Temporal IgG Subtype Changes in Recurrent Idiopathic Membranous Nephropathy

American Journal of Transplantation 2016; 16: 2964 2972 Wiley Periodicals Inc. Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13806 Temporal IgG Subtype Changes in Recurrent Idiopathic Membranous Nephropathy A. G. Kattah 1,, M. P. Alexander 2,, A. Angioi 3, A. S. De Vriese 4, S. Sethi 2, F. G. Cosio 1, E. C. Lorenz 1, L. D. Cornell 2 and F. C. Fervenza 1, * 1 Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 2 Department of Anatomic Pathology, Mayo Clinic, Rochester, MN 3 Universita degli Studi di Cagliari, Sardinia, Italy 4 Division of Nephrology, AZ Sint-Jan Brugge-Oostende AV, Brugge, Belgium *Corresponding author: Fernando C. Fervenza, fervenza.fernando@mayo.edu These two authors contributed equally to this manuscript. Determination of the IgG subtypes within the immune deposits in membranous nephropathy (MN) may be helpful in the differential diagnosis. IgG4 is the predominant subtype in idiopathic MN and recurrent MN, while IgG1, IgG2, and IgG3 subtypes are more common in secondary MN and de novo disease in the allograft. The temporal change of IgG subclasses in individual patients and its correlation with clinical variables have not been studied. We reviewed all posttransplantation protocol and indication biopsies (49) in 18 patients with recurrent MN who underwent transplantation at our center between 1998 and 2013 and performed IgG subtyping (IgG1 4). We tested serum for M-type phospholipase A 2 receptor (PLA 2 R) autoantibodies or performed PLA 2 R antigen staining on the kidney biopsy. IgG4 was the (co)dominant IgG subtype in 10 of 14 biopsies at the diagnosis of recurrence regardless of PLA 2 R association. In 8 of 12 transplantations with serial biopsies, the (co)dominant subtype did not change over time. There was a trend toward IgG1 and IgG3 (co)dominance in biopsies >1 year from recurrence and more IgG1 (co)dominant subtyping in the setting of more-advanced EM deposits. Treatment with rituximab did not affect the IgG subtype. In conclusion, the dominant IgG subtype did not change over time in recurrent MN. Abbreviations: CTLD1, C-type lectin domain 1 epitope; CTLD7, C-type lectin domain 7 epitope; CysR, cysteine-rich epitope; EM, electron microscopy; MN, membranous nephropathy; PLA 2 R, phospholipase A 2 receptor; THSD7A, thrombospondin type 1 domain containing 7A Received 12 February 2016, revised 17 March 2016 and accepted for publication 24 March 2016 Introduction Idiopathic membranous nephropathy (MN) is an organspecific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens. IgG4 is the predominant subtype of Ig found in the immune complexes in idiopathic MN, as opposed to IgG1, IgG2, and IgG3, which are found in cases of MN secondary to other disease processes or conditions, such as lupus erythematosus, malignancy, infection, or drugs (1 3). Similarly, IgG4 has been shown to be the dominant or codominant subtype in cases of recurrent MN after kidney transplantation, versus IgG1 in de novo or atypical posttransplantation MN (4). The predominant target antigen in primary MN is the M-type phospholipase A 2 receptor (PLA 2 R), and anti- PLA 2 R antibodies are found in the serum of 70% of patients with active idiopathic MN (5 10). The anti-pla 2 R antibody is mainly of the IgG4 subtype in native kidney disease (5). The autoantibody to thrombospondin type 1 domain containing 7A (THSD7A), the antigenic target in approximately 10% of patients with idiopathic MN, is also chiefly of the IgG4 subtype (11). Huang et al reported that in early MN, defined as the presence of stage I deposits on electron microscopy (EM) examination, IgG1 was the dominant subtype, whereas IgG4 was dominant in all later stages (12). Although no serial biopsy samples were studied, the authors suggested that the apparent temporal change in IgG subtypes may be due to immunoglobulin class-switching. In a few reported cases, anti-pla 2 R antibodies have recognized different epitopes over time in the same individual, a phenomenon known as epitope spreading (13). Identifying changes in the IgG subclass in recurrent MN may be indirect evidence of epitope spreading in the allograft, as well. The availability of posttransplantation protocol biopsy samples at our institution provides a unique opportunity to describe histologic features of recurrent MN from the earliest stages, follow histologic changes over time, and correlate these features with clinical data. In this study, we performed IgG subtyping on all available 2964

