Human C3 Deficiency Associated with Impairments in Dendritic Cell

Human C3 Deficiency Associated with Impairments in Dendritic Cell Differentiation, Memory B Cells, and Regulatory T Cells This information is current as of September 27, 2021. Arije Ghannam, Martine Pernollet, Jean-Luc Fauquert, Nicole Monnier, Denise Ponard, Marie-Bernadette Villiers, Josette Péguet-Navarro, Arlette Tridon, Joel Lunardi, Denis Gerlier and Christian Drouet

J Immunol 2008; 181:5158-5166; ; Downloaded from doi: 10.4049/jimmunol.181.7.5158 http://www.jimmunol.org/content/181/7/5158 http://www.jimmunol.org/ References This article cites 47 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/181/7/5158.full#ref-list-1

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human C3 Deﬁciency Associated with Impairments in Dendritic Cell Differentiation, Memory B Cells, and Regulatory T Cells1

Arije Ghannam,* Martine Pernollet,† Jean-Luc Fauquert,‡ Nicole Monnier,§ Denise Ponard,¶ Marie-Bernadette Villiers,ʈ Josette Pe´guet-Navarro,# Arlette Tridon,‡ Joel Lunardi,§ Denis Gerlier,* and Christian Drouet2**

Primary C3 deficiency, a rare autosomal inherited disease (OMIM 120700), was identified in a 2-year-old male suffering from recurrent pyogenic infections from early infancy with undetectable total complement hemolytic activity (CH50) and C3 values. The nonconsanguineous parents and the two patients’ two siblings had 50% normal serum C3 concentration. The molecular abnormality associated a paternal allele coding C3 with the missense mutation p.Ser550Pro and an apparently null maternal allele, Downloaded from with production of a defective protein that could no longer be secreted. Vaccination of the child did not induce a long-term Ab response. Accordingly, switched memory IgD؊CD27؉ B cells were barely detected, amounting to only 2.3% of peripheral blood -CD19؉ cells. Cells were significantly defective in stimulating alloreactive responses. The in vitro development of immature den dritic cells and their maturation capacity were greatly impaired, with decreased CD1a expression and IL-12p70 secretion ability. These cells were unable to induce autologous B cell proliferation and Ig secretion in the presence of CD40L and C3. Finally, the /regulatory T cell development ability of CD4؉ T cells after CD3 and CD46 activation in the presence of IL-2 was significantly http://www.jimmunol.org impaired. Thus, the association of important functional defects of dendritic cells, acquisition of B cell memory, and regulatory T cells with human C3 deficiency strongly supports a major role for C3 in bridging innate and adaptive immunity in humans. The Journal of Immunology, 2008, 181: 5158–5166.

