In recent years, acute renal allograft rejection associated with the development of donor specific antibodies (DSA) has emerged as a clinicopathological entity that carries a poor prognosis (acute humoral rejection, AHR) (1–7). Tubulitis and endothelialitis characterize T cell-mediated rejection (acute cellular rejection, ACR). In contrast, typical pathological features of AHR by light microscopy include neutrophils in peritubular capillaries, vasculitis, and fibrinoid necrosis in vessel walls (6–10) although no single histological feature is considered to be diagnostic.
The risk of graft loss has typically been high in recipients with AHR, with 1-year graft survival rates varying between 15 and 50% despite intensive conventional immunosuppressive therapy (1–4,11,12). We have recently reported that a new approach combining plasmapheresis and “tacrolimus-mycophenolate mofetil (MMF) rescue” can more consistently suppress DSA production and prevent allograft loss in these patients, and, therefore, has the potential to improve the outcome of AHR (4).
The significance of DSA in the diagnosis of renal allograft rejection has been unclear, with 38–100% of the reported patients with DSA suffering from rejection at the time (1,13,14). This variability is likely related to differences in the sensitivity and specificity of the assays used to detect DSA, and demonstrates the need for more specific assays for the diagnosis of AHR. Supporting the initial work of Feucht et al. (15), we recently found that peritubular capillary (PTC) staining for C4d, a durable split product of C4 that indicates activation of the classical pathway of complement, is the marker that best identifies AHR in renal allograft biopsies (10). C4d remains covalently bound to nearby endothelium or basement membrane collagen, thereby providing in situ pathological evidence of an anti-donor humoral response.
The purpose of our study was to determine the incidence of AHR in our transplant population, and to identify its clinical characteristics. We reviewed our experience over a 4-year period (1995–1999) and performed both sensitive and specific techniques for antibody detection, and correlated them with the presence of widespread C4d staining in PTC to identify patients with AHR.
PATIENTS AND METHODS
Patient population and immunosuppression.
Between July 1995 and July 1999, 232 kidney transplants (127 cadaver, 75 living related, and 30 living unrelated) were performed in 143 males and 88 females (1 patient received two transplants). Demographics of the population are shown in Table 1. We excluded 10 recipients who received combined kidney-pancreas (n=5) or kidney-liver transplants (n=5), and 2 patients who underwent tolerance-induction study protocols (16) from the analysis. Baseline immunosuppression included cyclosporine and prednisone, with or without azathioprine, between July 1995 and May 1997 (17); and cyclosporine, prednisone, and mycophenolate mofetil between June 1997 and July 1999. Clinically diagnosed episodes of acute rejection considered in this study were biopsy-confirmed in 89% of cases, using criteria published previously (18). AR was initially treated with daily intravenous methylprednisolone (500 mg), and if no significant improvement in allograft function was seen within 2 or 3 days (“steroid insensitive” rejection), monoclonal (OKT3 mAb or anti-CD2 mAb), or polyclonal antilymphocyte therapy (ATGAM) was administered for 7 to 15 days. Absence of improvement in allograft function was defined by no decrease in serum creatinine from its peak value during the rejection episode. Clinical information was collected by chart review and using the Transplantation Unit database.
Detection of DSA.
Pre- and post-TX DSA tests performed included complement-dependent-cytotoxic (CDC) and flow cytometric assays. For CDC, T and B cells were isolated using immunomagnetic beads (Dynal, Lake Success, NY). Four wash antihuman-globulin-enhanced (AHG) CDC assays were done with T cells and standard and four wash CDC at 37°C with B cells as previously described (19,20). IgG was differentiated from IgM using dithiothreitol (DTT). For patients receiving OKT3, the murine monoclonal antibody was removed using immunomagnetic beads bound with sheep anti-mouse Ig (21). For patients receiving ATG, cross-match tests were performed by two-color flow cytometry using phycoerythrin-labeled anti-CD3 (T cells) or anti-CD19 (B cells) mAb and affinity purified fluorescein-isothiocyanate-conjugated goat-antihuman IgG (Fab) that did not cross-react with horse or mouse immunoglobulin. Selected patients were tested by both cytotoxicity and flow cytometry assays. We have found that our AHG and flow cytometry assays have similar sensitivity (22).
