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B Cell Reconstitution after Rituximab Treatment in Idiopathic Nephrotic Syndrome Manuela Colucci,* Rita Carsetti,† Simona Cascioli,‡ Federica Casiraghi,§ Annalisa Perna,§ Lucilla Ravà,|| Barbara Ruggiero,§ Francesco Emma,* and Marina Vivarelli* *Division of Nephrology and Dialysis, †Immunology Unit, Immunology and Pharmacotherapy Area, ‡Research Center, and ||Clinical Epidemiology Unit, Medical Direction, Bambino Gesù Children’s Hospital, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; and §Istituto di Ricovero e Cura a Carattere Scientifico, Istituto di Ricerche Farmacologiche “Mario Negri,” Clinical Research Center for Rare Diseases “Aldo e Cele Daccò,” Ranica, Bergamo, Italy

ABSTRACT The pathogenesis of nephrotic syndrome is unclear. However, the efficacy of rituximab, a B cell–depleting antibody, in nephrotic syndrome suggests a pathogenic role of B cells. In this retrospective study, we determined by flow cytometry levels of B and T cell subpopulations before and after rituximab infusion in 28 pediatric patients with frequently relapsing or steroid–dependent nephrotic syndrome. At baseline, patients had lower median percentages of transitional and mature B cells than age–matched healthy controls (P,0.001). Rituximab induced full depletion of B cells (,1% of lymphocytes). At 1 year, most patients exhibited complete total and mature B cell recovery, whereas memory B cell subsets remained significantly depleted. Total T cell concentration did not change with rituximab, whereas the CD4+/CD8+ T cell ratio tended to increase. Fourteen patients relapsed within 24 months, with a median follow-up of 11.2 months (interquartile range, 8–17.7 months). We observed no difference at baseline between nonrelapsing and relapsing patients in several clinical parameters and cell subset concentrations. Reconstitution of all memory B cell subpopulations, number of immunosuppressive drugs, and dose of tacrolimus during the last 4 months of follow-up were predictive of relapse in univariate Cox regression analysis. However, only delayed reconstitution of switched memory B cells, independent of immunosuppressive treatment, was protective against relapse in multivariate (P,0.01) and receiver operator characteristic (P,0.01 for percentage of lymphocytes; P=0.02 for absolute count) analyses. Evaluation of switched memory B cell recovery after rituximab may be useful for predicting relapse in patients with nephrotic syndrome. J Am Soc Nephrol 27: ccc–ccc, 2015. doi: 10.1681/ASN.2015050523

Idiopathic nephrotic syndrome (INS), the most frequent glomerular disease in childhood, is characterized by intense proteinuria caused by modifications of the selective glomerular permeability barrier.1 The triggering event that induces glomerular damage and proteinuria remains unknown. Familial forms of INS may be caused by genetic abnormalities,2 but most steroid-sensitive forms are thought to have an immune pathogenesis through the production of a not yet defined circulating permeability factor. The current therapy of INS is on the basis of oral corticosteroids (prednisone [PDN]) given for 8–12 weeks, and complete remission is observed in up to 80% of children. However, 40%–50% of patients experience frequent relapsing-remitting episodes and acquire J Am Soc Nephrol 27: ccc–ccc, 2015

steroid dependency or rarely, resistance. Concurrent or substituting treatments with immunosuppressive agents, such as calcineurin inhibitors (CNIs) cyclosporin A (CsA), or tacrolimus, and the antiproliferatives azathioprine or mycophenolate mophetil

Received May 14, 2015. Accepted August 25, 2015. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Manuela Colucci, Division of Nephrology and Dialysis, Bambino Gesù Children’s Hospital, Istituto di Ricovero e Cura a Carattere Scientifico, Viale S. Paolo 15, 00146 Rome, Italy. Email: [email protected] Copyright © 2015 by the American Society of Nephrology

ISSN : 1046-6673/2706-ccc

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(MMF) are also effective, but 30%–40% of patients experience treatment-related toxicity, especially with CsA.3 Rituximab (RTX) is a chimeric murine/human mAb directed against CD20, an antigen expressed on all B cell subsets, excluding plasma cells. Because of its efficacy in inducing B cell depletion, RTX represents an eligible treatment for malignant lymphoma and autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.4–7 RTX has also been used to treat glomerular diseases in which circulating antibodies play a well described pathogenic role, such as idiopathic membranous nephropathy and antineutrophil cytoplasmic antibody–associated vasculitis.8,9 Although the role of B cells in the pathogenesis of INS is unclear, RTX has been shown to be effective in inducing prolonged remission and allowing tapering of concomitant immunosuppressive treatment in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid– dependent nephrotic syndrome (SDNS), with most evident benefits in children.10–12 Furthermore, it has been described that patients with INS treated with RTX may experience disease relapse after B cell recovery, whereas some treated patients maintain a long-term remission even after total B cell reconstitution.13 To date, the mechanism(s) responsible for RTX effectiveness remains elusive, and no biologic readout able to predict relapse after RTX treatment has been identified. The aim of this study is to determine if the reconstitution of different B cell subpopulations and/or the potential modification of T cell homeostasis can predict relapse after RTX administration in pediatric patients with FRNS or SDNS. During their ontogeny, bone marrow immature B cell precursors migrate in the periphery as transitional B cells, reach the spleen, where they acquire mature phenotype and functions, and can recirculate into secondary lymph nodes. Here, they are stimulated by encountered antigens in the presence of T helper cells and develop into short–lived antibody–producing plasma cells or long–lived memory B cells, which can produce IgM antibodies before isotype switching to IgG, IgA, or IgE on the basis of the nature of the antigen. Therefore, peripheral B cells in children are constituted by transitional B cells, mature naïve B cells (the prevalent population), and memory B cells, which can be subdivided into nonswitched (IgM memory) and isotype– switched memory B cells.14

