The role of alloantibodies in chronic renal allograft deterioration and the corresponding morphologic changes are increasingly recognized in recent years. In the Banff '05 meeting report, the criteria for late or chronic antibody-mediated rejection (CAMR) were defined to eliminate the unspecific term “chronic rejection” (1). The diagnostic criteria of CAMR include the following: (i) morphological features including transplant glomerulopathy (duplication or “double contours” in glomerular basement membranes) or basement membrane multilayering of peritubular capillaries (PTC) or interstitial fibrosis and tubular atrophy with or without PTC loss, or fibrous intimal thickening in arteries without duplication of the internal elastica; (ii) diffuse C4d deposition in PTC; and (iii) the presence of donor-specific antibody (DSA) (1).
The prognosis of untreated CAMR is unfavorable (2, 3). One study with 92 adult renal transplant recipients, for example, demonstrated a significant association between diffuse C4d staining, production of DSA, and graft failure (3). The grafts failed in 36% of the diffuse (P<0.025), 23% of the focal, and 7% of the negative group at between 1 month and 15 years posttransplantation. Only patients in the group with diffuse C4d positivity had concurrent DSA (5 cases, P<0.001). Of the five DSA-positive patients, three had type II acute rejection, and two of these transplants subsequently failed. The remaining two had chronic allograft nephropathy with features of alloimmune injury (3).
The poor outcome of CAMR underlines the need for effective therapies directed at the humoral response. Because there is no established treatment protocol for CAMR, we undertook the present pilot study to investigate the efficacy and safety of a therapeutic regimen comprising the use of high-dose intravenous immunoglobulin (IVIG) and the anti-CD20 chimeric antibody rituximab. IVIG is believed to work by blocking antihuman leukocyte antigen (HLA) antibody activity and through complement inhibition, whereas rituximab depletes B cells and interferes with antigen-presenting cell activity of B cells subsequently decreasing T-cell activation (4). These agents are currently used to desensitize patients in preparation for transplantation and to treat acute antibody-mediated rejection after renal transplantation (5).
PATIENTS AND METHODS
Patients and Study Protocol
From November 2001 to June 2006, 87 renal transplantations in pediatric recipients were performed at the University Hospital of Heidelberg. Six patients (two girls, four boys; 7% of the entire patient population), aged 10 to 26 years, developed CAMR according to Banff '05 criteria (1). Patient characteristics are given in Table 1. All patients had received their first transplant. The median time posttransplantation until the diagnosis of CAMR by renal graft biopsy was 44 months (range, 6–120 months). The initial immunosuppressive therapy posttransplant consisted of a triple regimen with a calcineurin inhibitor (CNI) (cyclosporine microemulsion [CsA] in four patients, tacrolimus [TAC] in two patients) in conjunction with mycophenolate mofetil (MMF) and methylprednisolone. All patients had been subjected to graft biopsy before index biopsy. None of these patients showed positive C4d staining in these previous biopsies. Patient 1 had acute T-cell-mediated rejection with mild to moderate arteritis (grade IIA according to Banff '05) at 1 month posttransplant. Patient 2 showed moderate benign nephrosclerosis at 1 month posttransplant. Patient 3 had a biopsy at 2 years posttransplant and showed borderline cell infiltrates, interstitial fibrosis, and tubular atrophy (grade II according to Banff ‘05). Patient 4 showed at 8 years posttransplant arteriolar hyalinosis and tubular atrophy/interstitial fibrosis. Patient 5 showed 3 years posttransplant chronic glomerulopathy with tubular atrophy/interstitial fibrosis (grade 1 according to Banff '05) and arteriolar hyalinosis. Patient 6 experienced one acute biopsy-proven T-cell-mediated rejection (grade IA according to Banff '05) at 1 month posttransplant.
The type of immunosuppressive therapy given at the time of index biopsy is presented in Table 1. Because of biopsy- proven chronic CsA-induced nephrotoxicity, the CsA dosage had been minimized previously in three patients (patients 2, 3, and 6); there was an additional presumed noncompliance in patient 6. One patient (patient 5) was on a CNI-free regimen based on MMF and methylprednisolone because of previous biopsy-proven chronic CsA-induced nephrotoxicity and a severe opportunistic infection (pneumocystis jirovecii pneumonia). Because of pronounced interstitial inflammation in the index biopsy (Table 2), this patient was subjected to a course of high-dose steroids before initiation of antihumoral therapy. During the study period of 12 months, the maintenance immunosuppression remained unchanged in five patients; in patient 6, the maintenance immunosuppressive therapy was switched from CsA to TAC because of concomitant borderline cellular infiltrates. There was no difference between CNI target levels before versus after index biopsy.
