the Ag of interest would be rare Although our experiments do not support a role for CD21 in at this stage of the immune response, whereas all of the B cells regulating CD86 expression, others have demonstrated that CD21 would express CD21 and be capable of binding complement- ligation results in up-regulation of both CD80 and CD86. One coated Ag. The participation of these nonspecific CD21-expressing study analyzed the effects of incubating complement-containing B cells in stimulating Ag-specific primed T cells would increase KLH immune complexes with human PBMC and found that ex- the likelihood that the immune response would be propagated. pression of CD80 was induced within 1 h (23). This effect was The absence of CD86 expression on B cells participating in thought to be due primarily to Fc␥RII ligation by the immune CD21-mediated Ag presentation may be important in maintaining complexes, with CD21 ligation playing a synergistic role. A sec- tolerance to self-Ag. Naive self-Ag-specific T cells that make their ond investigation analyzed the responses of resting murine splenic way into the periphery, having escaped central deletion in the thy- B cells to cross-linking of CD21 with mAbs. In these experiments, mus, would encounter soluble self-Ag on the surface of CD21- the expression of both CD80 and CD86 was up-regulated within positive B cells. Because CD21 ligation alone in the absence of B 14 h (42). There are several potential explanations for the discrep- cell receptor cross-linking does not induce CD86 expression, the T ancy between these results and our own. The first study used un- cells would receive only a single signal through their TCR and fractionated B cells, and it is possible that the B cell effect was would be tolerized. It has been suggested that resting B cells are indirect, being triggered by another cell population that actually important in the maintenance of T cell tolerance (35–37), and that bound the immune complexes. The results in the second study may CD21 in particular may play a role in this process (38). Interest- result from the use of a different population of B lymphocytes or ingly, expression of CD21 is markedly reduced on B lymphocytes an alternative method of ligating CD21 or from species specific of patients with systemic lupus erythematosus (SLE) (39, 40) variations in B cell responses. There is also an early and progressive decrease in this receptor in The studies described in this report support the role of resting B the MRL/lpr murine model for SLE (41). This decrease is detect- cells in Ag presentation by means of their ability to bind comple- able before clinical manifestations of nephritis occur, suggesting ment-coated Ag to surface complement receptors. The inability of The Journal of Immunology 6543

B cells to become fully activated in response to CD21 ligation 19. Matsumoto, A. K., D. R. Martin, R. H. Carter, L. B. Klickstein, J. M. Ahearn, and alone may provide the mechanism by which specific Ab responses D. T. Fearon. 1993. Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes. J. Exp. Med. 178:1407. are maintained, despite the provision of T cell help. Furthermore, 20. Carter, R. H., and D. T. Fearon. 1992. CD19: lowering the threshold for antigen the inability of the B cell to up-regulate CD86 may allow the cell receptor stimulation of B lymphocytes. Science 256:105. to stimulate previously activated T cells specific for foreign Ags, 21. Lanzavecchia, A., S. Abrignani, D. Scheidegger, R. Obrist, B. Dorken, and G. Moldenhauer. 1988. Antibodies as antigens: the use of mouse monoclonal while tolerizing naive T cells specific for self Ags. antibodies to focus human T cells against selected targets. J. Exp. Med. 167:345. 22. Arvieux, J., H. Yssel, and M. G. Colomb. 1988. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones. Acknowledgments Immunology 65:229. We thank Drs. P. Morganelli, J.van de Winkel, and Eric Brown for Abs; 23. Thornton, B. P., V. Vetvicka, and G. D. Ross. 1994. Natural antibody and complement-mediated antigen processing and presentation by B lymphocytes. J. Im- Dr. Valerie Pinet (National Institute of Allergy and Infectious Diseases); munol. 152:1727. Dr. James Miner (Pilgrim’s Pride Hatchery, Pittsburg, TX) for assistance 24. Boackle, S. A., V. M. Holers, and D. R. Karp. 1997. CD21 augments antigen with oviculture and propagation of influenza virus, and Dr. Laurie Davis presentation in immune individuals. Eur. J. Immunol. 27:122. for helpful discussions. 25. Liu, C., E. J. Gosselin, and P. M. Guyre. 1996. Fc␥RII on human B cells can mediate enhanced antigen presentation. Cell. Immunol. 167:188. 26. Greenman, J., A. L. Tutt, A. J. T. George, K. A. F. Pulford, G. T. Stevenson, and References M. J. Glennie. 1991. Characterization of a new monoclonal anti-Fc␥RII antibody, AT10, and its incorporation into a biospecific F(abЈ)2 derivative for recruitment 1. Ranheim, E. A., and T. J. Kipps. 1993. Activated T cells induce expression of of cytotoxic effectors. Mol. Immunol. 28:1243. B7/BB1 on normal or leukemic B cells through a CD40-dependent signal. J. Exp. 27. Madi, N., J. P. Paccaud, G. Steiger, and J. A. Schifferli. 1991. Immune complex Med. 177:925. binding efficiency of erythrocyte complement receptor 1 (CR1). Clin. Exp. Im-

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