he activation of C3 and subsequent production of various 120700) was recognized as being associated with a striking sus- proteolytic fragments are crucial and a strategic function ceptibility to pyogenic infections (1, 5). The rare cases of complete T for both innate and adaptive immune defense. The liver is C3 deficiency are associated with large deletions, microdeletions, the main site of C3 synthesis (1), which is increased during acute or mutations in splice sequences (for review, see Ref. 4). Het- by guest on September 27, 2021 inflammation. Diffusible amounts are also produced by activated erozygous individuals with missense mutations are at risk for non- monocytes, macrophages (2), and dendritic cells (DCs)3 (3, 4). infectious diseases, such as membranoproliferative glomerulone- Human complete C3 deficiency with two affected alleles (OMIM phritis (5), age-related macular degeneration (6), and atypical hemolytic and uremic syndrome (7). Evidence that C3 is involved in adaptive immunity was demonstrated in C3–/– animals, which *VirPatH, Universite´de Lyon, Universite´Lyon1, Centre National de la Recherche exhibited a substantial reduction in germinal center (GC) numbers, † Scientifique FRE3011, Lyon, France; Etablissement Français du Sang, Grenoble, low IgG Ab titers after virus infection (8), and impaired T-inde- France; ‡Centre Hospitalier Universitaire Clermont-Ferrand, Universite´d’Auvergne, Clermont-Ferrand, France; §Centre Hospitalier Universitaire Grenoble, Institut Na- pendent and T-dependent responses (9–12). The activation and the tional de la Santeét de la Recherche Me´dicale, Unite´836, Universite´Joseph Fourier, covalent attachment of C3 to an Ag enhance the specific B cell Grenoble, France; ¶Laboratoire d’Immunologie, Centre Hospitalier Universitaire Grenoble, Grenoble, France; ʈInstitut National de la Santeét de la Recherche Me´di- response by at least two mechanisms. On one hand, coligation of cale, Unite´823, Universite´Joseph Fourier, Grenoble, France; #EA 41-69, Universite´ the CD21/CD19/CD81 coreceptors lowers the threshold for B cell Lyon1, Hoˆpital E. Herriot, Lyon, France; and **GREPI/TIMC-IMAG Centre Na- activation both in vivo and ex vivo (13, 14), and the ligation of tional de la Recherche Scientifique Unite´Mixte de Recherche 5525, Universite´Joseph Fourier, Centre Hospitalier Universitaire Grenoble, Grenoble, France CD21 is required for the survival of activated B cells within the Received for publication May 27, 2008. Accepted for publication August 1, 2008. follicles and GCs (10). In contrast, the complement receptor-dependent retention of Ag on follicular DCs provides an Ag source The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance for the clonal selection within the GC (10, 15, 16). The direct with 18 U.S.C. Section 1734 solely to indicate this fact. coupling of C3d or C3b to a protein Ag lowers the amount of 1 This work was partly supported by Grant PHRC 2002 from the French Ministry of protein required for an optimal Ab response by as much as 10,000- Health (to C.D.). A.G. is a recipient of a fellowship from Tishreen University, Syria. fold (17, 18) and leads to more stable IgG production and better J.-L.F. took care of the family; A.G., M.P., N.M., and D.P. performed experiments; memory stimulation (19), resulting in an improved, long-lasting A.G., M.-B.V., J.P.-N., A.T., J.L., and D.G. analyzed results; A.G., D.G., and C.D. made the figures; and A.G., D.G., and C.D. designed the research and wrote the paper. response (20). Similarly, the coupling of C3d to the influenza hem- Part of this work was presented as an oral communication at the 11th European agglutinin in a DNA vaccine enhances the humoral response (21). Meeting on Complement in Human Diseases (Cardiff, U.K.), with the abstract iden- C3 also appears to be critical for optimal Ag presentation in allo- tification: Ghannam et al. 2007. Absence of memory B cells and unbalanced T cell responses in a C3 deficient patient. Mol. Immunol. 44: 3910 (Abstr.). reactive responses (3, 22, 23). Considering the importance of C3 in 2 Address correspondence and reprint requests to Dr. Christian Drouet, GREPI/ TIMC-IMAG CNRS UMR5525, Universite´Joseph Fourier, CHU Grenoble, PO Box complement-induced regulatory T cell; GC, germinal center; PPD, tuberculin purified 217, Grenoble, F-38043 France. E-mail address: [email protected] derivative; SNP, single nucleotide polymorphisms; Treg, regulatory T cell. 3 Abbreviations used in this paper: DC, dendritic cell; ACM, Ag preparation from Candida albicans; AT, tetanus toxoid; CH50, complement hemolytic activity; cTreg, Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 5159

the recovery from primary infection, the costimulation of B cells Ag proliferation assays via the CD21/CD19/CD81 coreceptors to recruit leukocytes at the PBMCs were prepared by density-gradient centrifugation over lymphocyte infection site has been suggested as a minor pathway (24). In con- separation medium (Eurobio). A total of 50 ␮g/ml tetanus toxoid (AT; ϩ ϩ trast, the ability of C3 to enhance specific CD4 and CD8 T cell Aventis Pasteur), 2500 UI/ml tuberculin purified derivative (PPD; Aventis responses can be critical in mediating antiviral protection (24, 25). Pasteur), or 25 ␮g/ml Ag preparation from Candida albicans (ACM; Bio- ϫ 5 ␮ All of these findings may have implications in vaccine Rad) was added to 1 10 PBMCs in triplicate in 96-well plates (200 l; BD Falcon). Mitogen (0.5 ␮g/ml)-activated cells and nonstimulated cells development. were used as positive mitogenic and negative control, respectively. The In the present study, we characterized a complete C3 deficiency proliferation was monitored after an 8-h [3H]thymidine (1.0 ␮Ci/ml) in- in a nonconsanguineous French family. In addition to a new mis- corporation at day 6 for each Ag. Tests were conducted in triplicate, and sense C3 mutant, our results identify several new immune dys- the results were expressed as mean net cpm Ϯ SD. Each experiment was conducted twice, and the experiments shown are representative of all functions (4). An important defect of memory B cells was found, the data. associated with the impairment of vaccine Ab production. The in vitro differentiation of myeloid DCs was greatly impaired. The Alloreactive mixed culture complement-induced regulatory T cells (Tregs) were lacking in the Responder and allogeneic irradiated (30 Gy) stimulator PBMCs were young patient and his heterozygous parents. Altogether, our find- cocultured at 1:1 ratio in 96-well plates (200 ␮l; BD Falcon); 5 ϫ 104 ings further emphasize the critical and multiple roles of C3 in the irradiated stimulator cells were cocultured with 5 ϫ 104 responder HLA- development of adaptive immunity in humans. nonmatched cells/well for 6 days. The proliferation was monitored after an 18-h [3H]thymidine (1.0 ␮Ci/ml) incorporation at day 6. Tests were conducted in triplicate, and the results were expressed as mean net cpm Ϯ SD. Downloaded from Materials and Methods Generation of myeloid DCs Case report and blood collections CD14ϩ cells were prepared, as described above, and cultured (1 ϫ 106 Since early infancy, a 2-year-old boy had suffered from recurrent pyogenic cells/ml) in six-well culture plates (BD Falcon) in RPMI 1640 medium infections, including severe meningitis, pneumopathy, otitis, and pyoeth- supplemented with 10% FCS (Invitrogen), 2 mM glutamine, and 25 mM moidis. The hepatic functions were normal. His two siblings (5 and 7 years HEPES, in the presence of 200 ng/ml human rGM-CSF (2 ϫ 106 U/mg) old) and parents (mother and father, 38 and 40 years old, respectively) were and 33 ng/ml human rIL-4 (20 ϫ 106 U/mg), which were gifts from Scher-