For pretransplant cross-match, sera from the previous 6 months (for sensitized, retransplant, or living donor recipients) or the most recent serum only (for unsensitized recipients of cadaveric transplants) were tested against peripheral blood lymphocytes from living donors or lymphocytes from cadaver lymph nodes or spleen. Panel-reactive antibodies (PRA) against T and B cells were determined by cytotoxicity using local frozen cell panels (40 and 20 cells, respectively).
Pretransplant PRA were determined monthly with T cells and quarterly with B cells. Posttransplant PRA were determined when donor or appropriate surrogate cells were not available for cross-match and to identify HLA specificity of DSA in select patients. In some cases, HLA specificities were determined or confirmed with purified HLA antigens using an ELISA technique (LAT, One Lambda Inc., Canoga Park, CA). Posttransplant assays for DSA testing were performed either prospectively (n=38 patients) using sera collected at the time of rejection or retrospectively (n=40 patients) with sera stored at −30°C at the time of rejection. Three patients did not have sera available for DSA testing. Donor cells were stored at −85°C at the time of the transplant or were collected fresh from some living donors.
Renal biopsies were obtained at the time of rejection from 72 patients and 70 had sufficient frozen tissue available for immunopathological studies. Paraffin-embedded tissue sections (2–3 μm) were stained with hematoxylin and eosin and periodic acid-Schiff stains. Direct immunofluorescence using mono-specific rabbit antisera against human IgG, IgM, IgA, C3, albumin, fibrin/fibrinogen, and a sensitive three-step immunofluorescence technique for C4d staining was performed on frozen kidney tissue as described (10). The details of the pathologic studies are described in a second report (Mauiyyedi et al., manuscript in preparation).
Parametric variables are expressed as mean±SD, and nonparametric variables are expressed as medians and ranges. Statistical tests were performed with the assistance of the SAS package program, and included χ2 for qualitative variables; ANOVA with test post hoc (Scheffe test) for the relationship between qualitative and quantitative parameters; and Mann-Whitney and Kruskal-Wallis H for nonparametric variables.
During the study period, 81 of the 232 renal allograft recipients (35%) suffered at least one episode of acute rejection within the first 3 months posttransplantation. The AR episode proved to be steroid insensitive in 51 of these 81 cases (sAR group: 63% of total AR cases and 22% of the total study population). Nine of these 51 patients suffered two episodes of acute rejection within the first 3 months posttransplant and at least one of the episodes was steroid insensitive. The remaining 30 patients (mild acute rejection group: 37% of AR) had mild, steroid-sensitive rejection episodes. As detailed in Table 1, there were no significant differences in sex, race, etiology of end-stage renal disease, and time on dialysis before transplantation between patients with no AR and those with sAR or mild steroid-sensitive AR. Age was lower in patients with mild AR versus those with no AR. Patients with sAR were on dialysis longer pretransplant than were patients with mild AR.
Steroid-Insensitive Acute Rejection Group
Pretransplant assays for DSA.
All patients (n=51) received their transplants with negative immediate pretransplant T cell cross-matches by AHG CDC. At the time of rejection in these recipients, we retested the pretransplant samples, 28 by AHG and 23 by both flow cytometry and AHG. We found that 2/51 patients had very weakly positive retrospective T cell cross-matches with pretransplant sera using frozen instead of fresh donor cells (23). Both flow cytometry and AHG CDC confirmed these. Forty-four of 51 patients had negative B cell CDC cross-match as well and the remaining patients had pretransplant PRA-B equal to 0%.
Two primary transplant recipients had very weakly positive flow cross-matches (one T, one B) but CDC was consistently negative and neither developed posttransplant DSA. One highly sensitized recipient had a borderline positive AHG cross-match, but was negative by flow with both T and B cells.
Posttransplant assays for DSA.
