Anti-Factor B and Anti-C3b Autoantibodies in C3 Glomerulopathy and Ig-Associated Membranoproliferative GN

CLINICAL RESEARCH www.jasn.org

† ‡ | Maria Chiara Marinozzi,* Lubka T. Roumenina,* § Sophie Chauvet,* Alexandre Hertig, †† ‡‡ Dominique Bertrand,¶ Jérome Olagne,** Marie Frimat, Tim Ulinski, || Georges Deschênes,§§ Stephane Burtey, Michel Delahousse,¶¶ Bruno Moulin,** † Christophe Legendre,*** Véronique Frémeaux-Bacchi,* and Moglie Le Quintrec*¶¶

*Team Complement and Diseases Cordeliers Research Center, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1138, Paris, France; †Assistance Publique-Hôpitaux de Paris, Service d’Immunologie Biologique, Hôpital Européen Georges Pompidou, Paris, France; ‡Université Paris Descartes Sorbonne Paris-Cité, Paris, France; §Université Pierre et Marie Curie, Paris, France; |Assistance Publique-Hôpitaux de Paris, Hôpital Tenon, Service de Néphrologie et de Transplantation rénale, Paris, France; ¶Service de Néphrologie et de Transplantation rénale, Hôpital Bois Guillaume, Rouen, France; **Service de Néphrologie et de Transplantation rénale, Strasbourg, France; ††Service de Néphrologie et de Transplantation rénale, Lille, France; ‡‡Assistance Publique-Hôpitaux de Paris, Service de Néphrologie, Hôpital Trousseau, Paris, France; §§Assistance Publique-Hôpitaux de Paris, Service de Néphrologie et de Transplantation rénale, Hôpital Robert Debré, Paris, France; ||Aix Marseille université, Assistance publique-Hôpitaux de Marseille, Service de Néphrologie et de Transplantation rénale, Marseille, France; ¶¶Service de Néphrologie et Transplantation rénale, Hopital Foch, Paris, France; and ***Assistance Publique-Hôpitaux de Paris, Service de Néphrologie et de Transplantation rénale, Hôpital Necker, Paris, France

ABSTRACT In C3 glomerulopathy (C3G), the alternative pathway of complement is frequently overactivated by autoantibodies that stabilize the C3 convertase C3bBb. Anti-C3b and anti-factor B (anti-FB) IgG have been reported in three patients with C3G. We screened a cohort of 141 patients with C3G and Ig-associated membranoproliferative GN (Ig-MPGN) for anti-FB and anti-C3b autoantibodies using ELISA. We identified seven patients with anti-FB IgG, three patients with anti-C3b IgG, and five patients with anti-FB and anti-C3b IgG. Of these 15 patients, ten were diagnosed with Ig-MPGN. Among those patients with available data, 92% had a nephrotic syndrome, 64% had AKI, and 67% had a documented infection. Patients negative for anti-C3b and anti-FB IgG had much lower rates of infection (17 [25%] patients with C3G and one [10%] patient with Ig-MPGN). After 48 months, four of 15 (26%) positive patients had developed ESRD or died. All 15 patients had high plasma Bb levels, six (40%) patients had low levels of C3, and nine (60%) patients had high levels of soluble C5b9. In vitro, IgG purified from patients with anti-FB Abs selectively enhanced C3 convertase activity; IgG from patients with anti-C3b/anti-FB Abs enhanced C3 and C5 cleavage. IgG from patients with anti-C3b Abs stabilized C3bBb and perturbed C3b binding to complement receptor 1 but did not perturb binding to factor H. In conclusion, the prevalence of anti-C3b/anti-FB Abs and alternative pathway activation is similar in Ig-MPGN and C3G, suggesting similar pathogenic mechanisms. Identification of the underlying defect in Ig-MPGN could lead to improved treatment. CLINICAL RESEARCH

J Am Soc Nephrol 28: 1603–1613, 2017. doi: https://doi.org/10.1681/ASN.2016030343

Received March 23, 2016. Accepted October 26, 2016. Blanc, Paris 75015, France, or Dr. Moglie Le Quintrec, Service de Néphrologie et Transplantation rénale, Hôpital Lapeyronnie, 371 Avenue Published online ahead of print. Publication date available at du Doyen Gaston Giraud, 34295 Montpellier cedex 5, France. Email: www.jasn.org. [email protected] or [email protected]

J Am Soc Nephrol 28: 1603–1613, 2017 ISSN : 1046-6673/2805-1603 1603 CLINICAL RESEARCH www.jasn.org