IgG Subtypes in Recurrent MN posttransplantation biopsy samples of patients with recurrent MN, to delineate the temporal changes of IgG subtypes in recurrent MN. Our goals were to identify the earliest subtype seen in recurrent MN and to describe the subtype change, if any, over time. In addition, we searched for a correlation of clinical parameters with IgG subtype changes, to add to the understanding of the pathogenesis of recurrent MN. Methods Samples Patients with idiopathic MN who underwent kidney transplantation at the Mayo Clinic between 1998 and 2013 underwent protocol biopsies at 4 and 1, 2, and 5 years posttransplantation, in addition to indication biopsies. Frozen tissue specimens from posttransplantation biopsies are saved for 7 years. Patients who underwent a biopsy within that time frame were included in the study. We selected biopsies with evidence of early recurrent MN by routine immunofluorescence, as defined as positive IgG in a granular, capillary wall pattern (14). The study was approved by the Mayo Clinic Institutional Review Board (15-003990). IgG subtyping We stained excess frozen tissue from the available biopsies for IgG subtypes (1 4). The renal pathologist (M.P.A.) reviewed each biopsy sample in a blinded fashion and assigned each IgG subtype a score for intensity (trace, 1 3+). Next, each biopsy sample was categorized based on the dominant subtype, when one subtype had the highest intensity, or based on the codominant subtype, when two or more subtypes had the highest intensity. Tissue staining for PLA 2 R Staining for the PLA 2 R antigen was performed as described previously (5,15). In brief, 3-lm serial sections were cut from formalin-fixed paraffin blocks and deparaffinized for antigen retrieval. PLA 2 R was detected in these paraffin-embedded sections by using rabbit polyclonal anti-pla 2 R antibodies (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:250, followed by polyclonal swine anti-rabbit IgG (Dako, Carpinteria, CA) at a dilution of 1:100. Each case was run with a positive and a negative control. The stain was evaluated with the use of standard immunofluorescence. A case was determined to be positive if there was positive granular capillary loop staining in the glomeruli and negative if there was no staining in the glomeruli. EM and immunofluorescence staining EM stages were determined by using the Ehrenreich Churg classification. When deposits were described as I II or II III, they were classified as the more-advanced stage. Stage III deposits with evidence of resorption were classified as stage IV. Biopsies with features of recurrent MN by immunofluorescence, but without EM deposits, were designated as having stage 0 deposits. A full immunofluorescence panel was routinely performed on all biopsy samples and included IgG, IgA, IgM, C1q, C3, and C4d, all recorded on a trace to 3+ scale. Disease characteristics Clinical information, including donor type, induction agent, and treatment for recurrence, was retrieved from the medical record. Early recurrence was defined as histologic recurrence within 6 of transplantation. PLA 2 R-associated MN was defined as a positive PLA 2 R staining in a posttransplantation biopsy with evidence of recurrence or the presence of anti-pla 2 R autoantibodies in the serum pretransplantation or posttransplantation. Patients with documented recurrent