Acute cellular rejection (ACR), an important event in renal transplants, is characterized by mononuclear cellular infiltrates in allograft (1). According to Banff classification, the principle diagnostic lesions of ACR include interstitial inflammation (i), tubulitis (t), and intimal arteritis (v), which are graded by arbitrary consensus rules (2). Borderline changes and T cell–mediated rejection (TCMR) are two categories of ACR, and TCMR is further graded into five types (3). Acute cellular rejection, therefore, encompasses distinct histologic features of different scored lesions and is related to the variable initial response to antirejection treatment, allograft function, and outcome (4, 5). Acute cellular rejection with intimal arteritis has been found to have poorer short-term graft survival compared to ACR with tubulointerstitial inflammation alone when antibody-mediated rejection (AMR) was excluded (6). Over the years, the description of each category has evolved based on continuous new research, and more areas are being investigated for further clarification. For example, “isolated v-lesions” was proposed in 2009 at the Banff conference (7), and the focus was placed on “isolated v1-lesion” in 2011(8). Its significance is still being studied by a Banff working group. However, it remains unclear how the updated classification and scoring relates to outcome and if the timing of rejection plays a role in graft survival.
Therefore, we performed this retrospective study to address the following issues in ACR only cases: 1) whether the severity of ACR is associated with the long-term graft loss, 2) whether the timing of the severest ACR episode correlated with graft survival, and 3) whether isolated intimal arteritis predicts better graft outcome.
Approximately 388 adult patients who had at least one episode of biopsy-proven acute rejection (BPAR) were considered. After excluding AMR (n=42), C4d or DSA positivity at biopsy (n=45), and co-existing MI lesions (n=31), 270 patients remained. Two hundred seventy ACR cases, which represented the highest severity of each patient, were chosen and classified into three groups: borderline (n=90, 33.3%), TCMR I (n=108, 40.0%), and TCMR II/III (n=72, 26.7%). Approximately 215 (79.6%) biopsies occurred within 6 months posttransplantation.
Patient Demographics and Clinical Characteristics at the Time of Biopsy
There was no significant difference in recipient and transplant characteristics among control and three ACR groups except for the significantly higher mean HLA mismatches in TCMR I and TCMR II/III groups, the statistically longer CIT in the borderline group and the demonstrable lower DGF incidence ratio in the control group (Table 1A).
The mean time from transplantation to enrolled biopsies were comparable among three ACR groups (median 12 days, P=0.21). At biopsy, the mean Scr concentration of TCMR II/III group was statistically higher than that of the TCMR I group (5.8 vs. 4.6 mg/mL, P=0.04). At 1 month postbiopsy, the mean Scr value of the borderline group (2.3 mg/mL) was statistically lower than that of TCMRI group (2.8 mg/mL, P=0.03) or TCMR II/III group (3.6 mg/mL, P=0.007). The borderline group showed the highest proportion of complete reversibility; in contrast, the TCMR II/III group presented with the highest fraction of partial/nonreversibility, although significantly more patients of TCMR II/III group received therapeutic PPH and antibody therapy (Table 1B).
All patients were followed up till the end of the study or graft failure. The incidence of de novo DSA after enrolled ACR was found evenly distributed in three groups (P=0.35). The de novo DSA occurred from 5.0 to 107.3 months after ACR (median 23.4 months; P=0.06). In addition, 26 patients had proteinuria (the amount of protein excreted in the urine≥300 mg/day) at biopsy in three groups (P=0.46); the incidence of proteinuria 2- and 5-year post ACR was similar among the three ACR groups (Table 1C).
Histologic Evaluation of Biopsies
Among the three ACR groups, the mean scores of Banff lesions ah and mm showed similar grades (Table 2A). The v-lesion occurred only in the TCMR II/III group, the mean t score of the TCMR I group was significantly higher than that of the borderline or TCMR II/III group; the mean i score of the TCMR I or TCMR II/III group was significantly higher than that of the borderline group; the mean ci/ct score of the TCMR I or TCMR II/III group was statistically higher compared with the borderline group. Compared with early overall ACR, the significantly higher scores of ah, cv, ci/ct, mm, t and i-lesions were found overall in late overall ACR (Table 2B). Within each ACR group, the mean ci/ct score of the late borderline was statistically higher than that of the early borderline. The mean ah and ci/ct scores of late TCMR I were statistically higher than those of the early TCMR I. The mean cg, mm, ah, cv, t, and ci/ct scores of the late TCMR II/III were statistically higher than those of the early TCMR II/III. Among the three early ACR groups, the mean i score of the early TCMR I and II/III was statistically higher than that of the early borderline; the mean t score of the early TCMR I was significantly higher than that of the early borderline or TCMR II/III. Among the three late ACR groups, the mean cv score of the late TCMR II/III was found to be significantly higher than that of the late borderline or TCMR I, and the mean ci/ct score of the late TCMR II/III was shown to be higher than that of the late borderline.