apparently healthy, but his twin brother had died in the first week of life ing-Plough Research Institute, Kenilworth, NJ. GM-CSF and IL-4 were http://www.jimmunol.org/ from fulminant meningitis (Fig. 1A). Blood was collected from all family added again at days 3 and 5. At day 7, DCs were checked for CD1ahigh, members for complement assays, cell phenotype, and function analyses. MHC class IIϩ, CD80low, CD14low phenotype (CD14 PE, HLA-DR PerCP, The study was conducted in agreement with the European Union and CD1a FITC), and IL-12p70 expression in supernatants (CBA; BD Bio- French ethical policies and was approved by the local ethics committee at sciences) after maturation by 0.1 mg/ml LPS for 24 h. the Centre Hospitalier Universitaire of Clermont-Ferrand. Coculture of B cells and DCs Complement assays and molecular analyses B lymphocytes were purified from PBMCs by negative selection using ϩ Complement hemolytic activity (CH50) was determined (26), and serum Dynal Biotech B Cell Negative Isolation Kit, with a purity of the CD19 concentrations of complement proteins and Igs were measured by laser population greater than 90%. B cell activation was evaluated in the pres- 3 ϫ by guest on September 27, 2021 nephelometry (detection limit for C3 ϭ 43 mg/L; Dade Behring). Comple- ence of DCs, according to Dubois et al. (29). Briefly, 2.5 10 irradiated mentation analyses were conducted using the human C3 protein, after pu- (80 Gy) CD40L-L-transfected cells (a gift from C. Caux, Lyon, France) ϫ 3 ϫ rification to homogeneity, as detailed by Al Salihi et al. (27). The C3 were seeded in the presence of 5 10 purified B lymphocytes, with 5 3 ␮ concentration in the supernatants of monocyte monolayers was measured 10 immature DCs harvested at day 7 in a 96-well culture plate (200 l; BD ␮ by ELISA, using purified C3 as standard, as described previously (20). At Falcon). When indicated, purified C3 was added (100 g/ml, final concen- 3 day 12 of the culture and after a 24-h activation by 0.1 mg/ml Escherichia tration, for 4 h). B cell proliferation was monitored after a 16-h [ H]thy- ␮ coli LPS, total RNA was prepared from monocyte lysates (TRIzol; Invitro- midine (1 Ci/ml) incorporation at day 6. Tests were conducted in tripli- Ϯ gen) and reverse transcribed using the Transcriptor system (Roche Bio- cate, and the results were expressed as mean cpm SD. To determine IgM chemicals). Seven overlapping fragments were amplified with primers cho- and IgG production, supernatants were recovered after 13 days and assayed sen using the cDNA reference sequence (GenBank accession by nephelometry (Dade Behring). NM_000064). Amplicons were directly sequenced using the ABI PRISM Tregs Big Dye Terminator Cycle Sequencing v3.0 reaction kit, and were analyzed on an ABI 3100 DNA Analyzer (Applera). CD4ϩ T cells were positively isolated from PBMCs using magnetic beads (CD4 isolation kit II, Dynabeads; Dynal Biotech), with greater than 90% Immunofluorescence analysis of intracellular C3 purity. CD4ϩ T cells were then cultured in 96-well plates precoated with the mAbs anti-CD3 (Hit3a; 10 ␮g/ml; BD Pharmingen), anti-CD28 Monocytes were prepared from PBMCs by negative selection (Dynabeads (CD28.2; 5 ␮g/ml; BD Pharmingen), or anti-CD46 (20.6; 5 ␮g/ml) (30) in MyPure Monocyte Kit2), with a purity of the CD14ϩ population greater ϩ the presence of human rIL-2 (40 U/ml; Genzyme) for 3 days. IL-10 se- than 95%, as assessed by flow cytometry. The CD14 monocytes/macro- cretion was analyzed in the supernatants using CBA (BD Biosciences). phages were grown for 12 days on LabTEK coverslips (Nunc-Fisher) in Cell surface expression of CD46 on CD4ϩ T cells was assessed by cy- ␮ culture medium and then stimulated for 24 h in the presence of 0.1 g/ml tometry using anti-CD46 FITC. E. coli LPS. The cells were washed with PBS and fixed with 4% parafor- maldehyde in PBS for 10 min; the cells were permeabilized with 1% Statistical analysis Tween 20 in PBS for 10 min. After three washes in PBS, the cells were incubated for 45 min at room temperature with the mouse anti-human C3 Two-tailed Student’s t test was used to compare patient and control data. Ϯ ϭ Ͻ ␤-chain Ab WMI (dilution 1/20 in PBS-1% BSA) (28). Following washes, All data are represented as mean SD; n 2 or 3. Value of p 0.05 was the cells were incubated with goat anti-mouse Ab conjugated with Alex- considered significant. afluor 488 (Invitrogen; dilution 1/100) for 45 min. Cells were washed twice in PBS and once with water, air dried, and mounted with Flourmount-G Results (Southern Biotechnology Associates). Fluorescence was observed using a Complement and vaccine Ab profiles laser confocal microscope (Zeiss LSM410). Fig. 1A illustrates the pedigree of the family and the symptomatic Cell phenotyping proband. The patient’s CH50 and C3 levels were below the detection limit (CH50 Ͻ 10%, C3 Ͻ 43 mg/L; Fig. 1B), whereas other Cells were typed using the following Abs: CD19 PC7, CD27 FITC, IgD PE, IgM APC, CD1a FITC, CD14 PE, HLA-DR PerCP, CD80 APC, and complement proteins, including C4, were within the normal range. CD46 FITC (BD Biosciences). The analyses were performed on a FAC- His parents and siblings displayed nearly half the normal C3 concen- SCalibur instrument using the CellQuest software (BD Biosciences). tration in serum (Fig. 1B). Complementation of the patient’s plasma 5160 C3 DEFICIENCY WITH DC, B CELL, AND Treg IMPAIRMENTS