MN on biopsy with consistently negative antibodies and tissue staining were considered to have non PLA 2 R-associated MN. Statistical analysis Dominant and codominant, henceforth referred to as (co)dominant, subtypes were grouped for the analysis, so the number of samples in each category (IgG 1 4) may sum to more than the total number of samples. We used the Fisher exact test to compare differences in categorical variables, and set a two-tailed a of.05. To evaluate the relationship between IgG (co)dominant subtypes and EM stages, we used the Cochran Armitage test for trend, treating EM stage as an ordinal variable. The relationship between C4d and IgG subtype was assessed by treating the intensity of each as continuous variables and evaluating correlation with the use of the Spearman coefficient. All analyses were performed with JMP statistical software, version 1. Results Number of samples Of the 62 patients identified with recurrent idiopathic MN, 18 patients (19 transplants) had posttransplantation biopsies with sufficient tissue to perform IgG subtyping. Among those 18 patients, there were 79 posttransplantation biopsies with recurrent MN, but 30 had no glomeruli left for additional staining, leaving a total of 49 biopsies to review. Seven patients had 1 posttransplantation biopsy available for subtyping (patients 12 18); the remainder had at least two biopsies for review. Patient and recurrent disease characteristics The clinical characteristics at kidney transplantation and at recurrence are listed in Table 1. Thymoglobulin was the induction agent in 16 of 19 transplants; basiliximab and alemtuzumab were used in the remaining three transplants. The median (interquartile range) time to recurrence was 4 (2 12), and 14 of 19 had early recurrence (<6 posttransplantation). The median (interquartile range) levels for serum creatinine, serum albumin, and 24-h urine protein at the time of initial histologic recurrence were 1.7 mg/dl (1.4 2.2 mg/dl), 4.1 g/dl (3.9 4.3 g/dl), and 0.44 g/24 h (0.18 1.44 g/24 h), respectively. Recurrent MN was diagnosed on the basis of protocol biopsy results, as opposed to indication biopsy results, in 14 of 19 cases and was treated with rituximab in 10 of 19 of cases. Testing for PLA 2 R-associated disease Fourteen patients were part of a previous study that analyzed the serum of patients with recurrent MN, in which 10 of 14 had a positive anti-pla 2 R antibody in the pretransplantation or posttransplantation serum (16). In the four patients with no serum available, excess renal tissue was stained for the PLA 2 R antigen, which was positive in three cases. Taken together, 13 of 18 patients had PLA 2 R-associated MN as determined either by anti- PLA 2 R antibody in the serum or by PLA 2 R antigen in the renal tissue. American Journal of Transplantation 2016; 16: 2964 2972 2965

Kattah et al Table 1: Patient, transplant, and recurrent disease characteristics Patient number Age at Tx/sex Donor type Induction agent Time to recurrence () Treated with RTX/ after Tx PLA2R () Rejection/ after Tx No. of biopsies (rmn +) Affected Tx/No. of Proteinuria Tx (total) 1 scr (mg/dl) 2 Alb (g/dl) 2 (g/24 h) 2 1 50/M LURD Alemtuzumab 0.5 No + ACR/12 6 1/1 1.7 3.5 0.75 2 44/M DD Thymoglobulin 0.5 Yes/0 + AMR/22 4 3/3 2.2 4.3 9 3 3 54/M LRD Thymoglobulin 2 No + No 3 1/2 2.2 3.5 0.43 3 4 LRD Thymoglobulin 4 No No 3 2/2 1.8 4.1 0.18 4 51/M LURD Alemtuzumab 4 No + No 4 1/2 1.3 0.15 5 55/F LURD Thymoglobulin 3 No No 2 1/1 1.5 4.3 0.54 6 70/F LRD Thymoglobulin 53 No + No 2 1/1 1.4 3.9 0.58 7 59/F LRD Thymoglobulin 2 No No 3 1/1 1.1 4.6 0.10 8 27/F LRD Thymoglobulin 0.3 Yes/31 + ACR/41 6 1/1 1.4 4.4 0.43 9 66/M LRD Thymoglobulin 4 Yes/21 and 46 No 5 1/1 3.6 3.1 10 63/M LURD Thymoglobulin 4 Yes/32 and 97 + No 2 1/1 2.1 0.4 11 55/M LRD Thymoglobulin 2 Yes/4 + No 2 1/1 2.5 2.5 0.44 12 28/M DD Thymoglobulin 4 No No 1 1/3 2.3 4.6 0.21 13 65/M LRD Thymoglobulin 4 Yes/3 + No 1 1/1 1.4 4.3 2.9 14 40/M LRD