Impact of ACR on Graft and Patient Survival Up to 8-Year Posttransplantation
Kaplan-Meier graft-survival analysis showed the control group had the best graft survival, and the patient survival was similar among four groups (Fig. 1). The DCGS rates of the control and the borderline groups were significantly higher than those of the TCMR I or TCMR II/III group (each pairwise comparison yields P<0.001), and the DCGS rate of the control group was statistically higher than that of the borderline group (P=0.03). Surprisingly, no statistical difference of DCGS rate was found between the TCMR I and TCMR II/III groups (P=0.20). Defined by the Banff scores of t and v-lesions, we divided TCMR I into Ia and Ib types and TCMR II into IIa and IIb types. The patients with TCMR Ia showed significantly better graft and patient survival than those with TCMR Ib or TCMR IIb/III; the patients with TCMR Ib and IIa had the same graft and patient survival; the lowest graft and patient survival was observed among patients with TCMR IIb/III.
Compared with late overall ACR, early overall ACR showed significantly better graft survival but same patient survival (Table 3A). The GS and DCGS rates of the early and late borderline group were similar; the graft survival was significantly higher in the early TCMR I or TCMR II/III group compared with the late TCMR I or TCMR II/III group. Either in the three early or late ACR groups, the graft survival of the TCMR II/III group was significantly lower than that of the borderline or TCMR I group, but there was no significant difference between the borderline and TCMR I groups.
The v1/v2-lesions were observed in 70 biopsies, of which, 11 met the criteria of isolated v-lesions showing minimal TI; the remaining 59 biopsies presented higher grade of TI. The graft and patients survival were similar between two groups (Table 3B). In addition, 48 biopsies showed v1-lesion, of which, 36 met the criteria of modified isolated v1-lesion, and the rest (n=12) had severe TI. No significant differences of graft and patient survival were found between the two groups (Table 3C).
Association of Histologic Factors With Graft Failure at 8-Year Posttransplantation
Cox-regression analysis of the whole population showed many histologic factors associated with graft failure (Table 4). Overall, inflammatory lesions (v and t) were independent predictors for graft loss at 8-year posttransplantation, especially in early ACR. V-lesion also appeared to be an adverse factor closely related to graft loss in late ACR and TCMR II/III. The mm-lesion of the borderline group was proven to be associated with graft loss. Surprisingly, no risk pathologic factor was found in the TCMR I group.
We retrospectively studied 270 biopsies, which showed the highest ACR severity of each patient and were negative for C4d and MC-lesions and attempted to grade borderline, TCMR I, and TCMR II/III as low, moderate, and high ACR severity. First, our data proved that any type of ACR, regardless of severity, was associated with deterioration of graft function overtime; even patients of the borderline group, in terms of the low ACR severity, could not sustain the same DCGS rate as the patients of the control group. The significant deterioration of graft survival might relate to partial ACR, which responds incompletely to antirejection treatment. The incompletely reversible ACR can lead to more indicative biopsies, increase the risk for subsequent late rejection, both of which result in persistent and progressive parenchymal damage and accelerate graft failure (9, 10).