total B cells (10.7% versus 13.3%) and total memory (2.6% versus 2.3%), IgM memory (1.268% versus 1.054%), and switched memory B cells (1.059% versus 0.919%). However, patients had a significantly lower median percentage of transitional (0.3% versus 0.8%; P,0.001) and mature B cells (4.5% versus 10%; P,0.001) compared with healthy controls. Complete depletion (,1% of total lymphocytes) of total B cells was observed 2–7 days after a single RTX infusion for all patients except four of them, who thus received a second infusion. One month after RTX treatment, all B cell subpopulations were completely depleted. Total B cells reappeared, on average, 6 months after RTX treatment (median, 1.5% versus 0.1% post-RTX; P,0.05), with an initial re-emergence of transitional B cells followed by mature B cells and finally, memory B cells (Figure 1), and completely recovered around 12 months after RTX administration (median, 8.9% versus 10.7% at baseline; P=NS). Transitional B cells were significantly higher compared with healthy donors at 12 months (median, 2.2% versus 0.8%; P,0.001), whereas mature B cells reappeared around 6 months after RTX treatment (median, 0.2% versus 0% post-RTX; P,0.01) and at 12 months, were comparable to baseline (median, 3.9% versus 4.5% at baseline; P=NS) but still significantly lower than median mature B cell percentage in healthy controls (10%; P,0.001). Interestingly, total memory, IgM memory, and switched memory B cells recovered very slowly and were still significantly lower compared with baseline values at 12 months (median, 0.3%, 0.183%, and 0.117%, respectively; P,0.001). Comparable results were obtained by analyzing the absolute number of each B cell subset (Supplemental Figure 2). Total CD3+ T cells and CD4+/CD8+ T cell ratio were determined at baseline and 1, 3, 6, 9, and 12 months after RTX administration in 20 patients (Figure 1, G and H). At baseline, the absolute count of CD3+ T cells fell within normal range of agematched controls (median, 1.43103 cells per microliter),15 and no significant variation was observed during the 12-month period (Figure 1G). On the contrary, the median CD4+/CD8+ T cell ratio, which was in the lower range of age-matched controls at baseline,15 tended to normalize after RTX treatment (1.6 at 12 months versus 1.1 at baseline; P,0.05) (Figure 1H). Baseline Characteristics

RESULTS Characterization of B and T Cell Subpopulations

Different B and T cell subsets were identified as described in Concise Methods by multicolor flow cytometry analysis (Supplemental Figure 1). The percentage of total lymphocytes for each B cell subpopulation at baseline and 1, 3, 6, 9, and 12 months after the first RTX infusion was analyzed in 28 patients with FRNS/SDNS, and baseline values were compared with values obtained for 28 age– and sex–matched healthy controls (Figure 1, A–F). At baseline, no difference between patients and healthy controls was detected in the median percentage of 2

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Table 1 reports characteristics of patients before RTX administration. There was no appreciable difference between nonrelapsing and relapsing patients in all of the analyzed demographic and clinical parameters, although MMF use during the last year before RTX treatment tended to be more frequent in nonrelapsing patients (P=0.08). Moreover, basal levels of B and T cell subpopulations were not associated with the risk of relapse (Table 1). Relapse-Free Survival

During 24 months after RTX infusion, 14 patients maintained a persistent remission (nonrelapsers), whereas 14 patients relapsed (relapsers), with a median follow-up of 11.2 months (interquartile range, 8–17.7 months): four patients relapsed before 9 months, J Am Soc Nephrol 27: ccc–ccc, 2015