The complement system is a major innate immune defense (AUs) of plasma from healthy donors. Plasma samples from 15 mechanism that can induce host tissue damage when uncon- (15 of 141; 10%) patients were positive for anti-FB and/or anti- trolled.1,2 Such uncontrolled activation of the alternative path- C3b Ab (Figure 1). Anti-FB IgG and anti-C3b IgG were de- way (AP) of complement gives rise to kidney diseases, such as tected in 12 and eight patients, respectively. Five (five of 15; C3 glomerulopathies (C3Gs).3 Complement is frequently 33%) patients were positive for anti-C3b and anti-FB IgG. overactivated by C3 nephritic factors (C3NeFs). C3NeFs Among the positive samples for anti-C3b, anti-C3b IgG levels bind to a neoepitope on the AP C3 convertase, C3bBb, without were higher in the group of patients negative for anti-FB Ab. interacting with its isolated components C3b and Bb and The interaction of the anti-FB and the anti-C3b Abs with their stabilize it.4 Classification on the basis of glomerular deposits ligands was dose dependent as shown by ELISA (Supplemental characterized by immunofluorescence (IF) distinguishes Figure 1, A and B). The level of binding detected by surface between C3G characterized by predominant glomerular C3– plasmon resonance (SPR) correlated with the ELISA results containing deposits and Ig-associated membranoproliferative (Supplemental Figure 1, C and D). GN (Ig-MPGN) characterized by mesangial and subendothelial deposits of any combination of IgG, IgM, or C1q with Characteristics of Patients with Anti-FB Ab and C3.5,6 An anti-factor B (anti-FB) autoantibody has recently Anti-C3b Ab been identified in a patient with Dense Deposit Disease The clinical characteristics of the 15 patients are summarized in (DDD), a subtype of C3G.7 This Ab has the same capacity as Table 1. Disease onset occurred during adulthood in 13 of 15 C3NeF to increase the C3 convertase half-life. Combined anti- (86%) patients, with a median age of 37 years old (range =7– FB and anti-C3b Ab–enhancing C3 convertase activity has also 65 years old), and 11 (73%) of the patients were men. At the been found in two other patients with DDD.8 In this study, we time of diagnosis on the native kidney, 13 of 14 (92%) patients retrospectively screened sera from patients with C3G and with available data had a nephrotic syndrome, and nine of 14 idiopathic Ig-MPGN for C3b and FB Ab using ELISA. We (64%) patients had renal failure. Six of ten (60%) patients with examined the effects of the positive IgG on the C3 and C5 Ig-MPGN had a history of addiction or hematologic diseases. activation and convertase regulation. Clinical data were col- An infectious event was in six of seven (87%) patients with Ig- lected. In vitro and in vivo experimental evidence indicates that MPGN with available data and two of five patients with C3G. The these autoantibodies may account for C3G and Ig-MPGN dis- types of infection were tooth infection (n=3), Staphylococcus sep- orders by overactivating the AP. ticemia (n=2), pneumonia (n=2), and pyelonephritis infection (n=1) due to Klebsiella pneumoniae. Two patients had a history of hepatitis B infection. Among 15 patients, one had anti-CR1 Ab RESULTS (patient 13), and two had anti-factor I Ab (patients 1 and 13; data not shown). The frequency of infection was significantly lower in Anti-FB and Anti-C3b Autoantibody Screening in the patients negative for anti-C3b and anti-FB with C3G (17 of 68; C3G and Ig-MPGN Cohorts 25%) or Ig-MPGN (one of ten; 10%). Patients with positive anti- Plasma samples from 141 patients with C3G or Ig-MPGN and C3b/anti-FB Ab were older at disease onset and more likely to 50 healthy donors were screened for IgG reactivity against C3b have infection as a trigger than patients without anti-C3b/anti-FB and FB using a homemade ELISA assay. A positive sample was Ab (Supplemental Tables 1 and 2). used as a standard, and the cutoff value between negative and Histologic renal lesions on native kidneys at the time of diagnosis positive samples was the mean with 2 SDs of the arbitrary units (n=14) were crescents (four of 14 biopsies), a mesangial proliferative

Figure 1. Detection of anti-FB and anti-C3b autoantibodies. Plasma samples from patients with C3G (n=118) and Ig-MPGN (n=23) were tested. Anti-FB and anti-C3b Abs were expressed in AUs. White circles represent samples with double positivity. The cutoff of positivity was determined with plasma samples from 50 healthy donors (HDs).

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pattern (14 of 14 biopsies), and glomerular inflammatory (14 of 14 biopsies). Inflammatory cell infiltration was significantly more fre-

3g/dl. quent in patients with anti-C3b/anti-FB Ab (92%) than in those . without (27%). No humps were seen. Using IF staining, ten of 15 Pos/Pos Pos/Pos Pos/Pos Pos/Pos Pos/Neg Pos/Neg Pos/Neg Pos/Neg Pos/Neg Neg/Pos Pos/Neg Neg/Pos Neg/Pos Pos/Neg (71%) biopsies were classiﬁed as Ig-MPGN, and ﬁve of 15 (29%)

Anti-FB/Anti-C3b were classified as C3G. C3 and IgM staining was highly positive in most of Ig-MPGN biopsies (Supplemental Tables 1 and 3). One patient had two biopsies in 6 months: the first was classified as Ig-

29 g/dl and proteinuria MPGN with similar intensity of IgM and C3, and the second was , Na Na classified as C3G defined by predominant C3 depositions. g, negative; Na, not available. 2 (36) 4 (48) 1 (144) 1 (56) 1 (112) 4 (6) C3andC5b9stainingwasperformedby immunohistochemistry CKD Stage at on six native kidney biopsies (illustrated in Figure 2). C3 staining ; stage 2, GFR between 60 and 89 ml/min per Last Follow-Up (mo) 2 was positive in these biopsies and localized within the basal membrane, in the subendothelial space, or in the mesangium. The C5b9 ned by albuminuria fi renal biopsy staining was positive in the mesangium and the subendothelial space for the isolated anti-C3b–positive patient but negative in the five anti-FB–positive patients tested. ESRD (mo .NSwasde 2 90 ml/min per 1.73 m after Diagnosis) etil;Pos,positive;F,female;Ne . Patient Outcome Two patients were lost to follow-up after diagnosis. Seven of 14 Na No NoNo Yes (24) Yes (120) No No Yes (48) No Yes (12) No Death (3) No No Yes patients with available data received immunosuppressive treatment. The median follow-up was 52 months (range =3–144 15 ml/min per 1.73 m

cation: stage 1, GFR months). One patient with severe thrombopenia died from cere- , fi bral hematoma, and five (five of 14; 35%) were put on dialysis. fi –

