As short-term allograft survival rates have steadily improved in recent years, chronic rejection, also termed “chronic allograft nephropathy,” has become the major limitation to long-term allograft function (1–4). Alloantibodies to donor HLA class I or class II antigens have been associated with chronic rejection of various transplanted organs (5–7). In renal transplant recipients, posttransplant production of anti-HLA class II alloantibodies is associated with chronic rejection, possibly as a manifestation of alloresponses via the indirect pathway (3,8). Posttransplant production of alloantibodies can predate the clinical manifestations of chronic rejection, suggesting that humoral mechanisms are a cause rather than a consequence of chronic rejection (6,9,10). More direct proof of the pathogenic role of alloantibodies has been obtained in animal experiments in which antibodies are necessary and sufficient for the development of chronic allograft rejection (11,12).
The pathological findings of chronic rejection in renal allografts are characterized by arterial intimal fibrosis, widespread glomerular basement membrane duplication (“chronic transplant glomerulopathy”), tubular atrophy, and interstitial fibrosis (13). Multilayering of the basement membrane in peritubular capillaries has been found to correlate with the chronic glomerular changes in some series, and this lesion appears to be a reflection of antibody-mediated rejection (13–15). We have shown recently that complement C4d deposits in peritubular capillaries of renal allograft biopsies are also typically found in patients with acute and chronic humoral rejection (but not with other causes of allograft dysfunction) and are associated with detectable donor specific alloantibodies in patients’ sera (16,17). Overall about 60% of patients with histological features of chronic rejection have C4d deposition in peritubular capillaries (17).
Clinically, chronic renal allograft rejection manifests as a slow and progressive decrease in renal function accompanied by hypertension. Low-grade or nephrotic-range proteinuria is often present. These clinical features can present months or years after transplantation (3,4,18,19), depending on many factors including the patient’s immunosuppressive regimen. The prevention or treatment of chronic rejection is currently the most important challenge in the management of not only renal but also heart and lung transplant recipients. To date, no immunosuppressive regimen has been shown to be consistently effective in humans. Over the last decade, newer immunosuppressive agents such as tacrolimus, mycophenolate mofetil, and sirolimus have become available but their efficacy in controlling the process of chronic rejection remains to be determined (20). Preliminary clinical studies have suggested that addition of mycophenolate mofetil with reduction of cyclosporine dosages (21–23), or conversion to tacrolimus (24), might be effective therapeutic strategies. However, in these limited studies, antidonor humoral responses were not studied, making evaluation of the diagnosis and clinical efficacy difficult.
Based on our previous experience with the treatment of acute humoral renal allograft rejection (25), we have evaluated tacrolimus-mycophenolate mofetil “rescue” in four consecutively identified allograft recipients with “chronic humoral rejection.” This was defined as chronic renal dysfunction associated with detectable levels of donor-specific alloantibodies in recipient’s serum and characteristic renal biopsy findings of chronic rejection with C4d deposits in peritubular capillaries. We report successful control of specific antidonor B cell responses with this regimen, a finding that may be important for the treatment and prevention of chronic allograft rejection in humans.
PATIENTS AND METHODS
In 1999, a total of 34 renal allograft biopsies were performed in 30 patients for clarification of the cause of chronic allograft dysfunction at our institution. In all patients C4d staining (see below) was performed. Four renal allograft recipients with chronic humoral rejection were prospectively selected for rescue therapy with tacrolimus and mycophenolate mofetil. Three patients had received initial immunosuppression with cyclosporine, prednisone, and azathioprine and one patient (transplanted in 1983) received prednisone and azathioprine only. The diagnosis of chronic humoral rejection was based on: (1) progressive rise in serum creatinine over 6 to 12 months (2); typical pathological features (“chronic transplant glomerulopathy” and transplant arteriopathy), with less specific features of interstitial fibrosis and tubular atrophy (3); widespread C4d deposits by immunofluorescence in peritubular capillaries; and (4) demonstration of previously undetectable donor specific antibody in recipient sera at the time of biopsy (i.e., all previous cross-matches for T and B cells were negative). Donor-specific antibody testing was requested by the clinical team in the presence of items 1, 2, and 3.