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Figure 1. RTX treatment affects levels of B and T cell subsets. Multicolor flow cytometry analysis of circulating B and T cell subpopulations at baseline and different time points after RTX treatment in patients with FRNS/SDNS. (A–F) Levels of B cells from patients (n=28) were compared with basal values of age- and sex-matched healthy donors (HDs; n=28). (A) Gated CD19+ B cells were identified on the basis of the expression of surface markers as depicted in Supplemental Figure 1: (B) transitional, (C) mature, (D) memory, (E) IgM memory, and (F) switched memory B cells were expressed as percentages of total lymphocytes. (G and H) T cells from patients (n=20) were compared with the normal range of agematched controls described by Bofill et al.15 (G) Gated CD3+ T cells were expressed as absolute numbers of cells per microliter of blood. CD4+ and CD8+ T cells were identified as depicted in Supplemental Figure 1, and (H) the CD4+/CD8+ T cell ratio is represented. Gray areas represent the normal range between the 2.5th and 97.5th percentiles (identified by dashed gray lines), and the bold gray line indicates the normal median. Horizontal lines indicate the medians. Differences with HDs were compared using the nonparametric unpaired Mann–Whitney U test. Levels of B and T cell subpopulations at different time points were analyzed using a nonparametric Friedman test, and pairwise comparisons were evaluated by a Wilcoxon signed–rank test with Bonferroni adjustment versus month 0 (0 m) or versus month 1 (1 m). *P,0.05; **P,0.01; ***P,0.001.

four patients relapsed during the 9- to 12-month period, and the remaining six patients relapsed after 12 months after RTX treatment (Figure 2A). The time to the first relapse did not significantly differ among subgroups, as shown in Figure 2, B–E. J Am Soc Nephrol 27: ccc–ccc, 2015

Previous and Concomitant Immunosuppressive Therapy

During the last year before RTX infusion, all patients were treated with oral PDN and at least one steroid-sparing agent (Table 1). After RTX administration, concomitant immunosuppressive B Cells after Rituximab in INS

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Table 1. Characteristics of the patients in the study group at baseline Parameter Demographics Age (yr), mean6SEM Male sex, n (%) Diseases characteristics Age at onset (yr), mean6SEM SDNS versus FRNS Previous relapses (12 mo pre-RTX), n (%) Two relapses Three relapses Four relapses Previous immunosuppressive drugs (12 mo pre-RTX), n (%) PDN CNIs Cyclosporin Tacrolimus Antiproliferative/cytotoxic agents Azathioprine MMF At least one steroid-sparing agent RTX doses, n (%) One infusion Two infusions B cell subsets (% of total lymphocytes), median (IQR) CD19 positive Transitional Mature Memory IgM memory Switched memory T cell subsets, median (IQR) CD3+ T cells (cells 3 103/ml) CD4+/CD8+ T cell ratio

P Value

Overall (n=28)

Nonrelapsers (n=14)

Relapsers (n=14)

13.6860.77 18 (64.3)

14.7460.84 10 (71.4)

12.6361.26 8 (57.1)

0.17 0.70

5.2260.72 26 versus 2

5.1460.97 12 versus 2

5.3061.10 14 versus 0

0.91 0.48

9 (32.1) 9 (32.1) 10 (35.7)

5 (35.7) 6 (42.9) 3 (21.4)

4 (28.6) 3 (21.4) 7 (50)

.0.99 0.42 0.24

28 (100) 17 (60.7) 11 (39.3) 6 (21.4) 22 (78.6) 1 (3.6) 21 (75) 28 (100)

14 (100) 6 (42.9) 4 (28.6) 2 (14.3) 13 (92.9) 0 (0) 13 (92.9) 14 (100)

14 (100) 11 (78.6) 7 (50) 4 (28.6) 9 (64.3) 1 (7.1) 8 (57.1) 14 (100)

.0.99 0.12 0.44 0.65 0.17 .0.99 0.08 .0.99

24 (85.7) 4 (14.3)

13 (92.9) 1 (7.1)

11 (78.6) 3 (21.4)

0.60 0.60

10.7 (7.7–16.9) 0.3 (0.1–0.8) 4.5 (2.1–7) 2.6 (1.1–4.2) 1.27 (0.66–2.75) 1.06 (0.43–1.83)

10.7 (8.2–17) 0.4 (0.1–0.9) 4.6 (2.4–7.2) 2.9 (0.9–4.4) 1.37 (0.42–2.55) 0.98 (0.29–1.6)

10.7 (7.1–17.1) 0.2 (0.1–0.7) 4.1 (2.1–7.3) 2.6 (1.6–3.8) 1.17 (0.76–3.04) 1.31 (0.62–3.14)

0.94 0.15 0.78 0.93 0.97 0.23

1.4 (1–2)a 1.1 (0.8–1.4)a

1.1 (0.5–2.1)b 1.1 (0.7–1.5)b

1.9 (1.1–2)b 1.1 (0.9–1.4)b

0.31 0.84

IQR, interquartile range. a n=20. b n=10.

drugs were gradually tapered or discontinued in all patients (Figure 3). Within 12 months, all of the nonrelapsers and eight relapsers discontinued PDN, two nonrelapsers and five relapsers discontinued antiproliferative agents, and five nonrelapsers and five relapsers discontinued CNIs. However, after recurrence of nephrotic syndrome (NS), some patients reintroduced immunosuppressive therapy (Figure 3A). In the last 4 months of followup (before recurrence for relapsers and months 21–24 for nonrelapsers), all nonrelapsers had discontinued PDN and CNIs, and three of them discontinued MMF, whereas only five relapsers had discontinued PDN, two relapsers had discontinued MMF, and four relapsers had discontinued CNIs before recurrence of NS (Figure 3B). At last observation, nine patients had stopped all of the immunosuppressive drugs (Supplemental Table 1), and five of them never relapsed during 24 months after RTX infusion. Predictors of Relapse