Unknown 132 Yes/151 + No 1 1/2 4.3 3.9 Alb/Cr 2.6 5 15 43/M DD Thymoglobulin 63 Yes/63 No 1 1/4 1.7 4.2 0.18 16 48/M LURD Thymoglobulin 111 Yes/112 + ACR/0 1 1/1 0.9 4.3 2.6 17 52/F LRD Basiliximab 12 No + No 1 1/1 1.4 3.9 1.4 18 47/M LURD Thymoglobulin 4 No + No 1 1/2 1.8 4.1 0.5 M, male; F, female; LRD, living related donor; LURD, living unrelated donor; DD, deceased donor; AMR, antibody-mediated rejection; ACR, acute cellular rejection; Tx, transplant; rmn, recurrent membranous nephropathy; PLA2R Ab, phospholipase A2 receptor antibody; RTX, rituximab; SCr, serum creatinine at time of recurrence; Alb, serum albumin at time of recurrence. 1 The Tx reviewed of the total number of transplants the patient received. 2 Values recorded at the time of the first biopsy available, diagnostic for rmn. 3 First Tx, recurrent disease. 4 Second Tx, recurrent disease. 5 Urine albumin/cr ratio. 2966 American Journal of Transplantation 2016; 16: 2964 2972

IgG Subtypes in Recurrent MN Table 2: Dominant IgG subtypes, electron microscopy deposit stages, and C4d from the time of first histologic diagnosis to time of last follow-up Diagnosis <12 1 2 years 2 3 years 3 4 years 4 5 years >5 years Patient PLA 2 R Subtype EM C4d Subtype EM C4d Subtype EM C4d Subtype EM C4d Subtype EM C4d Subtype EM C4d Subtype EM C4d 1 + IgG3 0 2 IgG3,4 I 3 IgG3 0 1 IgG1 I 2 IgG3 II III 3 2 + IgG4 I T IgG4 I 2 IgG4 I 2 IgG4 III IV 0 3 1 + IgG4 I 1 IgG4 0 0 IgG4 II 2 3 2 + IgG4 I 1 IgG4 II 2 IgG1,4 I II 3 4 + IgG2 I 2 3 IgG1, 2,3,4 I II 2 IgG1 II 2 IgG3 I II 1 5 IgG1 II III 3 IgG1 II III 3 6 + IgG3,4 I 3 IgG3 III IV 2 7 IgG4 0 3 IgG2 3 3 0 2 IgG1 4 0 2 8 + IgG4 I T IgG4 0 2 IgG4 II 1 IgG4 II 3 IgG4 II III 2 IgG4 II III 2 9 IgG4 I 2 IgG4 II 3 IgG4 I II 3 IgG1,4 II 1 IgG4 II 3 10 + IgG4 NA 3 IgG4 III IV 1 2 11 + IgG4 I II 1 2 IgG1 IV 1 2 12 IgG4 0 1 13 + IgG4 II III 2 14 + IgG3 0 2 15 IgG4 II III 2 16 + IgG4 II III 3 17 + IgG1,3 II III 0 18 + IgG4 I 0 Electron microscopy deposits staged I IV. C4d in capillary loops presented on a scale of trace (T) to 3+ intensity. PLA 2 R Ab, phospholipase A 2 receptor antibody; NA, not available. 1 First transplant. 2 Second transplant. 3 Immunofluorescence for IgG4 was not available. 4 Immunofluorescence for IgG4 and IgG3 was not available. American Journal of Transplantation 2016; 16: 2964 2972 2967

Kattah et al IgG subtypes at time of recurrence Table 2 shows the (co)dominant IgG subtypes in each patient at yearly intervals, starting from the time of first histologic recurrence. The first biopsy revealing recurrence was available for IgG subtyping in 14 of 19 transplants. IgG4 was the (co)dominant IgG subtype in 10 of 14, and IgG1, IgG2, and IgG3 were the (co)dominant IgG subtype in 1, 1, and 2 of 14, respectively. An example of an IgG4-dominant subtype biopsy is shown in Figure 1. The remaining five transplants only had biopsy results to review from later in the course of recurrent MN (patients 9, 10, 11, 14, and 17). IgG4 was the (co)dominant IgG subtype in 3, IgG3 was (co)- dominant in 2, and IgG1 was (co)dominant in 1 of 5 cases. A Temporal IgG subtype changes In 8 of 12 transplants with serial IgG subtyping (patients 1 11), the (co)dominant subtype did not change over time. IgG4 was the (co)dominant subtype on the earliest posttransplantation biopsy in seven of eight cases, and IgG1 was the dominant subtype in the remaining case. The patient with IgG1 dominant staining on serial biopsies (patient 5) had non PLA 2 R-associated MN, but no evidence for secondary MN (systemic lupus erythematosus, hepatitis B or C virus, or malignancy). In three patients, the (co)dominant subtype changed over time (patients 1, 4, and 11). Patient 1 had several changes in the IgG subtyping over time. His 4-month protocol biopsy results showed recurrence with IgG3 dominant subtyping, followed by IgG3/IgG4 codominance at 1 year. Subsequent biopsy samples revealed either IgG1 or IgG3 dominance. Patient 4 initially had IgG2 dominant