Inclusion criteria for this pilot study were (i) clinical evidence of slowly deteriorating graft function; (ii) deposition of the complement split product C4d in PTC as an immunologic footprint of complement activation and antidonor humoral activity; (iii) serologic evidence for anti-HLA antibodies or DSA at the time of biopsy; and (iv) informed consent for treatment with IVIG and rituximab. Patient 1 fulfilled the criteria (i) and (ii), but did not have DSA at the time of index biopsy; he was therefore diagnosed as “suggestive of CAMR,” as suggested by the Banff '05 Meeting Report (1). The treatment regimen for CAMR consisted of four weekly doses of IVIG (1 g/kg body weight per dose), followed by a single dose of rituximab (375 mg/m2 body surface area) 1 week after the last IVIG infusion.
Glomerular filtration rate (GFR) of renal allografts was assessed by creatinine clearance calculated according to Schwartz et al. (6). CsA and TAC blood concentrations were measured by a monoclonal immunoassay (EMIT, Dade Behring, Germany) according to the manufacturer's instructions. Plasma mycophenolic acid concentrations were measured by the EMIT immunoassay (Dade Behring) on a Cobas-Mira analyzer according to the manufacturer's instructions. Mycophenolic acid exposure was estimated by a validated algorithm based on a limited sampling strategy during the first 2 hr after MMF dosing (7).
Samples of venous blood in ethylenediaminetetraacetic acid were used for immunophenotyping by flow cytometry. CD20, expressed on all normal mature B cells, was used as specific B-cell marker.
Biopsies were evaluated using the Banff '05 classification (1). For detection of C4d, we used a polyclonal anti-C4d antibody (C4dpAb; Biomedica, Vienna, Austria), as described elsewhere in detail (8). The minimal threshold level to render a biopsy positive was a linear circumferential staining in at least 25% of PTC. Specimens stained with hematoxylin-eosin, periodic acid Schiff stain, methenamine silver, and the Masson trichrome stain were used to analyze the lesions in allograft biopsies according to the definitions provided by the Banff '97 working classification of renal allograft pathologic features (9). Staining for CD20 was performed using a mouse monoclonal antibody (Clone L26, Dako) and the streptavidin peroxidase technique using a biotinylated secondary antibody (Dako). Quantification of B lymphocytes was performed by counting positively stained cells per mm2 under 400× magnification.
Detection of Human Leukocyte Antigen-Specific Antibodies
Patients were tested repeatedly pre- and posttransplantation for anti-HLA-antibodies by the panel reactive lymphocytotoxicity assay and by solid-phase ELISA assays. For the determination of lymphocytotoxic panel reactivity, plates with isolated B lymphocytes were used in addition to plates with unseparated lymphocytes. For the determination of ELISA-reactive IgG-anti-HLA antibodies, AbScreen and AbIdent ELISA assays of Biotest (Dreieich, Germany) were used. Both ELISA assays are highly sensitive and specific for IgG and enable a differentiation between IgG antibodies directed against HLA class I and class II. While the AbScreen assay allows a general detection of IgG antibodies directed against pooled HLA class I or class II antigens, the AbIdent assay allows the assessment of panel reactivity as well as of the specificity of anti-HLA antibodies. Rituximab treatment does not interfere with test results obtained in ELISA.
Data are given as mean±SEM, if not indicated otherwise. The change of GFR before and after initiation of antihumoral therapy was analyzed by the Wilcoxon test for paired samples.
Histology and C4d Staining
The histopathology findings are shown in Table 2. All patients showed positive (≥40%) staining for C4d in PTC. Transplant glomerulopathy was present in three of six patients and was always associated with glomerular C4d deposition and more pronounced (≥60%) C4d deposition in PTC. None of these patients presented with glomerulitis. Peritubular capillaries basement membrane multilayering was present in only one case. All biopsies showed considerable amounts of interstitial inflammation and features of chronic deterioration such as tubular atrophy, interstitial fibrosis, and fibrous arterial intimal thickening. No patient fulfilled the criteria of acute T-cell-mediated rejection greater than or equal to type IA according to Banff '97 criteria (9). There were variable CD20-positive infiltrates ranging from less than 10% to 50%. Patients 5 and 6, who did not respond to therapy, had the highest degree of transplant glomerulopathy, the highest degree of C4d deposition in PTC and pronounced interstitial inflammation (Table 2).
Graft Function and Response to Therapy
The individual GFR values are depicted in Figure 1(A), the mean GFR values and the respective slope of GFR are depicted in Figure 1(B). All patients showed progressive deterioration of transplant function before therapy. The median absolute loss of GFR during 6 months before intervention was 25 (range, 11–26) mL/min per 1.73 m2 (P<0.05); the median relative loss of GFR was 40% (range, 24%–55%). Two of six patients had mild proteinuria at the time of biopsy, defined as a protein excretion more than 150 mg/m2 per day; patient 6 had heavy proteinuria (Table 1). All patients required antihypertensive therapy with at least one antihypertensive drug, two patients received treatment with the ACE inhibitor ramipril.