IS Relapse Four of ve combined anti-C3b/anti-FB Ab positive patients progressed to severe chronic renal insufﬁciency or ESRD or died within 48 months of diagnosis. Three patients received grafts (pa- ;andstage5,GFR 2 tients 5, 14, and 15). Two of these three patients (patients 5 and 14) had severe recurrence on the graft during the ﬁrst month after

CKD transplantation that resulted in graft loss. Patient 14 with anti-FB Stage Ab lost three kidney grafts due to early recurrence. Graft biopsy analysis from the third graft, performed under preemptive eculi- Renal

Failure zumab treatment, showed an increasing mesangial hypercellular- ity, inﬂammatory cells, occasional double contours, and C3 NS deposits in the glomeruli that increased over time persistently ale; HBV, Hepatitis B virus; Cs, corticosteroid; MMF, mycophenolate mof

dney Disease Outcomes Quality Initiative CKD classi without C5b9 deposits (Supplemental Figure 2). Na Yes Yes 3 NaNa Yes Na Yes Na 5 1 No Yes Yes 2 Cs/MMF/anti-CD20 Yes No Yes Yes 3 Cs/anti-CD20 Yes Yes (65) No No Yes 3 No Yes Yes 4 Cs Yes Yes No 1 Yes YesYes Yes Yes 5 Yes Cs/anti-CD20 4 No Yes Yes No 2 Cs Yes Yes No 2 Cs Yes Yes No 2

Trigger Complement Assessment in Patient Samples Positive Infectious for Anti-FB and Anti-C3b Autoantibodies At the time of the ﬁrst complement assessment, six (six of 15; ; stage 4, GFR between 15 and 29 ml/min per 1.73 m 2 40%) patients had low C3 levels (,660 mg/ml, with normal range from 660 to 1250 mg/L), with a median of 694 mg/L (range =60–1290 mg/L). The level of Bb was above the normal Ig-MPGN C3G Ig-MPGN Ig-MPGN Ig-MPGN C3G Ig-MPGN C3G C3G C3G range in all of the patients’ samples (15 of 15; 100%), with a median of 3517 ng/ml (range =1320–17,288 ng/ml, with nor- , brosis Ig-MPGN mal value 1275 ng/ml). The level of sC5b9 was elevated ﬁ in nine plasma samples (nine of 15; 53%), with a median of lymphoma – ,

Background Ig-MPGN/C3G 444 ng/ml (range =321 2844 ng/ml, with normal value 420

ned by the level of kidney function according to the Ki ng/ml) (Supplemental Tables 1 and 4). fi The patients were tested for complement biomarker analysis. C3 was decreased in three of five patients positive for the Age at Summary of the clinical characteristics of the patients with anti-C3b and anti-FB Abs Onset, yr two Abs, two of three anti-C3b–positive patients, and one of – fi

; stage 3, GFR between 30 and 59 ml/min per 1.73 m seven anti-FB positive patients. The C3 levels were signi - 2 cantly decreased in patients with isolated anti-C3b compared 9 F 9 8 M 63 Alcohol/HBV Ig-MPGN/C3G Yes Yes Yes 5 7 M 7 56 M M 49 32 4 M 40 Myelo 12 M3 F F 31 34 Drug/HBV 66 Anorexia Ig-MPGN Ig-MPGN Yes Yes No 1 Cs/MMF Yes 1 (48) Pos/Pos Pt Sex 1112 M M 34 38 Drug Ig-MPGN 10 M13 55 M 55 Crohn/B 1415 F M 32 18 Table 1. TheCKDstagesarede 1.73 m Pt,patient;NS,nephroticsyndrome;IS,immunosuppressivetreatment;M,m with the controls (Figure 3A). The levels of Bb in plasma were