FIGURE 1. Pedigree of the proband’s family and complement analyses. A, Pedigree: the arrow indicates the proband. B, C3 concentrations (mg/L in serum) and CH50 (percentage of the reference value) are indicated for each family member. C, Complementation of the patient plasma by increasing concentrations of puriﬁed human C3 from 43 (non- supplemented sample) to 1080 mg/ml (normal antigenic level). D, Secretion of C3 by monocytes. C3 was assessed

in culture supernatants from the wells Downloaded from of the patient (II.3), his parents (I.1 and I.2), and a healthy control (Ctrl) at day 12 (n ϭ 2, mean Ϯ SD). N, reference interval. http://www.jimmunol.org/

by increasing C3 concentrations with purified human C3 restored the nal cDNA at a homo- or hemizygous level. A c.1648T Ͼ C change hemolytic activity in a dose-dependent manner (Fig. 1C). was identified at a heterozygous level in the paternal transcript. This To confirm the diagnosis of C3 deficiency, we performed a bio- change led to a p.Ser550Pro substitution of a well-conserved residue synthesis experiment using monocyte monolayers prepared from located in exon 13, which was absent in 100 controls. The prolyl by guest on September 27, 2021 the patient, his parents, and a healthy control. As shown in Fig. 1D, residue is located within a ␤-strand at the C terminus of the MG5 C3 was found below the detection limit in the patient’s cell su- domain of C3 (␤-chain; Fig. 2C). Genomic sequencing of exon 13 pernatant and ϳ50% lower in parents’ cell supernatants than in the showed that both father and child were heterozygous for the control supernatants, suggesting a C3 deficiency, in agreement c.1648T Ͼ C (p.Ser550Pro) mutation. Notably, the analysis of the with the absence of plasma C3nef activity (data not shown). child’s cDNA showed that a single cytosine nucleotide was present at The ability of the proband’s macrophages to synthesize, but not position 1648, pointing out the instability or nonexpression of the to secrete C3 was then investigated. After intracellular immuno- maternal transcript (Fig. 2, C and D) confirmed by the absence of the staining, the macrophages from the proband exhibited a very bright two maternal SNPs, c.1692G and c.2745T (Fig. 2D). The maternal fluorescent intensity, with strong accumulation in a vesicular sys- null allele was not characterized at a genomic level. tem resembling the distribution of the Golgi apparatus (Fig. 2A, i The frequent infections in the patient raised the question of his and ii). In the macrophages from a healthy individual (control), a Ab protection toward vaccine Ags. When the proband was 5 years faint C3 labeling was found distributed throughout the cytoplasm, old, the adaptive immunity was evaluated. Total IgG level in se- from the nucleus to the plasma membrane (Fig. 2A, iii and iv). rum was found lower than normal with 3.7 g/L (IgG1 ϭ 3.5 g/L; Quantification of the fluorescence signal (mean fluorescence in- IgG2 ϭ 0.5 g/L; IgG3 ϭ 0.15 g/L; IgG4 Ͻ 0.07 g/L). Most of the tensity) from the individual cells ranked from fainter to brightest anti-vaccine Ag Ab titers were found to be lower than normally ones illuminates the overall much brighter C3 labeling of the pro- needed for protection. When positively detected after vaccination band’s cells (Fig. 2Aix). Remarkably, in the C3 labeling of the and tested 4 years after vaccination, the Ab titers lowered to near mother’s macrophages, C3 was identical with that of the control or under the threshold value, except for Streptococcus pneumoniae cells both morphologically (Fig. 2A, v–viii) and quantitatively (Fig. and Haemophilus influenzae, two pathogens that have reiteratively 2Aix). The father’s macrophages displayed a slightly brighter la- infected the proband (Table I). beling than mother’s and control’s macrophages (Fig. 2A, vii–ix). These data indicate that C3 was synthesized in the proband’s mac- Decreased ability of peripheral cells to stimulate alloreactive rophages, but most likely accumulated along the secretory path- cells in vitro way, an accumulation that also seems to occur, although at a much The potency of the patient’s cells to stimulate alloreactive cells lower level, in the father’s macrophages. was tested. For this purpose, PBMCs prepared from the patient, his To address the genetic abnormality, RT-PCR products were parents, and a healthy control were irradiated and cocultured in the prepared from monocyte RNA. The full-length C3 transcripts presence of allogeneic PBMCs. The patient’s cells exhibited lower were sequenced in the affected child and in his parents. Five stimulation capacity toward the control cells T1, T2, and T3, in com- single nucleotide polymorphisms (SNPs) (c.941T/C, c1692G/A, parison with the matching situations involving control cells ( p Ͻ c.2421C/G, c.2745C/T, and c.4896T/C) were detected in the mater- 0.05; Fig. 3A). In the reverse situation, the patient’s cells developed The Journal of Immunology 5161

