Rejection of kidney allografts occurs through alloreactive T cells and alloantibodies. Historically, alloantibodies were used as a diagnostic tool to measure overall or donor-specific sensitization using panel reactive antibody (PRA) and donor-specific crossmatch tests, respectively (1). The description of deposition of the complement component C4d on the endothelium of peritubular capillaries (PTC) eventually established acute antibody-mediated rejections (AMR) as a pathologically clearly defined entity (2). Later, solid-phase assays allowed more sensitive detection of donor-specific antibodies (DSA) previously not found using complement-dependent cellular assays (3). These diagnostic tools led to a new classification of acute kidney allograft rejection proposed by the Banff consensus conference (4). Patients suffering from acute AMR according to those criteria (rapidly deteriorating graft function, C4d positivity in PTCs with additional histologic criteria, and detection of DSA) have been successfully treated with plasmapheresis, intravenous immunoglobulins (IVIG), intravenous (IV) steroids, switch to a tacrolimus/mycophenolate mofetil, and anti-thymocyte globulin in various combinations. With this approach 70% to 80% of allografts could be saved (5).
Only in the past 5 years, a potential role of alloantibodies also for chronically deteriorating graft function has been postulated. Retrospective studies demonstrated that de novo appearance of DSA was associated with poor graft outcome (6). One study in more than 2000 patients prospectively established the risk of circulating alloantibodies for graft survival after 1 and 2 years (7). Also, antibodies against minor histocompatibility antigens such as MICA may be associated with a poorer graft outcome as demonstrated recently by Opelz and coworkers (8). Finally, it was again the deposition of C4d in chronically deteriorating grafts that allowed the definition of a new entity of chronic AMR (9). Histologic lesions associated with chronic AMR are transplant glomerulopathy and diffuse C4d deposition in PTCs. Transplant glomerulopathy seems to be associated with anti-class II DSA and has a poor graft outcome (10, 11). Also, chronic AMR is now included in the newest update of the Banff classification with the following criteria: (1) transplant glomerulopathy and severe PTC basement membrane multilayering, interstitial fibrosis and tubular atrophy with or without PTC loss, and fibrous intimal thickening in arteries without internal elastica duplication; (2) diffuse C4d deposition in PTCs; and (3) presence of DSA (12). Not all these criteria are always fulfilled in an individual patient at every given time point. Thus, Colvin (6) recently suggested four stages of chronic AMR with stepwise development of circulating DSA (stage I), C4d deposition in PTCs (stage II), allograft pathologic condition (stage III), and deteriorating graft function (stage IV). No therapeutic strategies for chronic AMR have been established. However, based on the pathophysiologic condition of this rejection process and efficacy of rituximab, a chimeric monoclonal anti-CD20 antibody directed against B cells, in antibody-mediated autoimmune diseases (13), a combination treatment with rituximab/IVIG represents a logical approach. A first pilot study using such an approach in pediatric renal allograft recipients was recently reported (14).
Here, we report on four consecutive patients with diagnosis of chronic AMR in different stages 1 to 27 years after kidney transplantation, who were treated with a combination of rituximab/IVIG. Patient characteristics are given in Table 1. A kidney biopsy and measurement of DSA (by Luminex technology) due to slowly deteriorating graft function was performed. On diagnosis of chronic AMR, all patients received IV steroid pulses (500–1000 mg once daily for 3 to 5 days) and rituximab (375 mg/m2 once on day 1), whereas IVIG (0.4 g/kg once daily on day 2 to 5) was given only to patient 1 to 3. The rationale for choosing this treatment was as follows: (1) steroid pulses were given to cover concomitant cellular rejection which was proven or suspected by histology in 3 of 4 patients (see below); rituximab was given to target the B-cell compartment, and the dose was chosen based on the experience with this drug during induction therapy in ABO-incompatible kidney transplantation, where complete depletion of peripheral B cells was described in 43 of 49 patients (15); IVIG was given because of its postulated immunomodulatory effects, and the dose was chosen according to the high IVIG desensitization protocols, where up to 2 g/kg were given (16).
Six months before biopsy, graft function significantly deteriorated in all four patients by 19% to 57% compared with baseline glomerular filtration rate (Table 1, Fig. 1A). On treatment with rituximab/IVIG, a significant improvement of graft function within the first 3 months posttreatment was observed in all four patients and was maintained until 6 months after therapy. Twelve months after rituximab treatment, patient 2 had acute vascular rejection with deterioration of graft function. He received another course of IV steroids and responded. The other three patients stabilized graft function on the level 6 months before rituximab/IVIG.
Patient 1 has had two previous biopsies 6 and 7 months after kidney transplantation showing acute T-cell-mediated rejection Banff IIA, transplant glomerulitis, peritubular capillaritis, chronic allograft arteriopathy, and negative C4d, which initially responded to treatment with steroids and anti-thymocyte globulin. A second deterioration of graft function at 1 year after transplantation led to the current biopsy, which again showed acute T-cell-mediated rejection Banff IIA (Fig. 2, Panel 1BB), chronic allograft arteriopathy (Fig. 2, Panel 1B), together with minimal transplant glomerulitis (Fig. 2, Panel 1AA) and peritubular capillaritis. Despite the fact that C4d was negative, chronic AMR was suspected based on detection of low-level class II DSA, deteriorating allograft function refractory to T-cell-targeted antirejection treatment with steroid pulses and thymoglobulin, and the mentioned biopsy findings (Table 1, Fig. 2).