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Figure 2. Biopsies on native kidney from two patients. Patients with (A1–A3) isolated anti-FB (patient 12) and (B1–B3) isolated anti-C3b (patient 9). (A1 and B1) Trichrome coloration (340), (A2 and B2) C3 staining (340), and (A3 and B3) C5b9 staining (340). significantly higher than in the controls in the three sub- compared with that in sera from healthy controls. FH- groups, without difference between the subgroups (Figure depleted serum served as a positive control (Figure 5). 3B). Soluble C5b9 was increased in all patients positive for anti-C3b and anti-FB Ab (n=5 of 5), two of seven patients Patient IgG Enhanced the C3/C5 Convertase Activity positive for anti-FB, and one of three patients positive for To find out whether the anti-C3b and anti-FB Abs were func- anti-C3b (Figure 3C). tionally relevant, their effect on the activity of the C3 convertase When available, a second plasma sample was analyzed, with was tested using SPR. Purified IgG from samples positive for the interval between the first and last samples ranging from 6 to anti-FB Ab (n=3), anti-C3b Ab (n=2), and both of them (n=3) 96 months. At the last biologic follow-up, three of ten patients bound to the C3 convertase and increased its stabilization had fluid-phase C3 consumption, and four of ten had increased (Figure 6, A and B). The presence of IgG during convertase sC5b9 (data not shown). formation enhanced the assembly of the complex and induced stabilization in six of eight tested patients. Epitope Mapping and Binding Properties of the Anti- Toassess whether IgG from positive patients influenced C3/ C3b and Anti-FB Abs C5 convertase activity, C3bBb convertase was assembled on We analyzed the reactivity of anti-FB– and anti-C3b–positive Ab-coated sheep erythrocytes in the presence of purified IgG IgG against the purified C3 and FB cleavage fragments (Figure from healthy donors and positive patients. IgG (from four 4). Anti-FB IgG recognized the Bb fragment of the protein but positive patients) induced the stabilization of the C3/C5 con- did not recognize the Ba cleavage product in all patients. vertaseformedonsheeperythrocytes(fourof15;26%)(Figure Isolated anti-C3b IgG recognized C3b and C3c but did not 6C). After anti-C3b/anti-FB Ab depletion from total IgG, recognize the C3d cleavage fragment. In the group of patients the percentage of the residual C3 convertase stabilization of with double positivity, anti-C3b IgG recognized C3b but did the same quantity of patient IgG dropped from 35% to 10% not recognize C3c. Weak reactivity toward C2 and C4 was for patient 6 and from 50% to 220% for patient 9, whereas detected in two patients, and weak reactivity toward C5 was the percentage of C3 convertase stabilization remained un- detected in one patients but not against properdin. changed for the C3Nef IgG subjected to the same procedure (Figure 6D). Positive Sera Induced C3 Deposition on Cells We assessed the capacity of the anti-C3b/anti-Fb Ab to influ- Anti-FB–Positive IgG Induces Fluid-Phase Complement ence the regulation of the AP on a cell surface expressing the Activation natural human complement regulators MCPand DAF (but not The interaction of anti-FB–positive IgG with the C3 conver- CR1) and binding factor H (FH). Serum samples were available tases C3bBb and C3(H2O)Bb was studied by SPR. The from four patients (one positive for anti-C3b, two positive four tested purified anti-FB–positive IgG bound to both con- for anti-FB, and one positive for anti-C3b/FB). When resting vertases, and the binding was stronger to the C3bBb in endothelial cells were incubated with the patients’ sera, C3 three patients and the C3(H2O)Bb in one patient (Figure deposition on the cell surface was significantly increased 7A, patients 13 and 14).

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Figure 3. Complement activation biomarker assessments. Plasma levels of (A) C3, (B) Bb, and (C) sC5b9 of 15 anti-C3b/anti-FB–positive plasma samples and 30 healthy controls. *P,0.05; **P.0.01; ***P.0.001. Ctr, control cohort of C3G without anti-C3b and anti-FB autoantibodies; HD, healthy donor.

To analyze the capacity of patient IgG to activate comple- the C5 convertases. The levels of released Bb and C3a were ment in fluid phase, six anti-FB–positive IgG samples were increased in the presence of patient IgG compared with IgG incubated in normal serum. The quantification of cleavage from healthy donors. No difference was observed for the for- fragments reflects the activation of the C3 convertase and mation of sC5b9 (Figure 7B).

Figure 4. Epitope mapping of the anti-FB and anti-C3b IgG. Heat map showing the binding levels of patient IgG to the different antigens. The intensity of the binding is represented by red shading. FP, factor properdine.