We then compared the recovery of the T and B cell subsets between relapsers and nonrelapsers. As shown in Figure 4, G 4

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and H, in any time point, there was no difference in the absolute number of CD3+ T cells or the CD4+/CD8+ ratio between relapsers and nonrelapsers. Furthermore, no difference in the reconstitution of total CD19+, transitional, or mature B cells was observed, whereas memory, IgM memory, and switched memory B cell subpopulations recovered later in nonrelapsers than in relapsers (Figure 4, A–F). To evaluate the potential role of each B cell subpopulation as a predictor of relapse, Cox proportional hazard regression analysis was performed for each B cell subset at 9 months after RTX infusion, because at this time point, most patients showed a reappearance of all B cell subsets, and 24 of 28 patients had not yet experienced a relapse. Furthermore, several demographic and clinical characteristics were considered as potential predictors of relapse. Univariate analysis showed that a delayed reconstitution of total memory (P=0.004), IgM memory (P,0.01), and switched memory B cell subpopulations (P,0.001), the number of immunosuppressive drugs (P,0.01), and the dose of tacrolimus (P=0.003) during the last 4 months of follow-up were significantly associated with the risk of relapse J Am Soc Nephrol 27: ccc–ccc, 2015

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under the curve, 0.814; P,0.01) and absolute count (Figure 5C) (area under the curve, 0.763; P=0.02). The best cutoff of switched memory B cells to discriminate between relapsing and nonrelapsing patients was 0.067% of total lymphocytes, with a sensitivity of 71% and a specificity of 93% (Figure 5A), or 1.65 cells per microliter, with a sensitivity of 64% and a specificity of 86% (Figure 5C). As shown, patients with switched memory B cells ,0.067% of total lymphocytes or ,1.65 cells per microliter at 9 months had a significantly lower risk of relapse within 24 months after receiving RTX (Figure 5, B and D) (P,0.001 and P=0.001, respectively). Predictors of Switched Memory B Cell Recovery

Because switched memory B cell reconstitution was predictive of relapse, we evaluated by linear regression model the contribution of several variables to this reconstitution (Table 3). By univariate analysis, baseline levels of memory (b, 0.019; P=0.02) and switched memory B cells (b, 0.036; P=0.03) and PDN (b, 0.005; P=0.02) and tacrolimus doses (b, 0.099; P=0.04) at 9 months significantly correlated with values of switched memory B cells at 9 months. However, multivariate analysis showed that only tacrolimus retained significant correlation with the switched memory B cells (b, 0.253; P,0.01).

DISCUSSION

The role of the immune system in nongenetic forms of INS has been investigated Figure 2. Half of patients relapsed within 24 months after RTX treatment. Kaplan–Meier in numerous clinical and experimental curves describing the relapse-free survival of patients (A) considered as a whole or grouped studies as recently reviewed.16 In particuaccording to (B) sex, (C) number of relapses, (D) number of immunosuppressive (IS) drugs lar, altered levels of T cell subpopulations, during the last year before RTX administration, or (E) number of IS drugs at last observation. such as a reduced CD4+/CD8+ ratio, as well The number of patients in the different subgroups is indicated every 6 months. Comparison as T cell–related cytokines have been debetween subgroups was performed using a log-rank test. scribed in patients with INS and animal models.16–18 On the contrary, the role of after RTX infusion (Table 2). By multivariate analysis, only the B cells in the pathogenesis of INS and how RTX works in INS are still widely discussed.19 The potential pathogenic reconstitution of the switched memory B cells retained a significant association (P,0.01) with the time to relapse, even when role of Igs in some forms of NS has been described. Experiadjusted for the other significant covariates (Table 2). mental data from Dantal et al.20 suggest that a permeability In addition, the recovery of switched memory B cells at factor inducing albuminuria may be or may bind to an Ig. The 9 months was also strongly predictive of relapse by receiver effectiveness of RTX in inducing and/or maintaining a longoperator characteristic (ROC) analysis for both percentage of term remission in INS has been described by several reports19 total lymphocytes (Figure 5A, Supplemental Figure 3) (area and recent randomized, controlled10,12 and uncontrolled J Am Soc Nephrol 27: ccc–ccc, 2015

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Figure 3. Concomitant immunosuppressive treatment was tapered or discontinued after RTX administration. (A) Number of patients treated with PDN, MMF, and CNIs up to 12 months post-RTX for all relapsing and nonrelapsing patients regardless of recurrence of NS. (B) Number of patients on immunosuppressive therapy in the 12 months preceding RTX infusion and the last 4 months of the follow-up (months 21–24 for nonrelapsers and before NS recurrence for relapsers). *P,0.05; **P,0.01; ***P,0.001.