FIGURE 2. Monocyte expression and molecular analysis. A, Cell expression of C3. Distribution of im- munoreactive C3 in macrophages was assayed after 24-h LPS stimulation. Macrophages were labeled with the anti-C3 ␤-chain Ab WMI and then with Alexafluor 488-conjugated anti-mouse Ab. The 25-␮m scale of the microscopy is indicated by Ⅺ (immersion objective lens magnification, ϫ63; aperture, 1.4). The excitation laser beam was at 488 nm, and selection dichroic filter for image caption at 510 nm (image acquisition by Zeiss). Control cells labeled with the second Ab alone (not presented) showed no fluorescence. Subpanels are identified as proband (A, i and ii), healthy control (A, iii and iv), mother (A, v and vi), and father (A, vii and viii). The mean fluorescence intensity (MFI) from the individual cells was ranked from fainter to brightest ones and in- dividually plotted to show the high, intermediate, and Downloaded from low levels of C3 labeling in cells from proband, father, and mother/control, respectively (Aix). B, Diagram of the C3Pro550 with identification of the structural domains (37). The position of the Ser550Pro mutation, thioester site (black diamond), and strand numbering (accession P01034) is shown. C, Sequence analysis. RT- PCR products were prepared from monocyte RNA and http://www.jimmunol.org/ sequenced. Both paternal and maternal alleles of the proband were identified using polymorphic markers at positions 1692 and 2745. Chromatograms of the proband gDNA and cDNA sequencing indicated the mono- allelic expression of the c.1648T Ͼ C (p.Ser550Pro) mutation (accession NM_000064). D, cDNA sequencing data. SNPs at positions 1692 and 2745 were used for characterization of parental alleles in the proband. by guest on September 27, 2021

normal responder properties (Fig. 3B). This indicates a possible im- control, significantly lower responses were found in the patient’s cell pairment of the Ag presentation ability of proband’s cells. culture stimulated by PPD and AT ( p Ͻ 0.02 and p Ͻ 0.01, respectively). No significant difference was found in the culture stimulated Decreased proliferation response to specific recalled Ags by ACM ( p Ͼ 0.05) (Fig. 3C). The response to mitogen was similar The cellular responses to soluble Ags such as PPD, AT, and ACM for the patient and the controls. These data further support an impair- were investigated using Ag proliferation assays. Compared with the ment of the patient’s PBMCs in Ag processing and/or presentation.

