Donor-specific HLA-DQ antibodies may contribute to poor graft outcome after renal transplantation
2012; Elsevier BV; Volume: 82; Issue: 5 Linguagem: Inglês
10.1038/ki.2012.190
ISSN1523-1755
AutoresJ. DeVos, A. Osama Gaber, Richard J. Knight, Geoffrey A. Land, Wadi N. Suki, Lillian W. Gaber, Samir J. Patel,
Tópico(s)Pregnancy and Medication Impact
ResumoIncreasing evidence suggests a detrimental effect of donor-specific antibodies directed against the human leukocyte antigen (HLA)-A, -B, and -DR loci on renal allograft outcomes. Limited data exist on the impact of de novo HLA-DQ antibodies. Over a 3-year period, we prospectively monitored 347 renal transplant recipients without pre-transplant donor-specific antibodies for their development de novo. After 26 months of follow-up, 62 patients developed donor-specific antibodies, of which 48 had a HLA-DQ antibody either alone (33 patients) or in combination with an HLA-A, -B, or -DR antibody (15 patients). Only 14 patients developed a donor-specific HLA-A, -B, or -DR antibody without a HLA-DQ antibody present. Acute rejection occurred in 21% of the HLA-DQ–only patients, insignificant when compared with 11% of patients without donor-specific antibodies. At the last follow-up, the mean serum creatinine and the fraction of patients with proteinuria were significantly higher in those that developed only HLA-DQ than those without antibodies. The 3-year graft survival was significantly worse when HLA-DQ antibodies were combined with non-DQ antibodies (52%) compared with HLA-DQ alone, non-DQ antibodies alone, or no antibodies (92–94%). Thus, our prospective monitoring study found that donor-specific HLA-DQ antibodies were the most common type detected and these antibodies may contribute to inferior graft outcomes. Ongoing surveillance is necessary to determine the long-term outcome of patients developing HLA-DQ donor-specific antibodies. Increasing evidence suggests a detrimental effect of donor-specific antibodies directed against the human leukocyte antigen (HLA)-A, -B, and -DR loci on renal allograft outcomes. Limited data exist on the impact of de novo HLA-DQ antibodies. Over a 3-year period, we prospectively monitored 347 renal transplant recipients without pre-transplant donor-specific antibodies for their development de novo. After 26 months of follow-up, 62 patients developed donor-specific antibodies, of which 48 had a HLA-DQ antibody either alone (33 patients) or in combination with an HLA-A, -B, or -DR antibody (15 patients). Only 14 patients developed a donor-specific HLA-A, -B, or -DR antibody without a HLA-DQ antibody present. Acute rejection occurred in 21% of the HLA-DQ–only patients, insignificant when compared with 11% of patients without donor-specific antibodies. At the last follow-up, the mean serum creatinine and the fraction of patients with proteinuria were significantly higher in those that developed only HLA-DQ than those without antibodies. The 3-year graft survival was significantly worse when HLA-DQ antibodies were combined with non-DQ antibodies (52%) compared with HLA-DQ alone, non-DQ antibodies alone, or no antibodies (92–94%). Thus, our prospective monitoring study found that donor-specific HLA-DQ antibodies were the most common type detected and these antibodies may contribute to inferior graft outcomes. Ongoing surveillance is necessary to determine the long-term outcome of patients developing HLA-DQ donor-specific antibodies. An increasingly popular area of research in renal transplantation is the contribution of the humoral immune system to post-transplant outcomes. In recent years, there has been much debate regarding the role and significance of de nosvo donor-specific antibody (DSA) development after renal transplantation. Several reports show an association between donor human leukocyte antigen (HLA)–specific antibodies and inferior graft outcomes, including decreased renal function, increased incidence of acute antibody–mediated rejection (AMR),1.Everly M.J. Everly J.J. Arend L.J. et al.Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss.Am J Transplant. 2009; 9: 1063Crossref PubMed Scopus (173) Google Scholar, 2.Zhang Q. Liang L.W. Gjertson D.W. et al.Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction.Transplantation. 2005; 79: 591Crossref PubMed Scopus (85) Google Scholar, 3.Worthington J.E. Martin S. Husseini A.L. et al.Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome.Transplantation. 2003; 75: 1034Crossref PubMed Scopus (260) Google Scholar and a higher rate of allograft failure.1.Everly M.J. Everly J.J. Arend L.J. et al.Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss.Am J Transplant. 2009; 9: 1063Crossref PubMed Scopus (173) Google Scholar, 4.Cooper J.E. Gralla J. Cagle L. et al.Inferior kidney allograft outcomes in patients with de novo donor-specific antibodies are due to acute rejection episodes.Transplantation. 2011; 91: 1103Crossref PubMed Scopus (97) Google Scholar, 5.Mao Q. Terasaki P.I. Cai J. et al.Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study.Am J Transplant. 2007; 7: 864Crossref PubMed Scopus (185) Google Scholar, 6.Slavcev A. Donor-specific antibodies and kidney transplant rejection.Ann Transplant. 2003; 8: 12PubMed Google Scholar Allograft loss following development of DSA may be a result of a higher frequency of chronic AMR and transplant glomerulopathy.3.Worthington J.E. Martin S. Husseini A.L. et al.Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome.Transplantation. 2003; 75: 1034Crossref PubMed Scopus (260) Google Scholar, 7.Piazza A. Poggi E. Borrelli L. et al.Impact of donor-specific antibodies on chronic rejection occurrence and graft loss in renal transplantation: posttransplant analysis using flow cytometric techniques.Transplantation. 2001; 71: 1106Crossref PubMed Scopus (123) Google Scholar, 8.Lee P.C. Zhu L. Terasaki P.I. et al.HLA-specific antibodies developed in the first year posttransplant are predictive of chronic rejection and renal graft loss.Transplantation. 2009; 88: 568Crossref PubMed Scopus (140) Google Scholar, 9.Eng H.S. Bennett G. Chang S.H. et al.Donor human leukocyte antigen specific antibodies predict development and define prognosis in transplant glomerulopathy.Hum Immunol. 2011; 72: 386Crossref PubMed Scopus (29) Google Scholar, 10.Lachman N. Terasaki P.I. Budde K. et al.Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection or renal allografts.Transplantation. 2009; 87: 1505Crossref PubMed Scopus (301) Google Scholar Controversy still exists as to whether the inferior outcomes associated with DSA occur independently or as a result of injury caused by acute rejections (ARs) incurred during DSA exposure. Regardless, recent evidence seems to favor a detrimental effect of DSA on long-term outcomes. Over the past decade, new and more sensitive methods of antibody and antigen detection have become commercially available. These have afforded the opportunity to more accurately detect specific antibodies, including those of the DQ locus. The current UNOS organ allocation system, which historically placed an emphasis on HLA-A and -B, has more recently awarded points for matching at the HLA-DR antigen, and does not take into consideration HLA-DQ matching. Minimal evidence exists regarding the impact of DQ mismatch on the rate of DQ DSA development and allograft outcomes, issues that remain controversial. Through the use of routine post-transplant DSA monitoring, we have noticed a predominance of class II DSA after renal transplantation, specifically those directed against DQ antigens. This observation is consistent with the current literature; nonetheless, few studies report on the specific incidence of DQ DSA.3.Worthington J.E. Martin S. Husseini A.L. et al.Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome.Transplantation. 2003; 75: 1034Crossref PubMed Scopus (260) Google Scholar, 10.Lachman N. Terasaki P.I. Budde K. et al.Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection or renal allografts.Transplantation. 2009; 87: 1505Crossref PubMed Scopus (301) Google Scholar, 11.Hourmant M. Cesbron-Gautier A. Terasaki P.I. et al.Frequency and clinical implications of development of donor-specific and non-donor-specific HLA antibodies after kidney transplantation.J Am Soc Nephrol. 