trials.21 Some of these reports show a correlation between post-RTX CD19+ reconstitution and relapses.22,23 However, persistent remission induced by RTX can be maintained in some patients with INS also after CD19+ recovery.13,24 This observation suggests a disease-modifying effect of RTX in INS that goes beyond total B cell depletion. A single study showed in vitro a direct effect of RTX on podocyte function.25 In this study, we have focused on the characterization of B cell subpopulations before and after RTX infusion in patients with FRNS/SDNS to analyze more in depth the role of B cells in INS pathogenesis. Also, we have characterized the effect exerted by RTX on T cell homeostasis. We observed that, at the time of RTX infusion, in the peripheral blood of patients, levels of total CD19+ and memory B cell subsets were comparable with those observed in age– and sex–matched controls. Reduced transitional and mature B cells at baseline were probably caused by the concomitant immunosuppressive therapy26–28 needed to maintain a persistent remission of NS. After complete depletion induced by RTX, total CD19+ B cells significantly increased at around 6 months as previously shown for 6

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RTX-treated patients with INS.10,13,21,23 At 12 months, we observed higher levels of transitional B cells and lower levels of mature B cells compared with healthy donors, suggesting that the development of transitional B cells into mature B cells is ongoing. Total memory B cells and IgM memory and switched memory B cell subsets also started to reconstitute around 6 months after RTX infusion in most patients. However, although total CD19+ and mature B cells reached baseline levels at 12 months, the memory B cell compartment still appeared significantly decreased at this time point, which was already observed after RTX treatment in other B cell–mediated autoimmune diseases29–33 and is in agreement with the physiologic B cell ontogeny, in which memory B cells are the last emerging B cell subset.14 Interestingly, RTX administration did not alter levels of total CD3+ T cells but led to a normalization of the initially low CD4+/CD8+ T cell ratio. We then analyzed the clinical response in the cohort of 28 patients with FRNS/SDNS subjected to RTX treatment. A persistent remission was observed in 14 patients, whereas 14 patients relapsed within 24 months. When we monitored the J Am Soc Nephrol 27: ccc–ccc, 2015

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Figure 4. Memory B cell recovery after RTX treatment was delayed in nonrelapsing patients. The percentage of total lymphocytes for (A) total CD19+, (B) transitional, (C) mature, (D) memory, (E) IgM memory, and (F) switched memory B cells as determined by multicolor flow cytometry at different time points after RTX infusion was compared between relapsers (n=14) and nonrelapsers (n=14). Levels of (G) total CD3+ T cells (expressed as cells per microliter) and (H) CD4+/CD8+ T cell ratio as determined by multicolor flow cytometry at different time points after RTX infusion were compared between relapsers (n=10) and nonrelapsers (n=10). Data are represented as means6SEMs. P values were calculated by comparing values of each cell subpopulation between relapsers and nonrelapsers using an unpaired t test and are indicated on the top of each graph.

reconstitution of each B cell subset, we observed that reconstitution of the memory B cell compartment was slower in nonrelapsers compared with relapsers. On the contrary, there was no difference in the number of CD3+ T cells or the CD4+/CD8+ ratio in the two groups after RTX infusion. After evaluating by univariate analysis the role of predictors of relapse of all B cell subpopulations and several clinical covariates, we observed that reconstitution of the memory B cells (both IgM memory and switched memory subpopulations) together with the number of immunosuppressive drugs and the tacrolimus dose during the last 4 months of follow-up were significantly predictive of the risk of relapse. However, multivariate analysis showed that only a delayed reconstitution of the switched memory B cells was significantly and independently protective against relapse. Furthermore, ROC analysis showed that the best cutoff to discriminate between relapsers and nonrelapsers was a level of switched memory B cells .0.067% of total lymphocytes or .1.65 cells per microliters at 9 months. These cutoff levels are not the values reached at the time to relapse, given that only four patients had J Am Soc Nephrol 27: ccc–ccc, 2015

already relapsed at 9 months, but indicate that higher levels of switched memory B cells at 9 months caused by a faster recovery of these cells predict recurrence of NS within 24 months post-RTX. Previous reports on RTX treatment in B cell–mediated autoimmune diseases showed that long-term remission can be observed in patients with a delayed memory B cell recovery.30,31 This population can remain reduced for several years, sometimes in association with a prolonged presence of transitional B cells in long–term nonrelapsing patients.30 Hypothetically, these results could suggest that RTX treatment may erase preexisting memory in some patients who do not relapse and favor a gradual development of a new nonpathogenic B cell memory compartment that has been correctly censored by the tolerance checkpoints.34 Recently, the importance of B cells with regulatory function has been shown in several autoimmune conditions.35,36 Regulatory B cells modulate pathogenic immune responses by producing the anti–inflammatory cytokines IL-10 and TGF-b and expressing inhibitory molecules.37 Although originally identified as a subset B Cells after Rituximab in INS