Second, based on the Banff scored lesions, the higher ACR severity predicted the poorer antitreatment response and graft survival. The patients of the borderline group showed significantly higher graft survival than those of the TCMR I or TCMR II/III group. However, there was no significant difference of graft survival between TCMR I and TCMR II/III. Our data were in accordance with a recent study of Lefaucheur et al., who reported that compared with acute cellular interstitial rejection, the risk of graft loss was 9.07 times higher in AMR with v-lesion and 2.93 times higher in AMR without v-lesion, although there was no significant increased in acute cellular vascular rejection (11). Overall, in ACR patients, the v and t-lesions showed significant association with long-term graft failure, and v-lesion was proven as an independent predictor of subsequent graft loss regardless of the timing of biopsy. Among the three types of interstitial rejection (borderline and TCMR Ia and Ib), patients with t3-lesion (TCMR Ib) had a demonstrable worse graft outcome than those with t1/t2-lesions (borderline or TCMR Ia). Although the severity of tubulitis has no significant adverse effect on graft outcome in patients with vascular rejection, the v-lesions plus minimal TI (isolated v-lesions) or intensive TI had an equally unfavorable impact on graft function. These data might seem to be at variance with prior studies in which the degree of graft dysfunction was associated with the extent of tubulitis (12, 13). One possible explanation for the different results is that partial antibody-mediated vascular rejection might be misdiagnosed as T cell–mediated vascular rejection because t and i-lesions are sensitive to high-dose steroid pulse therapy, whereas v-lesion do not recover after anti–T cell treatment and instead require potent antibodies therapy (14). Hence, we think the designation of isolated v-lesions may reflect distinct severity of acute vascular rejection or predict a favorable prognosis if the potential AMR cases are included.
Third, we observed the progression to graft failure occurred more frequently in patients with late ACR compared with those with early ACR. Moreover, patients with late vascular rejection showed a significantly higher likelihood of graft failure than those of patients with borderline or late TCMR I. The poor prognosis of late TCMR II/III is likely associated with the unrecognized AMR because the end-stage human kidney transplants with severe TCMR usually have concomitant AMR (15). To minimize any possible effect of AMR, we excluded any biopsies showing positivity of C4d or DSA or MC-lesions. Nevertheless, we cannot completely rule out the mixture of ACR with chronic active AMR because the multilayering of peritubular capillary basement membrane, which is only evident in electron microscopy or C4d-negative AMR, whereas C1q-fixing DSA (16) or non-HLA antibodies has not been routinely checked.
Late ACR is supposed to be more difficult to reverse and have a higher risk of subsequent graft loss than early ACR, considering of its higher scores of chronic scarring (ci/ct, mm), vascular diseases (ah, cv), and active immune inflammation (i, t). Histologically, interstitial fibrosis and tubular atrophy (ci/ct) are common in late allografts, indicating the cumulative burden of injury and diseases such as chronic allograft nephropathy (CAN), which has been defined as progressive allograft dysfunction occurring at least 3 months posttransplantation and is the cause of 44% of graft loss after the first year posttransplantation (17). However, ci/ct is not a disease itself but a feature of all progressive kidney diseases. We found that ci/ct combined with v and t-lesions was associated with graft failure in early ACR; early ci/ct appears to have an immune basis and shows subclinical rejection, tubulointestitial disease, and the residua of previous episodes of clinically evident acute rejection; whereas later ci/ct, combined with accelerated vasculopathy, glomerulosclerosis, and tubulointerstitial disease, is associated with nonimmune phenomena such as prolonged calcineurin inhibitor (CNI) exposure, donor disease, and hypertension (18).
In addition, chronic vascular damage (ah, cv) undoubtedly contributes to late graft loss, which also results from CNI use, as biopsy samples from both native and transplanted kidney exposed to CNI demonstrate that arteriolar hyalinosis (ah) eventually develops into an obliterative vasculopathy and finally leads to tubulointerstitial damage and striped fibrosis (19). Because of concern about poor reversibility, patients with TCMR underwent more intensive immunosuppression compared with the borderline patients; thus, later in the course, the mean grade of ah was obviously higher in the late TCMR I or TCMR II/III group than in the early TCMR I or TCMR II/III group, whereas the late borderline group maintained the similar low grade of ah with the early borderline group. Moreover, except for the infiltration of vessels by mononuclear cells, the histologic characteristics of vascular rejection, including endothelial-cell apoptosis and the synthesis of matrix proteins and collagens by intimal myofibroblasts, enhance the development of arteriosclerosis. This probably explains why the grade of cv in TCMR II/III group was significantly higher than that of early TCMR II/III group, whereas no prominent difference was found between early and late borderline as well as TCMR I group.
In summary, all types of ACR are associated overtime with renal allograft dysfunction; ACR with v-lesions and late occurring ACR predict poorer long-term graft prognosis. The extent of TI is of no prognostic significance for vascular rejection.