The next three patients showed diffuse C4d positivity in PTCs. At 27 years after transplantation, patient 2 had transplant glomerulopathy (Fig. 2, Panel 2B), focal segmental glomerular sclerosis, and hyaline arteriolar thickening, the latter consistent with calcineurin inhibitor arteriolopathy. He developed significant proteinuria (up to 5 g/day). In contrast, patient 3 at 2.5 years after transplantation showed interstitial fibrosis and tubular atrophy with accompanying infiltrates and focal minimal tubulitis in nonscarred areas (not diagnostic for tubulointerstitial rejection; Fig. 2, Panel 3A/B) and one artery with an adherent lymphocyte to an endothelial cell, suspicious but not diagnostic of acute vascular rejection. In addition, minimal PTC basement membrane multilayering was found. Patient 4 at 12 years posttransplantation showed mainly interstitial fibrosis/tubular atrophy with prominent lymphocytic B-cell rich infiltrates in lymphoid aggregates and plasma cells mostly in scarred areas (Fig. 2, Panel 4A–C), with focal edema, interstitial infiltrates and tubulitis (borderline changes according to Banff), and calcineurin inhibitor arteriolopathy.
Two patients showed sensitization by PRA, and all four had DSA: patient 1 had low-level [mean fluorescence intensity (MFI <500)] and patients 3 and 4 had intermediate-level (MFI <5000) anti-class II antibodies, whereas patient 2 had high-level (MFI >5000) anti-class I antibodies. On rituximab/IVIG treatment, DSA were unchanged in patients 1 and 4, whereas a significant drop in DSA levels was observed in patients 2 and 3 (Fig. 1B). PRA turned negative in patient 2.
Three patients underwent therapy with rituximab/IVIG without side effects. However, patient 3 develop progressive respiratory failure 6 weeks posttreatment, from which he became oxygen dependent for 4 months. Computed tomography showed acute interstitial lung disease. No infectious agent was identified despite two bronchoalveolar lavages. Therefore, the patient received presumable diagnosis of rituximab-induced interstitial pneumonitis (17), which was treated with steroids and ameliorated slowly over the next 6 months. Currently, the patient is still exercise limited but not oxygen dependent.
Safety of rituximab in this indication is indeed a concern. Most of the available data on side effects come from oncologic trials in patients receiving concurrent chemotherapy. However, the population of transplant recipients is heavily immunosuppressed (usually with a triple therapy) on the long run, and infectious complications such as progressive multifocal leucoencephalopathy may become a concern. A careful monitoring and reporting of such complications in transplant recipients is certainly necessary similar to patients with autoimmune diseases (18).
Rituximab is a useful therapeutic agent in a various autoimmune diseases mediated by B cells and antibodies (13, 19). In the context of transplantation, it was successfully used in occasional patients with steroid-resistant allograft rejection (20) and was studied in a small series of patients with acute AMR without clear proof of efficacy, because in this study the outcome was equal to the reported outcome of protocols without rituximab (21). This is not surprising for two reasons: (1) in acute AMR circulating DSA directly leads to allograft destruction by the complement activation; plasma cells producing those antibodies are CD20-negative and therefore not affected by rituximab therapy. (2) Rituximab efficiently depletes B cells in peripheral blood; in contrast, recent reports demonstrated that depletion in secondary and tertiary lymphoid structures is far less efficient and may not affect an ongoing localized humoral immune response (15, 22). Our patient 4 may serve as an example for this. However, B cells are not just plasma cell precursors, but represent an important population of antigen-presenting cells particularly efficient in the situation of a sensitized recipient, because they have specific immunoglobulin as an antigen-specific receptor on their surface, which leads to efficient uptake and presentation of donor antigens to T cells (23). Indeed, an increased frequency of alloantigen-specific B cells in sensitized recipients has been reported (24). Therefore, targeting these B cells will also interfere with activation of indirectly alloreactive T cells, which play an important role in chronic allograft rejection. In sensitized allograft recipients with DSA, sensitization has always occurred on the level of B and T cells, because B cells need T help to produce alloantibodies of IgG isotype as measured by the Luminex technology. Therefore, a combined pathogenesis of rejection must always be postulated, even if not all the pathologic criteria are fulfilled. Indeed in our study, patient 1, 3, and 4 had histologic signs compatible with combined chronic humoral and acute cellular rejection (the latter proven in patient 1, suspected in patient 3, and borderline in patient 4), and this may explain the efficacy of rituximab even without a measurable drop of DSA titers (in patients 1 and 4).