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Table 2. Cox proportional hazard regression Parameter B cell subsets 9 mo post-RTX CD19 positive Transitional Mature Memory IgM memory Switched memory Age at baseline Male sex Age at onset Previous relapses (12 mo pre-RTX) Three versus two Four versus two Previous ISs (12 mo pre-RTX) Three versus two RTX doses IS in the last 4 mo of follow-up PDN MMF CsA Tacrolimus

Units

Univariate Analysis

Multivariate Analysis

HR

95% CI

P Value

% of lymphocytes % of lymphocytes % of lymphocytes ‰ of lymphocytes ‰ of lymphocytes ‰ of lymphocytes Years Number Years

1.03 1.06 1.08 1.15 1.59 2.41 0.88 0.71 1.02

0.94 to 1.12 0.84 to 1.33 0.93 to 1.25 1.05 to 1.26 1.14 to 2.21 1.60 to 3.65 0.75 to 1.04 0.25 to 2.04 0.88 to 1.18

0.56 0.64 0.29 0.004a ,0.01a ,0.001a 0.14 0.52 0.78

Number Number

0.71 1.88

0.16 to 3.19 0.55 to 6.44

0.66 0.31

Number Number Number mg/m2/d mg/m2/d mg/m2/d mg/m2/d

1.17 1.58 2.18 1.13 1.00 1.01 2.86

0.41 to 3.37 0.44 to 5.68 1.22 to 3.90 0.98 to 1.31 1.00 to 1.00 1.00 to 1.03 1.42 to 5.76

0.77 0.48 ,0.01a 0.10 0.43 0.10 0.003a

HR

95% CI

P Value

0.53 3.45

0.21 to 1.30 1.39 to 8.54

0.16 ,0.01a

1.16

0.52 to 2.62

0.71

2.21

0.67 to 7.34

0.20

HR, hazard ratio; 95% CI, 95% confidence interval; IS, immunosuppressive drug. a Significant P value.

of transitional B cells, it has now been shown that regulatory B cells can acquire suppressive functions at different stages of development depending on environmental cues.38 Additional studies are necessary to explore the possibility that the reconstitution phase after RTX treatment may result in the expansion of regulatory B cells in nonrelapsing patients. Our data could explain why a prolonged remission can be maintained in some RTXtreated patients with INS also after CD19+ recovery13 and suggest that memory B cells and in particular, switched memory B cells rather than total CD19+ lymphocytes should be monitored and targeted to optimize RTX and other immunosuppressive therapy in FRNS/SDNS. Indeed, levels of switched memory B cells at 9 months were strongly predictive of relapse. Thus, monitoring switched memory B cells could be particularly useful to identify those patients who are at high risk of relapse and could benefit from an early retreatment with a second infusion of RTX. Next, we analyzed the effect of concomitant immunosuppressive medications on relapse and switched memory B cell reconstitution after RTX administration. The number of relapsers taking PDN and CNIs is higher during the follow-up, and comparing the year before RTX administration with the last 4 months of follow-up, nonrelapsing patients completely discontinued PDN and CNIs, whereas recurrence of NS impeded the interruption of these drugs in relapsing patients. This may explain why tacrolimus dose in the last 4 months of follow-up was significantly predictive of relapse by univariate analysis. Whereas MMF discontinuation was not frequent or significantly different in both subgroups in the months after RTX, in the year before RTX infusion, there was a trend toward more frequent MMF use in the 8

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group of nonrelapsers (92.9% versus 57.1% of relapsers). This observation could warrant additional investigation in a larger group of patients, especially because recent data in pediatric patients with INS have suggested that MMF use post-RTX may delay relapses.39 However, evaluation of the effect of all drugs received by each patient at 9 months showed a positive correlation only between PDN and tacrolimus dose and switched memory B cells by univariate analysis, and only tacrolimus maintained this positive correlation also by multivariate analysis, whereas MMF treatment does not affect switched memory B cell reconstitution. These results suggest no effect of such drugs in delaying switched memory B cell recovery but rather, that, in relapsing patients, tapering of concomitant immunosuppression was slower and less successful. It is currently not known whether B cells and in particular, the switched memory B cell subpopulation, other than representing a useful biologic readout, may play a direct role in the pathogenesis of INS. Conflicting results on the levels of total CD19+ B cells in steroid–sensitive nephrotic syndrome (SSNS) or steroid-resistant NS in pediatric patients have been described.40,41 Kemper et al.42 have shown an alteration of soluble CD23–mediated B cell activation in children with SSNS. Furthermore, indirect evidences provided by the association between variants in HLA-DQA1 and predisposition to SSNS43 and between prevalence of Epstein-Barr virus infection and INS onset44 have been recently published. However, to the best of our knowledge, the direct role of B cells and in particular, B cell subsets in the pathogenesis of SSNS has not yet been investigated. Rather than exerting a direct effect, B cells could also play a role in INS pathogenesis by modulating J Am Soc Nephrol 27: ccc–ccc, 2015