subtyping and progressed to codominance of all subtypes, followed by a switch to IgG1 dominance and finally to IgG3 dominance. Patient 11 switched from IgG4 to IgG1 dominance. In patient 7, IgG4 subtyping could not be performed on two of the later biopsy samples, so it remains unknown whether there was a subtype switch. We analyzed the change in (co)dominant IgG subtypes as a function of time from first histologic recurrence (Figure 2). IgG4 was the most common (co)dominant subtype, but there was a trend toward increasing IgG1 and IgG3 (co)dominant subtyping and decreasing IgG4 (co)dominant subtyping after the first year postrecurrence. The changes in intensity in IgG subtype staining over time in nine transplants (patients 1 8) are shown in Figure 3. Early postrecurrence, one IgG subtype was clearly the most intense (patients 2, 3, 4, 5, and 8). Over time, the intensity of the nondominant subtypes increased in many patients (patients 1, 2, 3, 4, 6, and 8), while the dominant subtype typically remained unchanged. The IgG subtype pattern did not change after administration of rituximab. B Figure 1: An example of IgG subtyping in a case of recurrent MN classified as IgG4-dominant subtype. (A) Granular IgG4 deposits are noted along the peripheral capillary loops. Direct immunofluorescence, 9200. (B) Ultrastructural studies demonstrate subepithelial deposits (arrows), stage I II, with rare spike formation. Uranyl acetate lead citrate, 97400. MN, membranous nephropathy. Clinical characteristics and subtype dominance Clinical characteristics were compared between patients with IgG4 (co)dominance and those with another dominant subtype on the earliest available posttransplantation biopsy (Table 3). As no switch from IgG1 3 (co)dominant to IgG4 (co)dominant subtyping was observed, we assumed that patients with IgG4 staining late in the posttransplantation course (patients 9 11) likely had IgG4 dominant staining immediately postrecurrence. Two patients (patients 14 and 17) had only a single posttransplantation biopsy available >2 years from the initial recurrence and did not have IgG4 dominant subtyping. As we could not be sure that they were not initially IgG4 dominant, we excluded them from this analysis. There was no difference in sex, timing of posttransplantation recurrence, or use of rituximab posttransplantation between those with IgG4 (co)dominant subtyping and those 2968 American Journal of Transplantation 2016; 16: 2964 2972

IgG Subtypes in Recurrent MN 100 90 80 70 Percentage of Biopsies (%) 60 50 40 30 IgG1 IgG2 IgG3 IgG4 20 10 0 Number of biopsies Diagnosis < 1 year 1-2 years > 2 years N=14 N=8 N=6 N=18 Figure 2: (Co-)dominant IgG subtypes according to time from diagnosis of MN recurrence. MN, membranous nephropathy. with other dominant subtypes. In addition, there was a similar percentage of patients with PLA 2 R-associated MN in the two groups (69.2% vs. 66.7%, respectively). However, those with (co)dominant subtypes other than IgG4 were more likely to have received alemtuzumab for induction therapy at the time of transplantation (p = 25). EM stages The relationship between IgG subtypes and EM stages was assessed in 49 biopsies (no EM available for 2 and incomplete IgG subtype staining for 2) (Table 4). No significant association was found between IgG (co)dominant subtypes and EM stages. However, there was a trend toward IgG1 (co)dominance in the more-advanced EM deposits. C4d staining C4d staining in the capillary loops was common (Table 2). We found a statistically significant correlation between C4d intensity and IgG1 subtype intensity (q = 0.41, p = 09) and the overall IgG intensity (q = 0.48, p = 01). There was no correlation between C4d intensity and IgG4 intensity (q = 043, p = 0.97) or IgG3 intensity (q = 0.29, p = 67). Discussion We present the first biopsy series delineating IgG subtypes in recurrent MN. Owing to the practice of protocol biopsies, we were able to detect recurrence of