The response of graft function to therapeutic intervention with IVIG and rituximab is depicted in Figure 1. GFR improved or stabilized in four patients during the 12 months after initiation of antihumoral therapy. In patient 5, GFR stabilized initially, but the graft was lost after 18 months of observation because of progressive chronic rejection. Patient 6 did not respond to therapy. The median absolute increase in GFR 6 and 12 months after initiation of therapy was 21 (range, −14 to +30) (P<0.05) and 19 (range, −14 to +23) (P=0.063) mL/min per 1.73 m2, respectively. The mean relative increase in GFR 12 months after initiation of therapy was 60% (range, −48% to +72%). Treatment with IVIG and rituximab was tolerated well in all patients; specific side effects were not observed.
The cell count of CD20+ cells in peripheral blood before and after initiation of antihumoral therapy with IVIG and rituximab is depicted in Figure 2. Five of six patients experienced complete depletion of circulating CD20+ cells (defined as <5 cells/μL) 3 to 6 months after rituximab, one patient (patient 3) had partial depletion. Nine to 12 months after rituximab, CD20+ cells remained completely depleted in three patients, partially depleted in one patient, and reappeared in two patients.
While none of the six patients had alloantibodies in their pretransplant serum (neither in the panel reactive lymphocytotoxicity assay with T or B lymphocytes nor in ELISA), de novo anti-HLA serum antibodies were detected in five of six patients at the time of biopsy (Table 3). Patient 2 had DSA in the AbIdent test while the test result in the less-sensitive AbScreen assay was negative. In four of these five patients (patients 2, 3, 5, and 6), the anti-HLA antibodies were DSA. Follow-up serum samples after antihumoral therapy were available in all patients; the antibodies persisted in three patients (patients 3, 5, and 6; Table 3) 12 months posttherapy and declined below the detection limit in one patient (patient 2). Patients 5 and 6, who did not respond to therapy, showed a high reactivity in the panel-reactive lymphocytotoxicity assay against B cells and DSA at the time of biopsy.
The time course of graft function and of circulating anti-HLA antibodies in two individual patients is depicted in Figure 3. In patient 4, circulating anti-HLA class I and class II antibodies peaked at the time of diagnosis of CAMR; the respective titers dropped approximately threefold in response to antihumoral therapy (Fig. 3A). Concomitantly, graft function improved markedly. In patient 5, the anti-HLA class I antibody titer dropped promptly in response to antihumoral therapy, whereas the anti-HLA class II antibody titer decreased only slowly (Fig. 3B). Graft function in this patient did not recover. After graft nephrectomy, 13 months after initiation of antihumoral therapy, the anti-HLA class I antibody titer sharply increased, presumably because this antibody was not absorbed any more by the rejecting graft.
The main finding of this prospective pilot trial is that CAMR leading to progressive loss of renal transplant function in pediatric recipients can successfully be treated with an antihumoral regimen consisting of IVIG and rituximab. Two of six patients did not respond; they showed the highest degree of transplant glomerulopathy and pronounced interstitial inflammation. This observation is consistent with findings in the literature that the development of glomerulopathy in the renal allograft is associated with poor graft outcome (10). The finding that the two nonresponders had the highest degree of transplant glomerulopathy may suggest that rituximab/IVIG may not be able to reverse more advanced CAMR. This may implicate that antihumoral treatment should be applied already at an earlier stage, for example, on the basis of antibody monitoring or protocol biopsies.
Our rationale for treatment of CAMR with the combination of IVIG and rituximab was the following: first, we sought to rapidly enhance the clearance of circulating DSA by IVIG, because rituximab does not appear on its own to reduce DSA titers (5). There are numerous proposed mechanisms how IVIG exerts its immunomodulatory action. They include modification of circulating alloantibody concentration through induction of antiidiotypic circuits, antigen binding through the Fab part of the immunoglobulin molecule, Fc receptor-mediated interaction with antigen-presenting cells to block T- and B-cell activation, and inhibition of complement activity (11). Second, we attempted to prevent new antibody production by rituximab, which depletes B cells as precursors of mature plasma cells in the circulation and the lymphoid tissue, prevents B-cell proliferation, and induces apoptosis and lysis of B cells through complement-dependent and -independent mechanisms (12). Consequently, it is expected that antibody formation will be suppressed. Rituximab- induced B-cell depletion generally lasts 6 to 12 months in more than 80% of patients, although the degree of depletion is highly variable and is observed for up to 24 months in some individuals (13). An additional potential mechanism of action of rituximab is the direct targeting of CD20-positive cells that infiltrate the graft (14). In four of six patients in our case series, more than 10% infiltrating CD20-positive cells were found in the biopsy. Our data do not allow us to differentiate to which extent these proposed mechanisms of action contributed individually to the overall therapeutic efficacy.