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CONCISE METHODS Study Patients Twenty-eight pediatric patients with FRNS/ SDNS followed at the Bambino Gesù Children’s Hospital were retrospectively selected for this study. Patients with FRNS were defined as patients with two or more relapses observed over the last 6 months or four or more relapses observed within any 12-month period. SDNS was defined as frequently relapsing NS, with relapses occurring while still on steroids or within 2 weeks of discontinuing steroids.21,49,50 Relapse was defined as proteinuria of at least 3+ for at least 3 consecutive days by urine dipstick as previously described.3 Eight of these patients have already been included in a recent randomized uncontrolled trial. 21 From April of 2009 to September of 2012, patients were treated with a single infusion of RTX (375 mg/m2) during steroid-induced complete remission of their NS followed by a second infusion at 7 days in case of noncomplete depletion of total B cells defined as CD19+ B cells .1% of the total peripheral blood lymphocytes assessed 2–7 days after the first infusion. RTX administration was chosen for patients who experienced Figure 5. Switched memory B cell reconstitution after RTX treatment was predictive of the frequent relapses despite double- or triplerisk of relapse. ROC curve analyzing switched memory B cell values expressed as (A) immunosuppressive therapy and/or a severe a percentage of total lymphocytes or (C) cells per microliter at 9 months after RTX infusion drug–related toxicity (Supplemental Table 2). and the risk of relapse within 24 months of treatment. The arrow indicates the best cutoff for The number of relapses in the 12 months preswitched memory B cells (0.067% or 1.65 cells per microliter, respectively). (B and D) ceding and the time to the first relapse during Survival analysis comparing patients with delayed (solid lines) and early (dashed lines) the 24 months after RTX treatment as well as recovery of switched memory B cells (log-rank test). AUC, area under the curve. the doses of immunosuppressive treatment were systematically registered starting from 12 months before RTX administration and throughout the entire follow-up. T cell function.19,45–47 RTX–induced B cell depletion could actu-

ally modify antigen presentation and cytokine production45 as well as alteration of T cell homeostasis and homing.48 Our results showed that total T cells were not affected by RTX, and whereas CD4+/CD8+ T cell ratio increased after RTX treatment, there was no difference between relapsers and nonrelapsers in the T cell subsets. The main limitation of our study is that it is a monocentric retrospective study: we have analyzed a relatively small number of patients, and our results could be difficult to generalize. However, patients were monitored systematically, and the evaluation of relapses continued until 24 months, allowing us to rigorously describe the long-term predictive role of B cell subset recovery. In summary, our study provides new insight into the heterogeneity of re-emerging B cell subpopulations in RTXtreated children with FRNS/SDNS, which reflects the physiologic development of peripheral B cells. More importantly, our data show that recovery of the memory B cell compartment and in particular, the switched population could be a strong predictor of the response to RTX, suggesting that this B cell subset may play an active role in the pathogenesis of INS. J Am Soc Nephrol 27: ccc–ccc, 2015

Concomitant Therapy and Follow-Up One month after RTX administration, concomitant immunosuppression was gradually tapered up to recurrence of NS if it occurred (Figure 3). After the initial infusion(s), RTX was no longer administered, despite the reappearance of CD19+ B cells. For eight patients (five relapsers and three nonrelapsers), CNIs and/or MMF were tapered first, and PDN tapering was started at a second time as previously described.21 For the remaining 20 patients (nine relapsers and 11 nonrelapsers), PDN was tapered first and discontinued in all patients, and it was reintroduced only in case of recurrence of NS, whereas CNIs and/or MMF were tapered later. The last observation was 24 months after RTX infusion for nonrelapsing patients or the time of relapse for relapsing patients.

Sample Procurement and Cell Isolation Blood samples were obtained from included patients according to our institutional guidelines for informed consent after approval from our local Ethics Committee and in compliance with the Declaration of Helsinki. Blood sampling was performed at baseline (time of the first B Cells after Rituximab in INS

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Table 3. Linear regression between parameters listed below and the percentage of switched memory B cells at 9 months post-RTX treatment Univariate Analysis Parameter B cell subsets at baseline CD19 positive Transitional Mature Memory IgM memory Switched memory Age at baseline Male sex Age at onset Previous relapses (12 mo pre-RTX) Previous ISs (12 mo pre-RTX) RTX doses IS at 9 mo post-RTX One Two Three PDN MMF CsA Tacrolimus

Units

b

95% CI Lower

Upper

Multivariate Analysis P Value

% of lymphocytes % of lymphocytes % of lymphocytes ‰ of lymphocytes ‰ of lymphocytes ‰ of lymphocytes Years Number Years Number Number Number