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available biopsy specimens were diagnosed as Ig-MPGN, because the C3 intensity of the staining evaluated by IF was identical to the other immune reactants. Our report highlights a new link between the histologic pattern of Ig-MPGN and the AP. Whatever the pathway involved in the initia- tion of the complement activation, the AP may play a major role in the exacerbation of the clinical phenotype of the Ig-MPGN. Interestingly, one half of our patients with anti-C3b/anti-FB Ab had a clinical his- Figure 5. C3 deposition on endothelial cells. (A) Histogram of the C3 deposition on the tory of infection and in particular, six of surface of resting endothelial cells incubated with the sera from patients 3, 9, 12, and 14, seven patients with Ig-MPGN and avail- fl which was measured by ow cytometry. Three serum samples from healthy donors served able clinical data. Infections are a well as controls. FH-depleted serum was used as a positive control for AP dysregulation. A known cause of Ig-MPGN.9,13 The patients representative histogram is shown. (B) QuantificationoftheC3depositiononresting presented a background of cancer, intra- endothelial cells. *P,0.05. iso, isotype; dpl, depleted; RFI, ratio fluorescency/intensity. venous drug addiction, alcoholism, or anorexia—well described predisposing Anti-C3b Autoantibodies Perturbed the Regulation of factors to infections. This association between infection and the C3 Convertase anti-C3b/anti-FB Ab-related GN was not fortuitous: the fre- To determine whether anti-C3b Ab binding altered the inter- quency of infection was significantly lower in patients negative actionofC3btoFHandCR1,C3bwasimmobilizedontheSPR for anti-C3b and anti-FB with C3G (23%) and Ig-MPGN chip, and IgG purified from patients or healthy donors was (10%). Although the origin of the autoantibodies remains un- flowed across the surface followed by purified FH and CR1. known, they may be linked to certain infectious events, be- The presence of patient IgG did not modify the interaction of cause we found similarities between the coding sequences of FH with C3b but resulted in a decreased capacity of C3b to C3 and Streptococcous epidermidis, Streptococcous capitis, bind to CR1 (Figure 8, A and B). FH dissociated the conver- K. pneumoniae, and Shigella flexneri, although not with CFB tase formed in the presence of patient IgG as normal IgG (data not show). Infection may also initiate glomerular inflam- (Figure 8C). mation with the accumulation of C3 fragments, leading to the immune response against complement proteins. Two of our patients had a history of hepatitis B viral in- DISCUSSION fection. Interestingly, a patient who had a history of hepatitis B and a complete CFH deficiency on the background developed In this study, we report for the first time a series of 15 patients an MPGN type 1–associating nephrotic syndrome and de- with anti-C3b and anti-FB Ab diagnosed with C3G or Ig- creased renal function.14 MPGN and show that these autoantibodies interfere with the Despite the severity of the inflammation within the glomer- activity of the C3 and C5 convertases of the AP of complement. uli at onset, the outcomes of our patients were heterogeneous, Wefound anti-C3b and/or anti-FB Abs in fewer than 10% of with minor persistent urinary abnormalities (four of 13), ne- our large series of patients with C3G and one half of a small phrotic syndrome with varying degrees of renal impairment cohort of patients with Ig-MPGN. The diagnosis of C3G is on (four of 13), and ESRD within 3 years of diagnosis (three of 13). the basis of a glomerular injury with membranoproliferative The prognosis of patients positive for anti-C3b and anti-FB GN (MPGN) patterns or nonspecific alterations sharing seems to be worse, because four of five patients with double C3-dominant glomerular staining. A better understanding positivity in our series died or progressed to CKD stage 4 within of the pathogenesis of MPGN has led to reclassification 2 years of diagnosis. Clinical recurrence after renal transplantation into Ig-MPGN and complement-mediated MPGN.5,9 In occurred in two of three patients. To our knowledge, only three Ig-MPGN, the Ig and complement-positive staining by IF sup- patients with anti-FB Ab have been reported in the literature port the hypothesis of an immune complex–mediated disease to date.7,8 Of these, one patient developed ESRD 3 years with activation of the classic complement pathway. However, after diagnosis. The clinical characteristics have similarities growing evidence underlines the potential role of AP in Ig- with postinfectious GN in the elderly. The patients had the MPGN. Two patients with homozygous CFH deficiency, the same immunocompromised background and high rate of severe main regulatory protein of AP, have been diagnosed with Ig- renal insufficiency with inflammatory cells in the glomerulus.15 MPGN.10,11 C3Nef was positive in 50% of patients in two large A recent study reported that a treatment of corticosteroids Ig-MPGN cohorts.4,12 Applying the current histopathologic and mycophenolate mofetil could be beneficial in C3 GN.16 definition of MPGN, ten of 14 patients in our series with Anticomplement drugs, such as anti-C5, have also been shown to

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In vitro, the addition of positive anti-FB IgG in normal serum increased the release of C3a and Bb but did not enhance the gen- eration of sC5b9. In vivo,C3andC5b9 staining in kidney biopsies revealed massive C3 staining, without detection of deposited C5b9. These results suggest a particular functional activity of these Abs, acting primarily on the level of the C3 convertase and not proceeding toward the C5 convertase. The relevance of the mechanism is illustrated by the history of patient 14. She received three grafts, because the first two were lost from recurrence. She failed to respond to a prophylactic anti- C5 therapy and lost her third graft. It would have been more effective in this patient to use an anti-C3 to block the C3 convertase. In the presence of anti-C3b–positive IgG, no effect was detected in the binding of FH to C3b, suggesting that the observed increased activity of the C3 convertase in plasma (low C3 level in patient plasma) and on the cell surface (C3 deposition on kidney biopsy and endothelial cells) is due to a Figure 6. Effects of positive patient IgG on the C3/C5 convertase of the AP. (A) Sensorograms illustrating real-time C3 convertase in the presence of positive patient direct overactivation and is not due to IgG. After coupling C3b on the biosensor chip, we flowed a mix of FB, factor D, and loss of regulation by FH. Evidence for the IgG across it. The sensorograms of the IgG of one patient from each group (combined overactivation of the C3 convertase in the anti-FB/anti-C3b–,anti-FB–, or anti-C3b–positive patients) and two IgG from healthy presence of anti-C3b Ab also comes from donors (NHIgGs) and in the absence of IgG were reported as examples. C3NeF-positive the increased Bb levels in the patient IgG was used as a positive control. IgG from patient 5 (combined anti-FB/anti-C3b Ab), plasma. However, only one of three pa- patient 13 (anti-C3b), and patient 8 (anti-FB) enhanced the formation of the C3 con- tients positive for anti-C3b Ab had in- vertase. FD, factor D. (B) Calculated off rate of the C3 convertase formed in the presence creased soluble C5b9 levels, suggesting of patient IgG. A lower off rate than controls signals an increased stabilization of the that the degree of dysregulation at the level complexes. The effect of the IgG seemed to be related to the stabilization of the con- of C5 convertases is inconstant in vivo.On vertase, because the off rate of the triple complex is lower in the presence of IgG from the contrary, in the presence of anti-FB Ab, our patients compared with IgG from healthy donors. Arrows indicate the Kd relative to the sensorograms shown in A. (C) Effect of positive IgG samples on the C3/C5 convertase the anti-C3b Ab enhanced the terminal stabilization. C3 convertase was assembled on the surface of sheep erythrocytes by in- pathway activation as shown by the sC5b9 cubating C3b-bound sheep erythrocytes with FB, factor D, and patient IgG. The addition levels, which increased in all of the patients’ of rat serum triggered lysis of cells, in which C5 lytic sites were still present after spon- plasma samples. This discrepancy suggests taneous decay. The results are expressed in percentages of residual C3/C5 convertase that there may be synergy between anti-FB sites. (D) Effect on the C3/C5 activation of the depletion of anti-C3b and anti-FB Ab from and anti-C3b IgG on their target antigens two positive samples (patients 6 and 9). Depleted anti-C3b and anti-FB Ab IgG from IgG on the C3 convertase to induce the switch positive reduced the percentage of the C3/C5 convertases compared with total IgG, of the enzymatic activity toward the C5 showing that they are responsible for this activity. The C3NeF sample, used as a positive convertase. The epitope mapping of control and treated as the anti-C3b IgG, maintained full activity after treatment. NH, theanti-C3bAbalsosuggestsadifferent normal human; Pt, patient; RU, response unit. P,0.05. binding area on the convertase. Isolated anti-C3b Ab recognized the C3b and C3c be effective in C3G or Ig-MPGN with highly soluble C5b9 or fragments, whereas in presence of anti-FB Ab, the anti-C3b Ab C5b9 depositions.17 In our series, all of the patients with infec- recognized C3b and in one case, C3d but did not recognize tious disease received appropriate antibiotic treatment, and im- C3c. However, in both types of anti-C3b Ab, the epitopes of munosuppressive drugs were used in seven of 15 (46%) patients. the anti-C3b Ab are predominantly located outside the ANA We mapped the binding sites of the autoantibodies on their and TED domains of C3, and hence, they will not interfere target. Interestingly, the anti-FB autoantibodies bind to the with the covalent binding of C3b to the cell surface and will not area exposed on the Bb fragment in the C3bBb complexes. prevent the proinflammatory action of C3a.18