Table I. Ab titres of the patient

Serum Titre 4 mo Serum Titre 4 Years Type, Unit after Vaccination after Vaccination Threshold

Mumps IgG, UI/L 500 230 230 IgM, UI/L 500 Ͻ230 230 Measles IgG, UI/L Ͼ300 300 300 IgM, UI/L Ͼ300 150 300 Poliovirus Type 1, dilution Ͻ10 Ͻ10 32 Type 2, dilution Ͻ10 Ͻ10 32 Type 3, dilution 160 80 40 Orthomyxovirus influenzae Dilution Ͻ20 Ͻ20 20 Orthomyxovirus parainfluenzae Types 1–3, dilution Ͻ20 Ͻ20 20 Tetanus toxoid kUI/L 0.10 Ͻ0.10 0.10 Diphteria toxoid kUI/L 0.18 Ͻ0.10 0.10 S. pneumoniae ␮g/mL 1.9 1.6 0.3 H. influenzae B IgG, ␮g/mL 5.8 1.0 Bordetella pertussis (immunoblot) Pertussis toxin Absence Absence Adenylate-cyclase Absence Absence Fimbrial hemagglutinin Absence Absence 5162 C3 DEFICIENCY WITH DC, B CELL, AND Treg IMPAIRMENTS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Allogeneic reactivity and Ag proliferation assay. Irradiated FIGURE 4. Differentiation of myeloid DCs. A, DC culture of the pro- PBMCs were cocultured with allogeneic PBMCs for up to 6 days, using the band and a healthy control at day 6 in the presence of GM-CSF and IL-4. patient’s stimulation capacity (A) and responder capacity (B) (C3–/–) and B, Histogram panels from cytometric analysis of immature DCs at day 6. control HLA-nonmatched cells (T1, T2, and T3). The proliferation was C, IL-12p70 in mature DC supernatants harvested after 24-h LPS activation measured by [3H]thymidine incorporation (n ϭ 3, mean Ϯ SD). Values of (n ϭ 2, mean Ϯ SD). C3–/–, Proband; Ctrl, healthy control; Fa, father; Mo, p (Student’s t test) for comparisons between data are given. When not mother. indicated, the differences were not significant. C, PBMCs were incubated with Ag, and their proliferation was measured using [3H]thymidine incorporation (n ϭ 3, mean Ϯ SD). Values of p (Student’s t test) for comparisons between data are given. Ctrl, cells without Ag. the parents’ monocytes, the total number of DCs that could be collected at the end of the differentiation process was much lower when derived from the patient monocyte culture, compared with the parents’ preparation (0.27 vs 0.90% of the monocyte popula- Decreased ability to differentiate myeloid DCs tion). Furthermore, the CD1aϩ expression on the DCs was dras- DC maturation was recently demonstrated to be C3 dependent (3, tically reduced to 550 of mean fluorescence intensity, compared 31), and we investigated the ability of patient cells to mature into with the high 1079 and 4980 expression levels on DCs from the DCs in vitro. Purified human monocytes were allowed to differ- parents and control, respectively. In addition, proband DCs ex- entiate into immature DCs by culture in the presence of GM-CSF pressed slightly lower HLA-DR (Fig. 4B). We next estimated the and IL-4 in FCS-supplemented medium (Fig. 4A). Compared with IL-12p70 secretion during the maturation step of the DCs upon The Journal of Immunology 5163

LPS activation. In the proband’s supernatants, IL-12p70 was nearly absent and parents’ DCs exhibited half the production found in the control (Fig. 4C). Thus, the ability of the patient’s monocytes to develop into immature DCs and their capacity to acquire the maturation phenotype were greatly altered.

Absence of memory B cells Because of the defective Ab responses to vaccine Ags, peripheral B cells were next analyzed. Memory B cells are distinguished in two subtypes according to the expression of CD27 and IgD with IgDϪCD27ϩ (as switched cells) and IgDϩCD27ϩ (as nonswitched cells) (32). The number of memory IgD-CD27ϩ B cells was greatly decreased in the patient (2.3% of the CD19ϩ cells), and was found in reduced amounts in his mother and father (13.1 and 14.2%, respectively; Fig. 5, A–C), as compared with the usual 30–40% in controls (33). The percentage of CD19ϩ B cells in the total lymphocyte population was within the normal value. Conse- quently, cells with naive IgDϩCD27– phenotype represented more than 95% of the patient’s circulating B cells, with rare memory Downloaded from CD27ϩCD19ϩ B cells belonging to the IgMϩIgDϩ phenotype (Fig. 5Aiii). Thus, the C3 deﬁciency was associated with a very low level of memory B cells.

Impaired B cell activation and differentiation in response to C3

In mixed cultures of DCs and B cells, the presence of CD40L has http://www.jimmunol.org/ been demonstrated to enhance both the B cell proliferation and Ig production (29), and the inﬂuence of exogenously added C3 on B cell functions was investigated in these conditions. In the absence of C3, the proliferative responses and Ig secretion of B cells from the proband, his parents, and the control were comparable (Fig. 5, D–F). In contrast, exogenous C3 increased the proliferation of B cells from the control and, to a lesser extent, the parents’ cells, but not those from the patient’s B cells (Fig. 5D). Furthermore, C3 induced IgM secretion in the control and parents’ and patient’s B by guest on September 27, 2021 cells, with isotypic commutation in control B cells, but not in the parents’ and patient’s B cells (Fig. 5, E and F). Thus, B cell activation and differentiation in response to C3 were impaired in both the patient and his parents.