A total of 165 serum samples from these 51 patients were tested. Sera were collected at the time of acute rejection, except for one patient tested within 2 weeks and one patient tested within 1 month of the rejection episode. All 51 patients had a DSA study performed: 26 by cytotoxicity, 14 by flow, and 11 by both techniques. We found DSA in serum samples at the time of rejection in 19 of these 51 recipients (37% of patients in the steroid-insensitive AR group). Seventeen of 19 developed IgG DSA. At the time of the first positive cross-match, eight were positive with both T and B cells, five were positive with T cells only (B cells were negative or untested), and four were positive with B cells only (T cells were negative). Two of 19 had IgM only against donor B cells. We could determine DSA specificity for 10/19 patients using PRA tests: 6 recipients had anti-HLA class I DSA, 3 had anti-HLA class II, and 1 had a positive T cell cross-match but only antibody specificity against class II antigens could be defined. These and other immunological data are shown in Table 2. The remaining 32 patients were not found to have DSA at the time of rejection. Thirty-one had negative T cell cross-matches and one had PRA-T equal to 0%, 26 had negative B cell cross-matches, and 5 had PRA-B equal to 0%.
The test was performed in biopsies taken to diagnose the cause of the renal allograft dysfunction, except for one patient whose only available frozen tissue was obtained approximately 1 month after the rejection episode. All 17 patients who developed IgG DSA at the time of rejection had widespread and diffuse C4d deposits in PTC in their biopsies. One of the two recipients who formed IgM DSA against donor B cells showed widespread C4d deposits in PTC. The other patient had typical pathological features of AHR by light microscopy (neutrophils in PTC and interstitium, fibrinoid necrosis of arterioles, and acute tubular injury), but only demonstrated focal C4d deposits in PTC.
In the 32 recipients who did not develop DSA after the transplant, two patients had widespread C4d deposits in PTC (6%). These two patients had no detectable serum antibodies against donor T or B cells as tested by flow cytometry and cytotoxicity, respectively. The serum from one of these patients was further tested against a panel of keratinocytes in an attempt to identify non-HLA antibodies, but no such antibodies could be detected (24). The renal biopsy pathology of both these patients was similar to the other C4d+ AHR cases, with prominent neutrophils in PTC, glomerular capillaries, and tubules. One of these patients lost the graft six weeks posttransplant, and the nephrectomy specimen had severe fibrinoid necrosis of arterioles and endarteritis, in addition to capillary neutrophils.
Clinical features of patients with acute humoral rejection versus acute cellular rejection.
The detection of both serum DSA and C4d deposits in PTC at the time of rejection allowed us to define 18 recipients as having primarily AHR (DSA+C4d+) and 30 recipients, with severe acute cellular rejection (severe ACR) who had no apparent humoral component (DSA−C4d−). Table 3 compares the clinical and immunological data in patients with AHR versus those with severe ACR, and also shows the characteristics of mild acute rejection patients. The number of HLA mismatches was similar. However, recipients in the AHR group were significantly more frequently sensitized, as measured by historical and current PRAs, (P <0.05), and more patients in this group were recipients of re-transplants (7/18 vs. 2/30, P <0.05). Cadaveric donors were more common in the AHR group than in the severe ACR group (P <0.05). The onset of rejection in the AHR group was somewhat earlier; however, this did not reach statistical significance. The percentage of recipients with delayed graft function and the percentage of patients receiving antilymphocyte therapy as induction immunosuppression did not differ in the two groups. Moreover, there was no significant difference in the incidence of AHR between patients receiving different baseline immunosuppression regimens (cyclosporine and prednisone, with or without azathioprine, versus cyclosporine, prednisone, MMF). Both of the AHR patients with a retrospective weak positive pretransplant cross-match (Table 2, patients 1 and 17) had primary nonfunction (the grafts never functioned).
Rejection was more often resistant to antilymphocyte therapy (no decrease in serum creatinine after 3 to 4 days of treatment) in recipients suffering AHR than in the severe ACR group (67 vs. 3%, P <0.001) and their graft outcome was significantly worse (78 vs. 97%, P <0.05). However, serum creatinine values at 6 months for functioning grafts were not significantly different.