MN at an early stage and report on temporal changes of the IgG subtypes. The salient finding is that IgG4 is the dominant IgG subtype in recurrent MN, regardless of time from recurrence or PLA2R-association, and in the majority of recurrent MN the (co)dominant subtype did not change over time. IgG1 (co)dominant subtypes appeared to be associated with more-advanced EM deposits. We also noted an increase in the intensity of staining of the nondominant subtypes later in the course of recurrence. The dominance of the IgG4 subtype is in line with the findings of Huang et al, who reviewed the native kidney biopsy results of 114 patients with idiopathic MN and 43 patients with secondary MN. In their study, 76% of patients with idiopathic MN had IgG4 dominant staining. The authors were unable to test for autoantibodies to PLA 2 R or THSD7A, both of which are predominantly IgG4 (5,11). They found that IgG1 was the dominant subclass in early MN with stage I EM deposits, as opposed to IgG4 in more-advanced deposits, suggesting a temporal IgG subtype switch. We found few cases of IgG subtype switching in the posttransplantation period. In addition, IgG1 was the dominant subtype in the more-advanced EM deposits. The discrepancy between our results and those of Huang et al can be explained by the different natures of recurrent and native MN. Even early recurrent MN still occurs late in the disease process compared with native MN. MN causing end-stage renal disease and recurring in the allograft has been immunologically active longer and likely behaves American Journal of Transplantation 2016; 16: 2964 2972 2969

Kattah et al 1 2 64 68 72 76 3* 3** 4 5 04812162024283236404448525660646872768084889296100104108112116120 6 8 Legend: Rituximab 1000 g 7 Figure 3: Changes in IgG subtype (1 4) intensity after diagnosis of MN recurrence in patients 1 8. Intensity is scaled 0 3. Values reported as trace, 1 2, and 2 3 are coded as 0.5, 1.5, and 2.5, respectively for purposes of graphical representation. Rituximab therapy is indicated with red arrows. For patient 3, *first and **second transplant. MN, membranous nephropathy. Table 3: Clinical characteristics in patients with IgG4 dominant staining on earliest biopsy postrecurrence versus any other dominant subtype Clinical characteristic IgG4 dominant/ codominant (n = 13) 1 Other dominant subtype (n = 3) p- value 2 Male sex 9 (69.2%) 2 (66.7%) Early recurrence 10 (76.9%) 3 (100%) (<6 ) Induction 13 (100%) 2 (66.7%) 25 thymoglobulin versus alemtuzumab PLA 2 R associated 9 (69.2%) 2 (66.7%) MN Treated with rituximab posttransplantation 9 (69.2%) 0 (0%) 63 PLA 2 R Ab, phospholipase A2 receptor antibody. 1 Two patients were excluded as they did not have subtyping available until late in the postrecurrence course. 2 Fisher exact test. differently from MN that will not lead to end-stage renal disease or does not recur in the allograft. In addition, the disease course and IgG subtyping may be different in native and transplanted kidneys, owing to the influence of the immunosuppressive drugs. A minority of patients changed (co)dominant subtype over time. Interestingly, all three of these patients had PLA 2 R-associated MN. Although the numbers are small, the changing IgG dominance may be explained by the type of immunosuppression that was given. Some of those patients received alemtuzumab for induction (patients 1 and 4), which may alter B and T cell populations after lymphocyte reconstitution (16 18). Another potential explanation is the phenomenon of epitope spreading, whereby an autoantibody recognizes a different epitope on the same disease-related antigen. An elegant study by Seitz-Polski et al determined the three main epitopes of the PLA 2 R antigen to which the anti-pla 2 R antibody can bind: cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) (13). Epitope spreading beyond the CysR domain was associated 2970 American Journal of Transplantation 2016; 16: 2964 2972