Renal allograft pathology.
Renal tissue was obtained by percutaneous biopsy. Paraffin-embedded tissue sections (2–3 μm) were stained with hematoxylin and eosin and by the periodic acid-Schiff method. Direct immunofluorescence for IgG, IgA, IgM, C3, fibrin/fibrinogen, and albumin was performed as previously described (26). Immunohistological evaluation of C4d deposition in peritubular capillaries was performed using a monoclonal antibody to C4d (clone 10–11; Biogenesis, Sandown, NH) with a three-step immunofluorescence technique (16). Electron microscopic studies were performed as described and examined using a Philips 301 electron microscope (27).
In each patient a T and B cell cross-match was performed at the time of the biopsy (within 48 hr of diagnosis). When donor cells were not available, a surrogate donor cell that shared HLA antigens with the donor was used. Subsequent serial serum samples were collected prospectively to monitor donor-specific antibody titers. Cytotoxic cross-matches were performed using patient sera and immunomagnetic bead-isolated donor lymphocytes. Both anti-human globulin-enhanced T cell and standard complement-dependent cytotoxicity B cell assays were used (28). Panel reactive antibodies were determined using a local frozen cell panel.
In 5 of 30 patients who received a renal allograft biopsy for clarification of the cause of chronic allograft dysfunction, widespread and diffuse C4d deposits in peritubular capillaries were present. In four of five cases with positive C4d deposits, circulating antidonor antibodies could be detected by performing a repeat cross-match with frozen donor lymphocytes. Thus 4 of 30 patients (13%) were found to have antibody-mediated chronic rejection, i.e., chronic humoral rejection (CHR). Repeat cross-matches were also performed in 13 patients (controls) who had no C4d deposits (3 with chronic calcineurin inhibitor toxicity, 4 with nonspecific interstitial fibrosis and tubular atrophy, and 6 with specific recurrent or de novo disease of the graft), and none was found to have circulating antidonor antibodies.
The clinical data of the four recipients with chronic humoral rejection are summarized in Table 1. The diagnosis of chronic humoral rejection was made between 3.9 and 15.7 years after transplantation. One patient (case 1) received a living-related renal transplant from his daughter (one haplotype match) and three patients (cases 2, 3, and 4) received cadaveric transplants (number of HLA A, B, DR mismatches were 5, 4, and 4). All patients were recipients of first transplants and were not sensitized at the time of transplantation (their respective T cell panel reactive antibodies were 0%). Pretransplant donor specific cross-matches were negative. All patients had excellent early allograft function. One patient (case 2) had an episode of steroid-resistant acute cellular rejection at day 6, requiring OKT3 therapy. The mean serum creatinine at the time of diagnosis was 3.9 mg/dl (range 3.3–5.4 mg/dl), and the mean urine protein excretion was 2 g/day (range 0.8–5 g/day). The mean serum creatinine values over the preceding 3 months before initiating rescue treatment were 3.0±0.2 mg/dl (case 1), 3.2±0.3 mg/dl (case 2), 3.3±0.2 mg/dl (case 3), and 5.1±0.5 mg/dl (case 4). In two patients a possible etiological factor in the development of “chronic humoral rejection” could be postulated, that is prednisone tapering (case 3) and noncompliance (case 4). In the three patients receiving cyclosporine, the serum trough levels prior to conversion (average of the last 4 cyclosporine levels), were 116 ng/ml (case 1), 171 ng/ml (case 2) and 81 ng/ml (case 4).
Renal biopsies revealed classic pathological features of chronic rejection by light microscopy (13), namely “chronic transplant glomerulopathy” (in allpatients), transplant arteriopathy (in 4/4) and variable degrees (60–75%) of interstitial fibrosis and tubular atrophy (Table 2 and Fig. 1). Intense staining for complement C4d by immunofluorescence microscopy was detected in a widespread distribution in the peritubular capillaries in all four cases (Fig. 4 A). Other findings by immunofluorescence included deposition of C3 and IgM segmentally in the arteries and arterioles (in four of four). Minimal IgM and C3 staining was noted along the glomerular basement membrane (in two of four) or in the mesangium (in three of four). Electron microscopy demonstrated global or segmental glomerular basement membrane duplication with cellular interposition (in four of four). Although none of the patients had immune complex deposition, rare subendothelial, intramembranous, or mesangial electron dense deposits were noted in two of four patients. Segmental (three of four) or widespread (one of four) foot process effacement was noted. The endothelium was focally reactive with loss of fenestrae in all four cases. Lamination or multilayering of the basement membrane of peritubular capillaries was present in all the cases.