0.005 20.031 0.005 0.019 0.018 0.036 20.004 20.027 0.008 0.031 20.060 0.037

,20.01 20.15 20.02 ,0.01 20.02 ,0.01 20.02 20.16 ,20.01 20.04 20.18 20.14

0.01 0.08 0.02 0.04 0.06 0.07 0.01 0.10 0.02 0.11 0.06 0.21

0.26 0.59 0.62 0.02a 0.34 0.03a 0.55 0.67 0.31 0.40 0.33 0.67

Number Number Number mg/m2/d mg/m2/d mg/m2/d mg/m2/d

20.013 0.174 0.049 0.005 20.051 0.001 0.099

20.19 20.02 20.28 ,0.01 20.16 ,20.01 0.01

0.17 0.36 0.38 0.01 0.05 ,0.01 0.19

0.88 0.07 0.76 0.02a 0.33 0.32 0.04a

95% CI

b

P Value

Lower

Upper

0.032

,20.01

0.07

0.10

20.019

20.09

0.05

0.59

0.023

20.09

0.14

0.68

0.253

0.07

0.44

,0.01a

95% CI, 95% confidence interval; IS, immunosuppressive drug. a Significant P value.

RTX infusion), after 2–7 days, and after 1, 3, 6, 9, and 12 months. PBMCs were isolated by Ficoll-Paque Plus (Amersham Biosciences) density–gradient centrifugation. Twenty-eight age- and sex-matched healthy individuals were included as controls.

RTX infusion and that 50% of the patients relapsed during 24 months after RTX treatment. Therefore, we calculated that the inclusion of 28 patients conferred a .90% power to show a statistically significant reduction (a=0.05; one-tailed test) in the number of patients with relapse after RTX administration.

Flow Cytometry TodiscriminatedifferentTandBsubpopulations,PBMCswere stainedwith fluorochrome-conjugated mAbs directed against CD3, CD4, CD8, CD19, CD24, CD27, CD38, IgD (BD Biosciences), and IgM (Jackson ImmunoResearch Laboratories) and then analyzed by multicolor flow cytometry (FACSCanto II; BD Biosciences). Subsets of gated CD19+ (total) B cells were identified on the basis of the expression of surface markers as follows: transitional (CD38highCD24high), mature/naïve (CD38intermediateCD24low), and memory (CD382CD24high), and they were expressed as the percentage of total circulating lymphocytes. Memory B cells were also defined as CD19+CD27+ cells, with results comparable with those obtained by CD24/ CD38 staining (data not shown), and memory subclasses were defined as IgM memory (IgM+IgDintermediate) or switched memory (IgM2IgD2) and expressed as the percentage of total circulating lymphocytes. All analyses were performed with the FACSDiva software. Gated events (50,000) on living lymphocytes were analyzed for each sample.

Estimation of Statistical Power

We considered the first relapse event as the main outcome to evaluate the efficiency of our study. We have observed that 100% of the patients selected for the study had at least one relapse during the year preceding 10

Journal of the American Society of Nephrology

Statistical Analyses Continuous data are expressed as means6SEMs if they passed the normality test (Shapiro–Wilk test) or medians and interquartile ranges otherwise; categorical data are represented as numbers and percentages. Variables were compared by an unpaired t test if normally distributed or a nonparametric Mann–Whitney U test; a chisquared test or Fisher’s exact test, when appropriate, was used for categorical variables. Monitoring of the reconstitution of each B and T cell subpopulation was analyzed using a nonparametric Friedman test, and pairwise comparisons were evaluated by a Wilcoxon signed– rank test with Bonferroni adjustment. The role of each B cell subset as a predictor of relapse and the effect of clinical parameters on risk of relapse were analyzed by ROC analysis, the Kaplan–Meier method, the log-rank test, and univariate and multivariable proportional hazard Cox models. The correlation of several parameters with the levels of switched memory B cells at 9 months was analyzed by linear regression model. Survival time was analyzed for the whole cohort of patients or by grouping patients according to sex, number of relapses or number of immunosuppressive drugs during the last year before RTX administration, and number of immunosuppressive drugs at the J Am Soc Nephrol 27: ccc–ccc, 2015

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last observation. Univariate Cox model and linear regression analyses tested the contribution of age at baseline, sex, age of onset, number of relapses during the last year before RTX infusion, number of RTX infusions, immunosuppressive treatment (PDN, MMF, CsA, and tacrolimus), and levels of different B cell subsets to the risk of relapse or the reconstitution of switched memory B cells, respectively. Covariates included in the multivariable models were those that reached a significant P value in univariate analysis. Proportionality assumptions were verified by tests (Grambsch and Therneau) and graphs on the basis of Schoenfeld residuals; Martingale residuals were used to graphically evaluate the assumptions of linearity of predictors. All P values are two sided and considered statistically significant with P,0.05. Analyses were performed through the software STATA 13.1 (StataCorp LP) and GraphPad Prism 6.

ACKNOWLEDGMENTS The authors thank Laura Massella and Giuseppe Remuzzi for helpful discussion. This work was supported by the Associazione per la Cura del Bambino Nefropatico–Onlus, by the Becton Dickinson Immunology Grant Program, and by the Ricerca Corrente of the Italian Ministry of Health (#201260X002808).

DISCLOSURES None.

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