Alteration of CD46-activated Tregs CD46, a receptor for C3b ligand, is expressed on all nucleated cells. It is a potent costimulator for human CD4ϩ T lymphocytes that leads them to acquire a distinct Treg-like phenotype called complement-induced Tregs (cTregs; phenotype Tr1) (34, 35). This prompted us to evaluate the CD46-costimulatory function in cells with C3 deficiency. Purified human CD4ϩ lymphocytes from the patient, his parents, and a healthy donor were stimulated with im- mobilized mAbs (anti-CD3, anti-CD46, anti-CD28), and their cy- tokine profile was analyzed. The CD3- and CD46-mediated stimulation of CD4ϩ T from the patient and his parents in the presence of IL-2 resulted in low IL-10 secretion (Fig. 6A), whereas no difference was found upon anti-CD28 and anti-CD3 costimulation. FoxP3ϩCD4ϩ T cells were within the normal range (8.15% of the CD4ϩ cells; reference 3–12%). Because CD46 was expressed at a normal level on the patient’s CD4ϩ T cells (Fig. 6B), the lack of

cultured with irradiated CD40L-L cells (left histogram) or together with FIGURE 5. B cell analysis. A–C, Phenotypes of peripheral blood B immature DCs (middle histogram). When indicated, purified C3 was added cells. Peripheral blood cells were stained with anti-CD19 PC7, anti-IgD to the culture (B ϩ CD40L ϩ immature DCs) for 4 h, and then the medium PE, IgM APC, and anti-CD27 FITC. The percentage of positive cells is was changed. Cells were incubated with [3H]thymidine uptake at day 6 indicated relative to the CD19-gated cells. Panels are identified as pro- (D), and the supernatants were harvested at day 13 and subsequently as- band (A, i–iv), father (B), and mother (C). D–F, B cell proliferation and sayed for their IgM (E) and IgG (F) contents (n ϭ 3, mean Ϯ SD). C3–/–, IgM and IgG secretion assays. Highly purified B lymphocytes were Proband; Ctrl, healthy control; Fa, father; Mo, mother. 5164 C3 DEFICIENCY WITH DC, B CELL, AND Treg IMPAIRMENTS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. IL-10 secretion upon CD46 activation of CD4ϩ T cells and CD46 expression. A, IL-10 production by purified CD4ϩ T cells from patient, parents, and healthy donor upon CD3 activation with CD28 or CD46 costimulation in the presence of IL-2. After 3 days, supernatants were harvested and IL-10 concentrations were quantified using CBA. B, CD46 expression by CD4ϩ T cells of the patient, parents, and healthy donor analyzed by flow cytometry (anti-CD46 FITC). Ig ctrl, Isotypic control; C3–/–, proband; Fa, father; Mo, mother.

Tr1 activation could result from an impaired signaling function of could be predicted to exhibit functional defects. However, there CD46 in C3 deficiency. were no clinical nor routine biological signs that could have alerted the clinician about liver or renal dysfunction in both proband and Discussion father. In the absence of thorough investigation of liver and renal Human primary C3 deficiency has been described to date in 28 functions, we cannot, however, exclude any dysfunctions of these patients from 20 families in the world. The deficiency combining organs. the p.Ser550Pro mutation and an apparently null allele further em- The lower ability of proband’s cells to stimulate allogeneic cells phasizes the heterogeneous characteristics of C3 deficiency. A is in agreement with recent findings in C3–/– mice (3, 22, 23). similar situation has been reported in C3 deficiency associated Mouse C3–/– cells displayed reduced cell surface expression of with connective tissue disease (36). In agreement with the first MHC class II and B7.2, with subsequent impairment of the Th1- descriptions (OMIM 120700), this novel French case has a long polarizing molecule IL-12 and reduction of alloreactive T cell history of recurrent infections, mainly associated with Gram-pos- priming. Thus, the data from mouse and human cells support the itive bacteria and H. influenzae; both parents and two siblings are role for C3 in the development of functional APCs. Indeed, mono- asymptomatic and phenotypically heterozygous, with the C3 con- cytes from the C3 deficiency poorly differentiate in vitro into DCs, centration reduced to approximately half of that normally present with reduced expression of CD1a and failed aptitude for secretion of in serum. The coding mutation is located in the middle of a ␤-sheet IL-12p70. In the absence of C3 from the culture medium, DCs ex- in the MG5 domain, as defined in the recently solved three-dimen- press a lower amount of the differentiation markers, including DC- sional structure (37). Because the C3Pro550 is not secreted by SIGN, HLA-DR, CD1a, CD80, and CD86, and produce many fewer monocytes, but accumulates intracellularly, the Ser550Pro mutation cytokines (31). Accordingly, in the mouse, C3 triggers the differen- most likely prevents the correct folding into the ␤-barrel structure tiation of monocytes into DCs (31). This suggests a novel role played of the MG5 domain. This intracellular accumulation could be del- by C3 as a critical cofactor in DC differentiation and maturation. eterious to some macrophage functions. Likewise, because liver The C3 deficiency was associated with a prominent B cell de- and kidney are the main source of C3 production, these organs fect, in agreement with the pioneering studies in mice, in which the The Journal of Immunology 5165