A limitation of our pilot study is the lack of an untreated control group for comparison. However, in view of the unfavorable outcome of untreated CAMR, we felt that it was ethically not justified to leave some patients untreated. Nevertheless, the beneficial clinical course reported for some of the included patients could provide a useful basis for the design of a randomized controlled study. Such a study will be necessary to clarify the actual efficiency of treatment and will therefore be a prerequisite for implementation of this or of similar antihumoral strategies for treatment of CAMR.
The precise mechanism leading to CAMR in renal transplant patients has not been elucidated. A potential reason for the development of this disease is a late alloimmune response as a result of under-immunosuppression with CNI or patient noncompliance, which occurs in about a fourth of recipients (15). In this context, it is noteworthy that four of six patients in our case series had low CNI exposure or were treated with a CNI-free regimen because of drug toxicity.
DSA against mismatched HLA specificities were detectable in the circulation in four of six patients in this series. However, failure to demonstrate DSA does not rule out a contribution of antibodies to the pathologic process, because absorption of antibodies by the allograft may result in a lack of circulating DSA. Martin et al. (16) investigated anti-HLA antibodies by FlowPRA in 20 kidney transplant recipients who underwent transplant nephrectomy. Whereas 42% had DSA in their sera at 1 year posttransplant and 32% at the time of nephrectomy, 74% of nephrectomy eluates and postnephrectomy serum samples showed DSA, indicating that in some cases anti-HLA antibodies were bound to the allograft and therefore were not detectable in the serum. In agreement with these findings, we observed an increase in HLA class I antibody reactivity in patient 5 after transplant nephrectomy 20 months after initiation of antihumoral therapy (Fig. 3B). Alternatively, DSA against non-HLA antigens or HLA-DP could explain the missing ELISA reactivity in the presence of increased cytotoxic anti-B-cell reactivity and ongoing antibody-mediated rejection, for example, in patient 1 (Table 3). In contrast to cytotoxicity testing, these two antibody specificities are not covered by the commercial ELISA assays, and there is increasing evidence that anti-DP and non-HLA immunity are involved in the rejection of kidney allografts (17–19).
Three of six patients in this case series of CAMR showed histologic lesions classified as transplant glomerulopathy. Hence, not all patients with de novo DSA develop transplant glomerulopathy, indicating not only that transplant glomerulopathy may develop through B-cell-mediated immune mechanisms but also other factors, such as T cells, may be involved in disease progression, along with antibody- mediated allograft damage (10).
Other authors have reported on specific therapeutic interventions for CAMR. Attempts of reduction of antidonor antibody synthesis by combined therapy with TAC and MMF is discussed controversially. Theruvath et al. (20) described four renal allograft recipients with chronic humoral rejection, based on the criteria (i) progressive rise in serum creatinine over 12 months; (ii) typical pathologic features by light microscopy, (iii) widespread C4d deposits in PTC by immunofluorescence, and (iv) detection of de novo DSA at the time of biopsy. Maintenance immunosuppression with CsA, prednisone, and azathioprine (n=3) or prednisone and azathioprine (n=1) was switched to rescue therapy with TAC and MMF, which was associated with a rapid and sustained decrease in antibody titers. In two patients, DSA became undetectable after 9 months, and a repeat biopsy performed after 12 months revealed a decrease in C4d deposition in PTC. Graft function stabilized in three patients, whereas one patient lost his graft 12 months later. In contrast, Schwarz et al. (21) reported on 11 recipients with C4d-positive chronic allograft nephropathy on CsA-based immunosuppression who were converted to TAC, and if not part of basal therapy to MMF. This treatment regimen (plus antirejection treatment with methylprednisolone in six recipients with additional signs of acute cellular rejection) failed to prevent progressive deterioration of graft function; four patients returned to dialysis after 4 to 18 months. Treatment with TAC and MMF did not affect the time course of alloantibody levels. The authors concluded that this drug regimen is not effective in established C4d-positive chronic allograft dysfunction.
There is only one anecdotal report suggesting efficiency of IVIG in the clinical setting of CAMR (22). This case report demonstrated a decrease in DSAs by IVIG treatment (200 mg IVIG/kg body weight on 2 consecutive weeks) and an improvement in kidney function in a 47-year-old woman with chronic allograft nephropathy, diffuse C4d staining of PTC, and de novo DSAs.
In conclusion, this pilot study demonstrates that CAMR in pediatric renal transplant recipients can be treated successfully and safely with a combination of IVIG and rituximab. This preliminary observation should encourage more extensive studies to evaluate this new treatment strategy.
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