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inhibited interactions of FH and CR1 with C3b and was correlated with the disease activity. Anti-C3b Ab found in patients with C3G displays distinct properties compared with those found in patients with lupus nephritis, and this study showed the heteroge- neity of autoantibodies reacting against C3b. Our study is limited due to the relatively small number of patients and by the quantity of available plasma samples to perform functional studies for all patients. However, it is the ﬁrst study to evaluate the presence of anti-C3b and anti-FB Ab in patients with Ig-MPGN. The presence of anti-FB and anti-C3b Ab needs to be evaluated in a larger cohort and in particular, PIGN diagnosed in adults. The identiﬁcation of these Abs could guide patient management in this setting, giving rise to a new treatment ap- proach with anti-infectious, immunosuppressive, or anticomplement agents.

Figure 7. Overall functional effect of the anti-FB–positive IgG. (A) Sensorograms illustrating real-time interaction between two examples of positive anti-FB IgG (patients 13 and 14) CONCISE METHODS with C3(H2O)Bb (gray line) or C3bBb convertase (black line). Patient IgG is able to bind to its target, even when it is complexed in the convertase. (B) Complement activation after addition of purified positive anti-FB IgG to normal human serum or IgG from healthy donors Cohort Description One hundred forty-one patients with Ig-MPGN (NHIgG). Release of C3a, Bb, and sC5b9. Statistical analyses were performed by GraphPad software using a Mann Whitney test. *P,0.05.FD,factorD;NH,normalhuman;RU,re- without coexisting disease (n=23) or C3G sponse unit. (n=118) referred to the Immunology Laboratory at Hôpital Georges Pompidou (Paris, France) for Some anti-C3b/anti FB Abs enhance the convertase forma- complement assessment were enrolled in the study. At diagnosis, the tion and stabilize the C3 convertase as shown by SPR and median age was 30612 years old, 58% (75 of 129) had a nephrotic functional assay. We showed that the functional effect is due syndrome, and 35% (65 of 129) had acute renal failure. Forty percent to the Ab, because anti-C3b/anti-FB–depleted IgG did not in- of patients had low C3 levels (Supplemental Table 1). fluence the dissociation of the C3 convertase. Therefore, some All of the clinical and biologic data from the patients were retro- anti-C3b/anti-FB Abs share the same functional consequence spectively collected. Renal failure was defined as a creatinine plasmatic with C3NeF. level .150 mmol/L or a clearance of creatinine by the Modification of Nevertheless,we cannot exclude thepossibility that the anti- Diet in Renal Disease equation ,60 ml/min at the time of diagnosis. C3b and anti-FB Ab may not stabilize the convertase directly Renal failure persisting for .3 months was considered chronic renal but rather, may change the binding characteristics between failure. The five stages of CKD are on the basis of eGFR: stage 1, .90 C3b and FB before convertase formation and/or influence ml/min; stage 2, between 90 and 60 ml/min; stage 3, between 59 the susceptibility of FB to be cleaved. and 30 ml/min; stage 4, between 15 and 29 ml/min; and stage 5, More interestingly, we showed that the anti-C3b Ab per- ,15 ml/min. Nephrotic syndrome was defined by albuminuria turbed the binding of CR1 to the C3b. CR1 is a specific com- ,30 g/L and proteinuria .3g/24h.Classification of histopathology plement regulator expressed by podocytes in the kidney. A patterns follows the recent C3G classification.6 Patients were classi- growing body of data suggests that CR1 may be involved in fied Ig-MPGN or GC3 on the basis of pathologic IF. C3G is charac- the physiopathology of C3G. A recently described C3 mutation terized by exclusive or predominant glomerular C3 deposits with or in a familial form of C3 GN was associated with decreased CR1 without dense deposits. Kidney biopsies with a similar intensity or binding, whereas the C3 mutations found in atypical hemolytic less than two orders of magnitude difference between Ig and C3 uremic syndrome did not alter CR1 interaction.19,20 CR1 staining in the glomeruli were classified as Ig-MPGN. All renal bi- plays a major role in the elimination of the micro-organisms opsies from anti-FB– and anti-C3–positive patients were reviewed and the immune complexes. The interference of the anti-C3b by a renal pathologist. The renal biopsy report from the local renal IgG in the C3b-CR1 interaction may participate in the phe- pathologist was used for the control cohort. notypic expression of the disease. Anti-C3b Abs have been All of the patients had previously provided informed written con- identified in patients with lupus who experience nephritic sent for gene complement screening, complement assessment, and flares.21,22 In this context, IgG containing anti-C3b Ab renal biopsies.