IgG Subtypes in Recurrent MN Table 4: Association of IgG dominant/codominant subtype and electron microscopy stages IgG subtype Electron microscopy stage, n 1 0(n= 7) I (n = 12) II (n = 13) III (n = 9) IV (n = 4) p-values Comparing proportions in all groups 2 Test for trend 3 IgG1 versus other 0 1 (8.3%) 4 (30.8%) 3 (33.3%) 1 (25%) 0.28 58 IgG2 versus other 0 1 (8.3%) 1 (7.7%) 0 0 0.71 IgG3 versus other 3 (42.9%) 2 (16.6%) 2 (15.4%) 3 (33.3%) 1 (25%) 0.63 0.81 IgG4 versus other 4 (57.1%) 9 (75%) 11 (84.6%) 4 (44.4%) 2 (50%) 0.31 0.42 1 Among 49 biopsy samples analyzed, six had codominant deposits of IgG subtypes: five represented a paired codominance, while one biopsy sample showed all the four subtypes equally represented in the deposits, so numbers sum to >100%. Two biopsy samples were not considered for the analysis because EM was not available, and two were not considered as IgG subtyping was not complete on two later biopsy samples (patient 7). Stages are reported according to highest Ehrenreich Churg classification. 2 Fisher exact test. 3 Cochran Armitage test for trend. with a worse prognosis. Antibodies against the CysR and the CTLD1 epitope were predominantly IgG4 but could also be IgG1, IgG2, and IgG3. In contrast, anti-ctld7 antibodies were always of the IgG4 subtype. The IgG subtype profile may be influenced by induction immunosuppression, the donor antigen, and other immunologic factors, such as rejection, infection, and additional immunosuppressive medications. How these factors determine IgG subtypes in the posttransplantation period and affect prognosis remains elusive. We found that C1q and C3 were less commonly positive in recurrent MN, compared with C4d (data not shown), which is consistent with the findings of Rodriquez et al (14). IgG4 is the dominant subtype in MN but is not able to bind and activate the classic complement pathway (1,17,18). Others have proposed that IgG4 anti-pla 2 R may be able to activate the mannose-binding lectin pathway in MN, and this could explain the C4d positivity (14,19). An alternative hypothesis is that the IgG1 and/or IgG3 anti-pla 2 R subtypes that coexist with the dominant IgG4 subtype are able to bind and activate complement (18). While our results are limited, we did find a correlation between IgG1 and C4d intensity, which may support this latter hypothesis. IgG subtyping may help to determine whether the disease is recurrent or de novo in the posttransplantation period. Kearney et al compared the IgG subclasses in recurrent MN, de novo MN, and atypical/indeterminate MN (4). IgG4 was (co)dominant in all cases of recurrent MN and IgG1 was (co)dominant in the de novo cases. In accordance with our study, codominance was commonly seen, suggesting that the IgG subtype pattern alone cannot be used to differentiate these entities. Although PLA 2 R status may help discriminate between idiopathic and secondary MN, anti-pla 2 R antibodies have also been described in secondary MN due to hepatitis C virus infection, sarcoidosis, and neoplasm (15). In some cases of MN, anti-pla2r -antibodies became positive only after prolonged follow-up, while the PLA2R antigen staining on the biopsy was positive at the time of diagnosis (20,21). The combination of both IgG subtyping and testing for PLA 2 R-assocation may improve diagnostic accuracy before transplantation. Our study has notable limitations. We did not have serial biopsy samples in every patient, nor did we have tissue available at diagnosis of recurrence in every patient. This is the first study to perform IgG subtyping on posttransplantation biopsies in subjects with known PLA 2 R status. However, we did not have serum samples in all patients to compare the peripheral IgG subtypes or the anti- PLA 2 R or anti-thsd7a antibody titers with the biopsy pattern. In conclusion, we found that the dominant subtype in recurrent idiopathic MN is IgG4 and remained unchanged over time in the vast majority of cases. Further studies comparing IgG subtyping on both native and posttransplantation biopsy samples in the same patients may elucidate the roles of time, immunosuppression, and other clinical factors in the change in IgG subtype profile and advance the understanding of the pathogenesis of recurrent MN. Acknowledgments The research was supported by the Fulk Family Foundation. Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. References 1. Doi T, Mayumi M, Kanatsu K, Suehiro F, Hamashima Y. Distribution of IgG subclasses in membranous nephropathy. Clin Exp Immunol 1984; 58: 57 62. American Journal of Transplantation 2016; 16: 2964 2972 2971

Kattah et al 2. Kuroki A, Shibata T, Honda H, Totsuka D, Kobayashi K, Sugisaki T. Glomerular and serum IgG subclasses in diffuse proliferative lupus nephritis, membranous lupus nephritis, and idiopathic membranous nephropathy. Intern Med 2002; 41: 936 942. 3. Ohtani H, Wakui H, Komatsuda A, et al. Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy. Nephrol Dial Transplant 2004; 19: 574 579. 4. Kearney N, Podolak J, Matsumura L, Houghton D, Troxell M. Patterns of IgG subclass deposits in membranous glomerulonephritis in renal allografts. Transplant Proc 2011; 43: 3743 3746. 5. Beck LH Jr, Bonegio RG, Lambeau G, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 2009; 361: 11 21. 6. Hofstra JM, Beck LH, Jr., Beck DM, Wetzels JF, Salant DJ. Anti-phospholipase A(2) receptor antibodies correlate with clinical status in idiopathic membranous nephropathy. Clin J Am Soc Nephrol 2011; 6: 1286 1291. 7. Hoxha E, Harendza S, Zahner G, et al. An immunofluorescence test for phospholipase-a(2)-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant 2011; 26: 2526 2532. 8. Debiec H, Martin L, Jouanneau C, et al. Autoantibodies specific for the phospholipase A2 receptor in recurrent and de novo membranous nephropathy. Am J Transplant 2011; 11: 2144 2152. 9. Qin W, Beck LH, Jr., Zeng C, et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 2011; 22: 1137 1143. 10. Oh YJ, Yang SH, Kim DK, Kang SW, Kim YS. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy. PLoS ONE 2013; 8: e62151. 11. Tomas NM, Beck LH Jr, Meyer-Schwesinger C, et al. Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy. N Engl J Med 2014; 371: 2277 2287. 12. Huang CC, Lehman A, Albawardi A, et al. IgG subclass staining in renal biopsies with membranous glomerulonephritis indicates subclass switch during disease progression. Mod Pathol 2013; 26: 799 805. 13. Seitz-Polski B, Dolla G, Payre C, et al. Epitope spreading of autoantibody response to PLA2R associates with poor prognosis in membranous nephropathy. J Am Soc Nephrol 2015; 27: 1517 1533. 14. Rodriguez EF, Cosio FG, Nasr SH, et al. The pathology and clinical features of early recurrent membranous glomerulonephritis. Am J Transplant 2012; 12: 1029 1038. 15. Larsen CP, Messias NC, Silva FG, Messias E, Walker PD. Determination of primary versus secondary membranous glomerulopathy utilizing phospholipase A2 receptor staining in renal biopsies. Mod Pathol 2013; 26: 709 715. 16. Kattah A, Ayalon R, Beck LH, Jr., et al. Anti-phospholipase A(2) receptor antibodies in recurrent membranous nephropathy. Am J Transplant 2015; 15: 1349 1359. 17. Aalberse RC, Schuurman J. IgG4 breaking the rules. Immunology 2002; 105: 9 19. 18. Beck LH, Jr., Salant DJ. Membranous nephropathy: From models to man. J Clin Invest 2014; 124: 2307 2314. 19. Ma H, Sandor DG, Beck LH, Jr. The role of complement in membranous nephropathy. Semin Nephrol 2013; 33: 531 542. 20. van de Logt AE, Hofstra JM, Wetzels JF. Serum anti-pla2r antibodies can be initially absent in idiopathic membranous nephropathy: Seroconversion after prolonged follow-up. Kidney Int 2015; 87: 1263 1264. 21. Ramachandran R, Kumar V, Nada R, Jha V. Serial monitoring of anti-pla2r in initial PLA2R-negative patients with primary membranous nephropathy. Kidney Int 2015; 88: 1198 1199. 2972 American Journal of Transplantation 2016; 16: 2964 2972