Characterization of the donor-specific antibodies in the four patients revealed IgG directed against HLA class II (n=3) or HLA class I (n=2). One patient (case 4) had both anti-HLA class I and class II antibodies. The antibody specificities are shown in Table 1. Pretreatment antibody titers (in dilution of tested sera) were 1:16 (case 1), 1:8 (case 2 and case 3), 1:64 (case 4, anti-HLA class I), and 1:128 (case 4, anti-HLA class II). In two patients (cases 1 and 2) two different serum samples, drawn within 1 week before starting rescue treatment, were tested for circulating donor-specific antibodies and both sera samples showed the same pretreatment antibody titers (1:16 and 1:8, respectively).
After the diagnosis of “chronic humoral rejection,” rescue therapy with tacrolimus (initial mean dose 0.05 mg/kg/day, aiming for levels of 5–7 ng/ml) and mycophenolate mofetil (initial mean dose 2 g/day) was initiated in all patients, after informed consent was obtained. We intentionally aimed to maintain relatively low serum levels of tacrolimus, in accordance with the concept that mycophenolate mofetil allows a safe reduction of the dose of calcineurin inhibitors in this patient population (4,21–23). Cyclosporine and azathioprine were discontinued 12 hr before starting tacrolimus and mycophenolate mofetil. Prednisone at a dosage of 10–15 mg/day was continued in three patients, and in one patient (case 1), who was not on maintenance steroids at the time of diagnosis, prednisone therapy was reinstituted. The dose of mycophenolate mofetil was subsequently reduced to 1 g/day in three patients, due to gastrointestinal side-effects (n=3) and anemia (n=2). There were no infectious or neoplastic complications. As shown in Figure 2, renal function initially improved in all patients and remained stable in three. In one patient (case 4), who suffered from advanced renal failure with nephrotic-range proteinuria at the time of diagnosis, progressive symptoms of uremia (asthenia, anorexia) 12 months later led the clinical team to discontinue tacrolimus and mycophenolate mofetil and return the patient to hemodialysis.
Serial monitoring of antibody titers revealed that introduction of tacrolimus and mycophenolate mofetil rescue therapy was associated with a progressive and sustained decrease in antidonor antibody titers (Fig. 2). A 50 to 90% fall in antibody titers was observed after 2 months of therapy, which continued thereafter. In two patients (cases 2 and 3), who had initial antibody titers of 1:8, cross-matches became negative after 9 to 12 months, i.e., donor-specific antibodies became undetectable. In case 4, both anti-HLA class I and anti-HLA class II antibodies decreased in parallel after starting rescue therapy. Of note, antibody titers progressively increased in this patient after discontinuing tacrolimus and mycophenolate mofetil although maintaining low dosage prednisone during dialysis therapy (Fig. 3). In one patient (case 2) a surveillance biopsy was performed after 12 months that revealed a significant decrease of C4d deposits in peritubular capillaries, with most areas (more than 80%) demonstrating complete absence of C4d (Fig. 4 B).
The four renal transplant recipients reported herein presented with classic clinical and pathological features of chronic rejection. The detection of donor-specific antibodies in recipient sera and C4d deposits in peritubular capillaries of the biopsy specimens strongly implicate humoral mechanisms in the pathogenesis of the rejection process, a condition that we have recently termed “chronic humoral rejection”(17). The alloantibodies detected in these patients proved to be IgG directed against HLA class I or HLA class II specificities, with serum titers ranging from 1:8 to 1:128. Three of the four patients had circulating anti-HLA class II alloantibodies, a finding that is consistent with previous studies in which it was found that posttransplant production of anti-HLA class II antibodies is associated with chronic rejection (3,8).