GC reaction was inhibited after depletion of C3 by cobra venom Collectively, our data further emphasize the major role of com- factor (38), and with the more recent observations made in C3–/– plement in bridging innate and adaptive immunity. C3 directly or mice (9) (for reviews, see Carroll (16, 39)). Surprisingly, the indirectly provides a costimulatory signal or sets a threshold C3nullC3Pro550 proband lacks much of the expected switched needed for monocyte maturation into DCs, B cell isotypic switch- memory B cells (IgD–CD27ϩ) in the periphery, with as little as ing and differentiation into memory cells, and cTreg activity. The 2.3% of switched IgG memory B cells among the CD19ϩ cells, importance of complement in regulating cell-mediated immunity is and the IgMϩIgDϩCD27ϩ B cells were nearly the only circulating likely to have a more central role in adaptive immunity than has memory B cell population. Because B cells from the present previously been suspected. C3 deficiency were almost pure nonswitched memory IgMϩIgDϩCD27ϩ B cells, the response to exogenously added C3 Acknowledgments observed in vitro, that is increased IgM secretion without stimu- We are indebted to Agne`s Colomer, Françoise Csopaki, and Sylvie Glaizal lation of IgG secretion and B cell proliferation, can be considered (Grenoble) for skillful technical assistance; Professor Jean-Claude Weill as specific properties of this B cell memory subset. Whether these (Paris) for helpful discussion; Dr. Yves Usson (Grenoble) for confocal microscopy; Dr. Christine Gaboriaud (Grenoble) for structural analysis; properties are independent of the presence or absence of C3 in vivo and Dr. Jean-Charles Renversez (Grenoble) for Ig assays. or resulted from a lack of proper B cell education due to a C3-free environment remains to be determined. The absence of switched Disclosures IgG memory B cells in the proband correlated with the low Ab The authors have no financial conflict of interest. titers after vaccination. This indicates that C3 should deliver a third signal required for the accumulation of proliferating B cells, iso- References Downloaded from typic switching, and differentiation into Ab-secreting cells. 1. Alper, C. A., A. M. Johnson, A. G. Birtch, and F. D. Moore. 1969. Human CЈ3: Overall, this B cell phenotype strongly suggests a defect in the evidence for the liver as the primary site of synthesis. Science 163: 286–288. 2. Einstein, L., P. J. Hansen, M. Ballow, A. E. Davis, III, J. S. Davis, IV, development of the GC, a failure in the maturation process C. A. Alper, F. S. Rosen, and H. R. Colten. 1977. Biosynthesis of the third within the GC, and/or a failure in the transfer process to the component of complement (C3) in vitro by monocytes from both normal and homozygous C3-deficient humans. J. Clin. Invest. 60: 963–969. marginal zone from where originate the memory B cells that 3. Peng, Q., K. Li, H. Patel, S. H. Sacks, and W. Zhou. 2006. Dendritic cell syn- http://www.jimmunol.org/ secrete IgM (33, 40, 41). thesis of C3 is required for full T cell activation and development of a Th1 The impairment of IL-10 secretion by T cells from the proband phenotype. J. Immunol. 176: 3330–3341. 4. Reis, E., D. A. Falcaõ, and L. Isaac. 2006. Clinical aspects and molecular basis and his heterozygous parents upon anti-CD46 activation suggests of primary deficiencies of complement component C3 and its regulatory proteins that the Tr1 phenotype could be dependent on a critical expression factor I and factor H. Scand. J. Immunol. 63: 155–168. 5. Pussell, B. A., E. Bourke, M. Nayef, S. Morris, and D. K. Peters. 1980. Com- level of C3 for development. This lack of regulatory function in the plement deficiency and nephritis: a report of a family. Lancet 1: 675–677. absence of the natural CD46 ligand is in agreement with the 6. Yates, J., T. Sepp, B. K. Matharu, J. C. Khan, D. A. Thurlby, H. Shahid, sharply decreased IL-10 secretion and abnormal T cell response D. G. Clayton, C. Hayward, J. Morgan, A. F. Wright, et al., and Genetic Factors in AMD Study Group. 2007. Complement C3 variant and the risk of age-related observed in the contact-hypersensitivity reaction by CD46 trans- macular degeneration. N. Engl. J. Med. 357: 553–561.

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