1610 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1603–1613, 2017 www.jasn.org CLINICAL RESEARCH

and 0.05% Tween 20 and added to the wells for 1 hour. Bound IgG was revealed by an anti-human IgG Ab conjugated with HRP (Southern Biotech) diluted 1:1000 in PBS and 0.05% Tween 20 followed by the TMB substrate system. A titration curve was included on all plates in this assay using a positive plasma sample. A 1:1000 dilution of this sample was given an AU of 1000. The OD value was converted into AUs using a standard curve of reference positive plasma. A cutoff was used to qualitatively differentiate between positive and negative results. The cutoff value of positivity was determined by taking the mean +3 SD of titers using plasma samples from 50 healthy donors (130 AU/ml for anti-C3b and 170 AU/ml for anti-FB Abs). The anti-CR1 and antifactor I were screened by ELISA. Brieﬂy, 10 mg/ml soluble CR1 or factor I wascoatedontheplates,andsimilarstepswere followed as for the anti-FB anti-C3b ELISA detection. For the epitope mapping, we tested the re- Figure 8. The anti-C3b autoantibodies decreased the binding of the regulator CR1 but activity against different C3 fragments (C3a, did not decrease the binding of FH to C3b. (A) Binding of CR1 to C3b pre-exposed to C3b, C3c, and C3d), FB fragments (Ba and patient IgG. (B) Binding of FH to C3b pre-exposed to patient IgG. (C) Dissociation of Bb), or other complement proteins, such as the C3 convertase formed in presence of patient IgG by FH. IgG from a C3NeF-positive C4, C5 C2, and properdin (Complement Tech- patient served as a positive control for a convertase, resistant to decay by FH. Anti- nologies, Tylor, TX), by ELISA. All samples were C3b–positive IgG is in black, IgG from healthy donors is in light gray, and dissociation tested simultaneously. Values obtained from the in the absence of IgG is shown as a dashed line. IgG from a C3NeF-positive patient, tested samples were displayed in a heat map, in stabilizing the convertase and making it resistant to FH decay, is shown as a dark which the intensity of the binding is represented dashed line. Pt, patient; RU, response unit. by red (positive) to white (negative) shading.

Immunohistochemistry for C3 and C5b9 Staining on IgG Purification fi Renal Biopsies Puri cation of IgG was performed from the plasma of patients or C3 and C5b9 were analyzed for all of the patients with available healthy donors using Protein G Beads (GE Healthcare) as recom- paraffin-embedded native kidney or graft biopsy sections using poly- mended by the manufacturer. Anti-FB or C3b IgG was depleted clonal rabbit anti-C3d (Abcam) and anti-C9 neoepitope (gift from from the total IgG as follows. CNBr Resin (GE Healthcare) was cou- PaulMorgan, Cardiff, United Kingdom) as described.17 The streptavidin- pled with FB or C3b following the manufacturer’s instructions. Total biotin-peroxydase complex system was used for signal amplification. purified IgG was incubated for 1 hour at 4°C with the resin. After time Amino-9 Etylcarbazole and 3–39-Diaminobenzidine Biosystems Visions for binding, we applied the resin to a column and collected the flow Kits were used for the anti-C3d and anti-C5b9, respectively. Peritumoral through containing IgG without IgG specific for FB or C3b. We kidney tissue served as a negative control, and class 4 lupus nephritis washed the column and finally, collected purified anti-FB or anti- served as a positive control. C3b IgG by acidic elution with Gly-HCl Ph 2.8. To ensure that the depletion had occurred, we performed an ELISA coating FB or C3b Complement Assays and adding total IgG or IgG present in the flow through. We revealed Complement assessment was performed on EDTA plasma samples at IgG binding as for the ELISA screening already described. the immunology laboratory of the Georges Pompidou European Hos- pital. The Bb and soluble C5b9 were measured by ELISA (Quidel). Characterization of the Interaction of the Anti-FB and Complete exon sequencing of CFH, MCP,andCFI genes was undertaken Anti-C3b Autoantibodies with Their Antigens by SPR using direct sequencing analysis as described. All of the patients had The interaction of the anti-FB and anti-C3b autoantibodies with previously provided informed written consent for gene screening.23 immobilized FB, Bb, C3, and C3b was analyzed in real time using ProteOn XPR36 SPR equipment (BioRad, Marne-la-Coquette, ELISA for Identification of Anti-FB and Anti-C3 Abs France) as described.21 The proteins (Complement Technologies) The screening for anti-FB and anti-C3b Ab was performed as pre- were covalently immobilized on a GLC Sensor Chip (BioRad) follow- viously described.21,22 Briefly, microtiter ELISA plates were coated with ing the manufacturer’s procedure. Protein G–purified IgG from the 10 mg/ml C3/C3b or FB (Calbiochem) in PBS for 1 hour followed by a positive patients or healthy donors (at 100 mg/ml) were injected for blocking by PBS and 0.25% Tween 20. Plasma was diluted 1:100 in PBS 300 seconds in PBS and 0.005% Tween 20 containing running buffer.