We found that three patients with steroid-insensitive acute rejection did not fulfill the strict definition of either AHR (DSA+C4d+) or severe ACR (DSA−C4d−). One patient developed an IgM DSA but did not have widespread C4d deposits in PTC (Table 2, patient 11), and two patients had widespread C4d deposits but no detectable serum DSA. The patient who developed IgM DSA of unclear specificity presented with refractory acute rejection and pathological features of AHR, and he was treated according to the protocol below. Of the two patients with C4d and no DSA, one lost the graft due to ongoing rejection at 6 weeks despite OKT3 and ATG treatment, although the other patient has good allograft function 16 months posttransplant (serum creatinine 1.6 mg/dl).
Treatment of rejection associated with DSA.
Thirteen of the 19 patients (67%) found to have circulating DSA at the time of acute graft dysfunction had a clinical picture of refractory acute rejection, that is a rejection episode resistant to both steroid boluses and antilymphocyte therapy. Primary nonfunction occurred in 2 of the 13 patients (as mentioned above). One patient, the initial case to be identified, did not receive the protocol treatment described below and lost the graft at 6 months due to ongoing rejection despite sequential courses of therapy with OKT3 and ATG.
In 10 consecutive recipients with refractory AHR, a regimen of plasmapheresis (PPh) with “tacrolimus-mycophenolate mofetil (MMF) rescue” was initiated (4). Daily PPh (five sessions, 1.3 volume per exchange), followed by alternate day PPh (five sessions) if necessary, were combined with tacrolimus (mean 0.11 mg/kg/day, with target plasma trough levels of 10–15 ng/ml) and MMF (2 g/day initial dose). Sequential DSA titers were measured throughout the treatment regimen. This therapeutic strategy significantly decreased circulating DSA over 2 to 4 weeks, with reversal of the rejection process in 9/10 patients. One patient (Table 2, #4) suffered from recurrent AHR, i.e., another episode of renal dysfunction with increase in DSA titers, and was successfully treated with additional PPh (4). At the end of PPh, polyclonal immunoglobulin (0.4 g/kg) was administered i.v. to limit the risk of infectious complications. The total number of PPh treatments was guided by response to therapy (improvement in allograft function) and serum levels of DSA. Of note, in one patient low levels of DSA were detected for 6 months after PPh, yet renal function had returned to normal.
Currently, with a mean follow-up of 29 months (range 9 months to 4.2 years), patient and graft survival are 100 and 80% (mean serum creatinine 1.5±0.4 mg/dl), respectively. The only graft losses were due to refractory AHR and antiglomerular basement membrane disease at day 10 (third transplant in a recipient with underlying Alport’s disease) and to allograft glomerulopathy associated with CMV viremia at day 290. Pulmonary cryptococcal infection occurred in one patient, and was successfully treated by lobectomy and prolonged fluconazole administration at 3 years posttransplant. There were no neoplastic complications in patients who received PPh and tacrolimus-MMF rescue.
Six of the 19 patients with AHR responded to i.v. methylprednisolone boluses and antilymphocyte therapy and therefore did not receive the therapeutic rescue regimen. Five of the six currently have good allograft function and one died (with a functioning graft) of metastatic bladder cancer 40 months posttransplant. Of note, low titers of DSA were present at the time of rejection in five of these six patients. This was in contrast to patients with refractory AHR, where only 2 of 13 had low titer antibodies.
Steroid-sensitive acute rejection group
Thirty of 81 patients with AR presented with steroid-sensitive acute rejection episodes (Table 1). Sera taken at the time of rejection were available in 27 patients; 23 had negative T cell cross-matches and 4 had PRA-T equal to 0% at the time of rejection; 18 had negative B cell cross-matches, and 9 had PRA-B equal to 0%. A total of 21 recipients had allograft biopsies at the time of rejection, and 19 had frozen tissue available for C4d staining. One biopsy showed C4d staining in PTC, but unfortunately there was no available serum sample to test for DSA in this patient.
Clinical and immunological data for this group are shown in Table 3. Patients with mild acute rejection were similar to the steroid insensitive ACR group. However, none had delayed graft function, an unusual finding because the incidence of DGF in our renal transplant population has always approximated 20% in cyclosporin A-treated patients.
In this study, we determined the incidence and clinical characteristics of AHR in kidney transplant recipients over a 4-year period, by using sensitive and specific cross-match techniques and analyzing C4d deposits in renal allograft biopsies from patients with AR. We have also extended our previously reported observations with the use of PPh-tacrolimus-MMF rescue therapy in the subset of patients who present with refractory AHR, a condition that typically carries a poor prognosis (1–4,11,12).