Rescue therapy with tacrolimus and mycophenolate mofetil was associated with a sustained decrease in antidonor antibodies, which was evident as soon as 2 months after starting therapy and continued progressively over a 12-month period. In two patients, antidonor antibodies became undetectable and in one patient who underwent a second biopsy 1 year later, the absence of antidonor antibodies in serum was associated with a decrease of C4d deposits in the peritubular capillaries. During treatment with tacrolimus and mycophenolate mofetil, a decrease in total plasma globulin of approximately 15% (not shown) was also detected, suggesting that control of antidonor antibody production was associated with a nonspecific modest decrease in total immunoglobulin synthesis. Interestingly, a tendency to hypogammaglobulinemia has been recently reported in heart and renal allograft recipients treated with tacrolimus and/or mycophenolate mofetil (29,30), indicating that combining these two drugs results in a powerful immunosuppressive regimen that limits both T cell and B cell responses. This is not unexpected since previous studies have reported that mycophenolate mofetil can inhibit in vitro antibody production and in vivo humoral responses (31,32) and, when used in combination with tacrolimus, can limit B cell responses in renal allograft recipients with acute humoral rejection (25). Interestingly tacrolimus, but not cyclosporine, appears to interfere with the metabolism of mycophenolate mofetil, which may increase the biological effects of this drug on alloantibody production (33). In our studies, we have not yet determined whether tacrolimus or mycophenolate mofetil is the more effective agent for inhibiting antidonor antibody synthesis.
The treatment of chronic rejection has remained one of the most important challenges in organ transplantation. Until recently, no drug regimen has been shown to effectively control humoral responses in allograft recipients suffering from this condition (4). In 1997, however, Weir et al. suggested that conversion to mycophenolate mofetil from azathioprine, together with a reduction in cyclosporine dosage, might improve chronic renal allograft dysfunction (21). We and others have confirmed those earlier clinical observations (22,23), but in these studies antidonor humoral responses were not assessed. Furthermore, prospective controlled clinical trials that demonstrate consistently improved graft survival in patients with chronic rejection are still lacking. Tacrolimus rescue (without mycophenolate mofetil) has also been evaluated as treatment for chronic rejection. In a small, nonrandomized study, tacrolimus rescue appeared to stabilize, but not improve, renal function in a majority of patients (24). The lack of significant improvement of allograft function may have been related to relatively advanced lesions of chronic rejection, resulting in serum creatinine levels of more than 3 mg/dl at the start of therapy in most of these patients. In our patients, rescue therapy with tacrolimus and mycophenolate mofetil appeared to stabilize renal function in the short-term, but one patient, who had clinical and pathological evidence of advanced chronic rejection, had to initiate dialysis after 12 months of therapy.
The observations in these four consecutively studied patients suggest that with newer immunosuppressants effective control of antidonor antibody production can now be achieved. This was illustrated in case 4, in whom donor-specific alloantibody titers were significantly reduced after initiation of tacrolimus and mycophenolate mofetil therapy; but after drug discontinuation, there was a marked increase in titers to equal (anti-HLA class I) or higher (anti-HLA class II) than pretreatment levels (Fig. 3). Such limitation of antibody synthesis may be clinically relevant for the treatment of the subset of patients with chronic humoral rejection, however, more work is needed to determine whether combining tacrolimus and mycophenolate mofetil will improve long-term renal allograft function.
It should be emphasized that prevention of chronic rejection remains the priority in organ transplantation, and defining the optimal maintenance immunosuppressive regimen (among other measures such as blood pressure control or lipid lowering) appears to be critical toward achieving this goal. Recently, the immunosuppressive efficacy of tacrolimus and mycophenolate mofetil in combination has been further demonstrated in two prospective randomized clinical trials in renal transplantation, in which acute rejection rates were lowered to approximately 8–15%(34,35). In these studies, however, antidonor humoral responses were not monitored. In the future, immunosuppressive regimens that will specifically control both T cell and B cell responses (i.e., limit antidonor antibody production) may improve long-term graft survival, if the immunoregulatory efficacy of such regimens is not hampered by an increase in infectious, neoplastic or cardiovascular complications.
The authors thank Donna Fitzpatrick and Jessica Soter for their help and their laboratory expertise in the cytotoxicity assays.
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