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The dissociation was followed for 300 seconds. The background sig- Effect of the Anti-C3b Autoantibodies on the nal from the noncoated surface (the interspots of the GLC Biosensor Interaction of FH and Soluble CR1 with C3b Chip; BioRad) reflecting the background binding was subtracted as Patient or healthy donor IgG was injected at 300 mg/ml over a C3b- recommended by the manufacturer. Because the exact proportion of coated SPR chip in PBS with 0.005% Tween 20 as described before. the anti-C3b and anti-FB Ab in the patients’ IgG was unknown, After 300 seconds of dissociation, CFH (Complement Technologies) kinetic analyses that determine the precise binding constants and at 50 mg/ml or soluble CR1 (R&D) at 10 mg/ml was injected. Back- affinity were unfeasible. To evaluate the stability of the formed com- ground signal from the interspots was subtracted by ProteOn Manager plexes, the dissociation rate (off rates) was calculated by ProteOn Software. The CFH or sCR1 injectionwas aligned at 0 RUat the moment Manager Software. of injection to evaluate the effect of IgG on FH and sCR1 binding. Toevaluate the abilityof FH to dissociate a C3 convertase formed in fi Effect of the Anti-FB and Anti-C3b Autoantibodies on the presence of patient IgG, rst FB, factor D, and IgG were injected as the Formation of a C3 Convertase and C5 Convertase described above. After 300 seconds of spontaneous dissociation, CFH The effect of positive IgG on the formation of the C3 convertase was was injected to accelerate the decay. IgG from healthy donors or buffer studied by SPR. Protein G–purified IgG from positive patients, served to evaluate normal decay. IgG from one C3Nef-positive patient C3NeF-positive samples or healthy controls at 300 mg/ml, or buffer with DDD served as positive control, which stabilized the convertase were mixed with FB at 80 mg/ml and factor D at 8 mg/ml (Comple- and rendered it resistant to decay by FH. ment Technologies) and injected for 300 seconds over a C3b-coated GLC chip in 10 mM Hepes, 75 mM NaCl, 1 mM MgCl, and 0.005% Tween 20 (pH 7.4) running buffer. The dissociation was followed for ACKNOWLEDGMENTS 300 seconds. The background signal from the noncoated surface (interspots) was subtracted. We used hemolytic sheep erythrocyte assay We thank all pathologists for kidney biopsies analysis: Marion Rabant to detect the capacity of purified IgG to stabilize a membrane-bound (Hôpital Necker, Paris, France), Laurent Daniel (Hopital La Timone, C3/C5 convertase (C3bBb/C3bBbC3b). Marseille, France), Magali Colombat (Hôpital Foch, Suresnes, France), The functional assay occurs in three steps: (1) the formation of the Isabelle Brocheriou (Hôpital Tenon, Paris, France), and Arnaud Fran- AP C3 convertase with sheep erythrocytes bearing C3b with FB çois (Hopital Bois Guillaume, Rouen, France). We also thank Prof. Paul (Complement Technology) and factor D (Sigma-Aldrich), (2)the Morgan (Cardiff, United Kingdom) for the gift of anti-C9 neoantigen. interaction between sheep erythrocytes bearing the C3bBb and a This work was supported by European 7th Framework fixed concentration of 400 mgIgGpurified from healthy donors Programme grant 2012‐305608 (EURenOmics) and Fondation du and patients during 20 minutes to evaluate the ability of IgG to sta- Rein (FDR) grant Prix 2012 FDR. bilize the convertase, and (3) the formation of the C5 convertase and This work was presented as an abstract at the 2015 15th European the lytic complex from the residual convertase sites with the addition MeetingonComplementinHumanDiseaseinUppsala,Sweden, of 0.3 ml rat serum diluted 1:15 in GVB/EDTA as a source of C3/C9 June 27–30, 2015. for 45 minutes at 37°C. Because rat FH is four times less effective than human FH in dissociating the human C3 convertase, rat serum is added in the last step to provide an effective C3/C5 convertase. After DISCLOSURES addition of l.5 ml isotonic saline, the extent of hemolysis was determined, and the average number of hemolytic sites per cell (Z)was V.F.-B. has received lecture, consultancy, and travel honoraria from Alexion Pharmaceuticals, Inc. M.L.Q. has received consulting fees from Alexion Phar- calculated. The amount of hemolysis is dependent on the amount of maceuticals, Inc. convertase stabilized by IgG. In the presence of IgG from healthy donors, the residual convertase after 20 minutes of decay was ,20% of the initial convertase. The same experimental procedures REFERENCES were used for the C3NeF screening.24

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