MATERIALS AND METHODS
Patient and Data Collection
Between January 1, 1996, and December 31, 2010, approximately 1083 adult (≥18 years) patients received only kidney transplantation and followed up for at least 6 months in our center. After excluding 443 patients on the criteria detailed in Figure 2, this study enrolled 640 patients, who were further classified into two groups: 270 patients who had at least one episode of ACR were chosen as study group; 370 patients who had no biopsy were employed as the control group. Graft loss was defined as returning to chronic dialysis or death with functioning grafts. Death-censored graft failure was defined as returning to chronic dialysis. All clinical and laboratory data were recorded in our transplant database system (TBase) at each visit. This study was approved by the institutional review committee.
Pathologic Review of Biopsies
An ultrasound-guided graft biopsy was performed when clinically indicated, that is, elevation of serum creatinine (Scr). All patients with DGF, defined as needing dialysis within 1 week posttransplantation (20), underwent protocol biopsy on the seventh day posttransplantation.
Biopsy specimens were processed with standard techniques in the Department of Pathology, Charite Campus Mitte. Adequate sample involved minimal of seven glomeruli and one artery. Indirect immunofluorescent staining of C4d was performed on paraffin sections (polyclonal anti-C4d antibody, Dianovo, Germany). Biopsies from the pre-C4d era were retrospectively tested for C4d. Diffuse linear C4d deposition was interpreted as positive. All light microscopy slides were reviewed by two pathologists (B.R. and K.Y.) and graded according to the 2009 Banff classification (3). Each sample was scored on the following: glomerulitis (g), peritubular capillaritis (ptc), transplant glomerulopathy (cg), intimal arteritis (v), interstitial inflammation (i), tubulitis (t), mesangial matrix increase (mm), vascular intimal fibrosis (cv), arteriolar hyaline thickening (ah), interstitial fibrosis (ci), and tubular atrophy (ct). Borderline referred to histologic indices t1/i1-2 or i1/t1-2 (21). TCMR I was defined as v0, t2-3, i2-3, and TCMR II/III defined as v1-3, t0-3, and i0-3. Borderline, TCMR I, and TCMR II/III groups represented low, middle, and high ACR severity in this study. The MC lesions included g≥1, or ptc≥1, or cg≥1. The isolated v-lesions were defined as v1-2, i0-1, t0-1, and the modified isolated v1-lesion was defined as v1, t1-2, i1-2. Diagnosis of AMR was based on simultaneous presence of DSA, C4d positivity, and allograft pathology. The biopsies meeting AMR criteria plus t- or v-lesions were called mixed AMR plus ACR. “C4d-negative AMR” was considered if C4d was negative, but DSA and morphologic MC-lesions were present, Nevertheless, the biopsies were chosen, which showed the highest ACR severity of each patient, free of MC-lesions and negative for C4d or DSA.
Donor-specific antibody level was monitored as previously described (22). All serum samples, which were collected once a year or at biopsy were qualitatively screened for HLA antibodies using two ELISA-based screening systems (PRA-STAT and LAT) from 1996 to 2006 or the Luminex-based bead assay LABScreen Mixed (One Lambda, Canoga Park, CA) from 2007 on. All tests were performed according to the manufacturer’s guidelines (23).
Immunosuppressive Protocol and Antirejection Treatment
The immunosupression protocol comprised of cyclosporine A (CyA)/tacrolimus (Tac), mycophenolate mofetil (MMF)/azathioprine (Aza), and methylprednisolone since 1996. The doses of CyA and Tac were adjusted according to whole blood trough levels. Antirejection therapy involved two broad steps: the pulse therapy of corticosteroids and steroid-resistant ACR received antibody therapy (ATG or Rituximab) plus additional therapeutic PPH. Complete, partial, and no reversibility of ACR were defined by Gaber et al. (24) by comparing 1 month postbiopsy Scr with prebiopsy level.
All data were assessed for completeness by a single investigator (D.S.). Continuous variables were expressed as mean±standard deviation. Categorical variables were expressed as N and percentage of total. Student t test was used to compare two groups of continuous variables and chi-square for categorical data. The survival curves were analyzed using Kaplan-Meier graphs and statistically compared using log-rank test. To test putative risk factors for long-term graft loss, Banff scored lesions were tested in univariate Cox-regression analysis; those of which with significant association (P<0.05) were then entered into multivariate analysis. All statistics were performed using SPSS16.0 (SPSS Inc., Chicago, IL). P<0.05 was considered significant.
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