We found an overall incidence of AHR of 7.7% in our renal transplant recipients, as defined by acute rejection associated with DSA. Of the 19 patients who were found to have DSA in posttransplant sera, 18 (95%) also had prominent and diffuse C4d deposits in allograft biopsy PTC, a marker of antibody-mediated rejection (10). This contrasts with patients who developed AR but without serum DSA, who were rarely (6% of biopsies) found to have C4d in PTC. These results confirm that C4d staining of allograft biopsies provides a useful molecular tool to identify the presence of humoral mechanisms of rejection at the time of graft dysfunction. Previous studies have noted the central role of complement activation in allograft damage mediated by DSA (25–27) and therefore the presence of C4d deposition in the biopsies can be taken as evidence supporting a pathogenic role of the circulating DSA.
The typical clinical presentation of AHR was that of early (within the first week) severe rejection (n=11), poorly responsive to both methylprednisolone boluses and antilymphocyte therapy. This is consistent with previous observations, in which AHR carried a poor prognosis with a risk of early graft loss varying from 50 to 85%(1–3,6,11,28). The second most frequent clinical presentation in our series was that of “classic” acute rejection (n=7), i.e., allograft dysfunction occurring after the first week posttransplant.
Four AHR cases had delayed graft function or primary nonfunction. We and others have suggested that delayed graft function or primary nonfunction due to AHR is probably associated with low levels of DSA undetected by the pretransplant cross-match (5,7,29), but that become apparent in recipient sera a few days posttransplantation. This was confirmed in the two patients with primary nonfunction, in whom retrospective cross-matches using frozen cells demonstrated very low levels of DSA (23). These two graft failures did not meet the definition of “hyperacute rejection” (graft loss occurring in minutes or hours), as renal scans revealed persistence of renal blood flow in the early days posttransplant. However, the pathogenic mechanisms of hyperacute rejection or AHR presenting as primary nonfunction are likely to be similar, that is local complement activation after DSA binding to the graft vasculature.
There were also rare cases of primary transplant recipients with no apparent evidence of sensitization but who developed humoral rejection early posttransplant. One example is patient 8, an apparently unsensitized male who received a kidney transplant from his mother, and who was diagnosed with severe rejection and high titer DSA within the first week. Another example is patient 4, who received a kidney from her 32-year-old daughter and developed DSA 7 days posttransplant despite having no detectable antibodies pretransplant. This case is similar to reports of accelerated rejection in previously pregnant women who received cadaver donor kidneys that shared mismatched paternal antigens (30).
One striking finding of our study is that more than one-third of patients with steroid-insensitive acute rejection had evidence of AHR. This emphasizes that screening for de novo production of DSA should be performed in all renal recipients with steroid-insensitive AR. Six of the 19 patients with DSA were identified retrospectively, demonstrating that this type of AR is often associated with acute cellular rejection and therefore is not always detected by classic pathology (see companion paper, Mauiyyedi et al., manuscript in preparation).
Higher historical and pretransplant sensitization, and a previous failed allograft, were found to be risk factors for AHR, suggesting that an anamnestic humoral response against donor antigens may play a role in the pathogenesis of this type of rejection. In addition, it also suggests that an overall state of enhanced humoral alloreactivity may facilitate a donor-specific response, or alternatively such patients may be “high responders” who are more likely to develop antibody in response to an allograft. More cadaver donors were in the AHR group compared to the other two rejection groups, consistent with the hypothesis that ischemic damage increases the immunogenicity of the transplanted organ (31).
In our series, 6 of the 19 patients with DSA initially presented with B cell positive/T cell negative cross-matches. Some authors have noted that anti-HLA class I DSA are predominantly involved in causing AHR (1,2,7). However, we could determine the specificity in three of the six, and all were directed against mismatched donor class II antigens, suggesting that in at least three cases, anti-HLA class II DSA played a major role in the AHR process. One of the donors was mismatched only for class II antigens, confirming that class II DSA were the only alloantibodies involved in this case. However, half (three of six) of these patients developed weak positive T cell cross-matches within a few days, thus making it impossible to discern if anti-HLA class I DSA were or were not also playing a role in the initial rejection process.