The immunomodulatory effects of IVIG are multiple, and the exact mechanisms are not elucidated. However, effective alloantibody inhibition by IVIG was shown in the context of desensitization protocols only relying on high dose IVIG treatment (25). Recently, it has been reported that the combination of rituximab/IVIG for desensitization of kidney allograft recipients on the waiting list is effective and can increase the rate of transplantation by 100% (26). These data are in line with our observations in the context of chronic AMR.
Taken together, we report on four patients with chronic AMR, who on therapy with rituximab/IVIG significantly improved their graft function 3 and 6 months after treatment. In two patients DSA levels significantly dropped, whereas in the other two patients no change was observed. One patient had possibly severe rituximab-associated lung toxicity. Thus, rituximab/IVIG may be a useful strategy for treatment of chronic AMR, but further randomized multicenter studies are necessary to establish its efficacy and safety profile for this indication.
1. Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med
1969; 280: 735.
2. Feucht HE, Schneeberger H, Hillebrand G, et al. Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int
1993; 43: 1333.
3. Patel AM, Pancoska C, Mulgaonkar S, et al. Renal transplantation in patients with pre-transplant donor-specific antibodies and negative flow cytometry crossmatches. Am J Transplant
2007; 7: 2371.
4. Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteria—An addition to the Banff 97 classification of renal allograft rejection. Am J Transplant
2003; 3: 708.
5. Venetz JP, Pascual M. New treatments for acute humoral rejection of kidney allografts. Expert Opin Investig Drugs
2007; 16: 625.
6. Colvin RB. Antibody-mediated renal allograft rejection: Diagnosis and pathogenesis. J Am Soc Nephrol
2007; 18: 1046.
7. Terasaki PI, Ozawa M. Predictive value of HLA antibodies and serum creatinine in chronic rejection: Results of a 2-year prospective trial. Transplantation
2005; 80: 1194.
8. Zou Y, Stastny P, Susal C, et al. Antibodies against MICA antigens and kidney-transplant rejection. N Engl J Med
2007; 357: 1293.
9. Solez K, Colvin RB, Racusen LC, et al. Banff ‘05 Meeting Report: Differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (‘CAN'). Am J Transplant
2007; 7: 518.
10. Cosio FG, Gloor JM, Sethi S, et al. Transplant glomerulopathy. Am J Transplant
2008; 8: 492.
11. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: Subclinical incidence and association with alloantibody. Am J Transplant
2007; 7: 2124.
12. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: Updates and future directions. Am J Transplant
2008; 8: 753.
13. Levesque MC, St Clair EW. B cell-directed therapies for autoimmune disease and correlates of disease response and relapse. J Allergy Clin Immunol
2008; 121: 13.
14. Billing H, Rieger S, Ovens J, et al. Successful treatment of chronic antibody-mediated rejection
with IVIG and rituximab
in pediatric renal transplant recipients. Transplantation
2008; 86: 1214.
15. Genberg H, Hansson A, Wernerson A, et al. Pharmacodynamics of rituximab
in kidney allotransplantation. Am J Transplant
2006; 6: 2418.
16. Vo AA, Toyoda M, Peng A, et al. Effect of induction therapy protocols on transplant outcomes in crossmatch positive renal allograft recipients desensitized with IVIG. Am J Transplant
2006; 6: 2384.
17. Liu X, Hong XN, Gu YJ, et al. Interstitial pneumonitis during rituximab
-containing chemotherapy for non-Hodgkin lymphoma. Leuk Lymphoma
2008; 49: 1778.
18. Calabrese LH, Molloy ES. Progressive multifocal leucoencephalopathy in the rheumatic diseases: Assessing the risks of biological immunosuppressive therapies. Ann Rheum Dis
2008; 67 (suppl 3): iii64.
19. Eisenberg R, Albert D. B-cell targeted therapies in rheumatoid arthritis and systemic lupus erythematosus. Nat Clin Pract Rheumatol
2006; 2: 20.
20. Becker YT, Becker BN, Pirsch JD, et al. Rituximab
as treatment for refractory kidney transplant rejection. Am J Transplant
2004; 4: 996.
21. Faguer S, Kamar N, Guilbeaud-Frugier C, et al. Rituximab
therapy for acute humoral rejection after kidney transplantation. Transplantation
2007; 83: 1277.
22. Thaunat O, Patey N, Gautreau C, et al. B cell survival in intragraft tertiary lymphoid organs after rituximab
2008; 85: 1648.
23. Noorchashm H, Reed AJ, Rostami SY, et al. B cell-mediated antigen presentation is required for the pathogenesis of acute cardiac allograft rejection. J Immunol
2006; 177: 7715.
24. Zachary AA, Kopchaliiska D, Montgomery RA, et al. HLA-specific B cells. II. Application to transplantation. Transplantation
2007; 83: 989.
25. Jordan SC, Vo AA, Nast CC, et al. Use of high-dose human intravenous immunoglobulin
therapy in sensitized patients awaiting transplantation: The Cedars-Sinai experience. Clin Transpl
26. Vo AA, Lukovsky M, Toyoda M, et al. Rituximab
and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med
2008; 359: 242.