Two patients (nos.11 and 15) developed IgM antibody of unclear specificity against donor B cells. These antibodies may not have been directed against any HLA or other antigens present on the kidney, and therefore it cannot be concluded that the IgM was directly involved in the rejection process. These patients, as well as the two patients with widespread C4d deposition yet no detectable DSA may instead have had non-HLA antibodies against endothelial specific antigens (24,32–34) that could have caused the humoral rejection and complement deposition. However, another explanation may be that these patients had HLA antibodies below the level of detection of our assays, perhaps because most were absorbed by the kidney and therefore not present in the circulation.
In 10 patients with refractory AHR identified prospectively, we used PPh and tacrolimus-MMF rescue in an attempt to remove and suppress production of DSA. With this combined therapeutic approach, successful initial reversal of AHR was achieved in 9 of 10 patients. It should be noted that at the end of the PPh treatment, polyclonal i.v. immunoglobulin (IVIg; 0.4 g/kg) was administered to prevent infectious complications. As other groups have reported that high-dose (2 g/kg) polyclonal immunoglobulin can control AHR (35), a contribution of the IVIg cannot be ruled out.
We have found that the combination of tacrolimus and MMF is highly effective in preventing a rebound in antibody synthesis after the initial removal of DSA by PPh. This observation in patients with refractory AHR has been confirmed in the treatment of chronic rejection associated with DSA, in which rescue with tacrolimus and MMF alone (without PPh) can effectively suppress anti-donor antibody production (Pascual M, unpublished results). Interestingly, MMF has been shown to inhibit Ab production by B cells in vitro and in vivo, and this property appears to be useful in the control of humoral responses in humans (36).
From these observations, a strict definition of AHR in renal transplantation can be proposed which would incorporate the following features. 1) Evidence of acute allograft dysfunction, typically steroid-insensitive rejection, requiring the addition of more intensive immunosuppression, such as antilymphocyte therapy, and often resistant to it. 2) The presence of widespread C4d deposits in peritubular capillaries (10). In addition, other pathological features such as neutrophils in peritubular capillaries and interstitium, neutrophilic tubulitis or glomerulitis, microthrombi in arterioles and glomeruli, arteritis with fibrinoid necrosis, thrombosis, and infarction are more prevalent in this kind of rejection (5,6,8,9) (Maiuyyedi et al., manuscript in preparation). 3) Demonstration of previously undetected DSA in the recipient serum at the time of rejection. Most frequently DSA are IgG anti-HLA class I, but IgG anti-class II or IgM DSA may also be associated with AHR. In some instances low titers of DSA, which were undetected at the time of transplant with routine cross-match techniques, become apparent in serum in the early days posttransplant. Rarely, DSA not reactive with HLA antigens on donor lymphocytes may cause a clinicopathological picture consistent with AHR (24,32–34).
However, we suggest that from a clinical and therapeutic perspective, the presence of DSA in serum only or C4d deposits in PTC only in a patient with severe allograft dysfunction may justify an aggressive approach, because in these patients ongoing humoral mechanisms of rejection are likely. Combining DSA testing in serum and C4d staining of biopsy tissue provides a useful approach to diagnose AHR, a condition that often necessitates an intensive therapeutic rescue regimen. Currently at our institution, as soon as the diagnosis of AHR has been established (steroid-insensitive acute rejection, C4d+, DSA+), we initiate a therapeutic regimen that includes antilymphocyte therapy, tacrolimus-mycophenolate rescue and daily PPh for 5 consecutive days, followed by alternate day PPh if necessary for up to five additional procedures.
The authors thank the Brigham and Women’s Hospital Tissue Typing Laboratory for some of the flow cross-matching, Dr. Peter Stastny for antikeratinocyte antibody testing, and Natividad Cuende, MD, PhD, and José Francisco Cañón, MD, for help with the statistical analysis. The authors also thank the staff of the Histocompatibility Laboratory at the Massachusetts General Hospital for all of their technical assistance and the staff of the Blood Transfusion Service for their help in the management of the patients.
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