2005; 16: 2804Crossref PubMed Scopus (257) Google Scholar While it has generally been accepted that DSA directed toward HLA-A, -B, and -DR loci are clinically relevant, limited data exist regarding the clinical significance of DSA directed against the DQ antigen. Thus, the purpose of our study was to report the incidence and impact of de novo donor–specific DQ antibodies on outcomes following renal transplantation. We reviewed 389 consecutive kidney and combined kidney–pancreas transplants from July 2007 to July 2010. A total of 37 patients were excluded owing to pre-transplant DSA and/or the need for desensitization therapies. In addition, four patients who lost their graft within the first post-operative month and one patient who transferred care within the first month were also excluded from the analysis. Patient disposition is shown in Figure 1. Of the 347 remaining patients, the majority were male (60%), Caucasian (43%), and received deceased donor transplants (61%). About 25 patients (7%) received a simultaneous pancreas–kidney transplant. Median follow-up was 26 months (range, 3–47). Baseline characteristics are shown in Table 1.Table 1Baseline characteristics of patients according to DSA groupNo DSADQ onlyNonDQDQ+NonDQP-valueTotal number of patients, n (%)285 (82)33 (10)14 (4)15 (4)Recipient Gender—male, n (%)169 (59)22 (67)7 (58)10 (59)0.87 Age at transplant, mean±s.d.49±1450±1348±1945±140.74Ethnicity Caucasian, n (%)131 (46)9 (27)3 (21)6 (40)0.06aP<0.05 for no DSA vs. DQ only. African American, n (%)66 (23)12 (36)7 (50)6 (33)0.08 Hispanic, n (%)71 (25)8 (24)2 (21)4 (27)0.99Retransplant, n (%)24 (8)01 (7)1 (7)0.14aP<0.05 for no DSA vs. DQ only.Mean HLA mismatch/6, mean±s.d.3.6±1.84.1±1.34.6±1.24.7±1.10.01bP<0.05 for no DSA vs. nonDQ.,cP<0.05 for no DSA vs. DQ+nonDQ.Mean DQ mismatch/2, mean±s.d.0.9±0.71.3±0.61.4±0.61.1±0.60.06aP<0.05 for no DSA vs. DQ only.,bP 80%, n (%)30 (11)2 (6)1 (3)0 (0)0.27Donor Gender – male, n (%)144 (53)14 (44)9 (64)8 (57)0.59 Age, mean±s.d.39±1637±1640±1741±150.86 Deceased donor, n (%)168 (59)23 (70)9 (75)12 (71)0.36Induction therapy IL2RA, n (%)119 (42)9 (27)2 (14)6 (40)0.07bP<0.05 for no DSA vs. nonDQ. ATG, n (%)164 (58)23 (70)12 (86)8 (53)0.08bP<0.05 for no DSA vs. nonDQ.Outcomes Delayed graft function, n (%)25 (9)2 (6)3 (21)1 (7)0.49Abbreviations: ATG, human antithymocyte globulin; DSA, donor-specific antibody; HLA, human leukocyte antigen; IL2RA, interleukin 2 receptor antagonist (i.e., basiliximab and daclizumab); PRA, panel reactive antibody.a P<0.05 for no DSA vs. DQ only.b P<0.05 for no DSA vs. nonDQ.c P 80%) among the groups (Table 1). DSA detection occurred at a median of 6.1 months (range, 0.4–44) and a mean of 8.1±8.8 post transplant (Figure 2). In the DQ-only group, 75% (24/33) of DSA occurred within 6 months of transplant. While there was a tendency for later detection of DSA in the DQ+nonDQ group, there was no statistical difference in mean time to DSA development among groups (7.2±11.6 months for DQ-only, 6.5±7.4 months for nonDQ, and 11.9±7.8 months for DQ+nonDQ; P=0.17). Recipients in the DQ+nonDQ group were more likely to have persistent DSA (DSA present at more than one time point) compared with those in both the DQ-only and nonDQ groups (87% vs. 45% and 43%, respectively, P=0.01). Strength of the peak DSA in each group is shown in Figure 3a. There were significantly more weak and moderate DSA and fewer strong DSA in the DQ-only group when compared with the DQ+nonDQ group (P=0.001) and no difference between DQ-only and nonDQ groups.Figure 3Mean fluorescence index and outcomes. (a) Fluorescence index of DSA according to group. (b) Clinical outcomes based on MFI. DSA, donor-specific antibody; MFI, median fluorescence index.View Large Image Figure ViewerDownload (PPT) DSAs were detected at a median of 4 days (range, 229–195) and mean of 2.3±82 days following an AR diagnosis, with no differences observed between groups (Table 2). The overall incidence of biopsy-proven AR during the follow-up period was 15% (52/347), with a higher rate in the nonDQ and DQ+nonDQ groups compared with the no-DSA group. Although not statistically higher, the incidence of AR in DQ-only patients was double that of the no DSA group (21% in DQ-only vs. 11% in no DSA; P=0.11).Table 2Rejection and renal functionNo DSADQ onlyNonDQDQ+nonDQP-valueTotal number of patients, n (%)285 (82)33 (10)14 (4)15 (4)Acute rejection (AR), n (%)31 (11)7 (21)4 (29)10 (67)<0.001c,d,fAntibody-mediated rejection (AMR), n (%)1/285 (0.4)*3/33 (9)1/14 (7)9/15 (60)<0.001a,c,d,fTime from AR to DSA detection—4±100-10±335±870.95Serum creatinine (mg/dl) 6 months after transplant1.3±0.4 (n=236)1.3±0.1 (n=29)1.8±0.2 (n=12)1.3±0.2 (n=13)0.09b,e Most recent (end of study)1.3±0.6 (n=250)1.6±0.8 (n=33)1.7±0.9 (n=14)2.6±1.2 (n=14) 0.5, n (%)** 6 months after transplant15/195 (8)5/24 (21)3/10 (30)3/12 (25)0.03b Most recent (end of study)23/251 (9)7/29 (24)2/13 (15)4/13 (31)0.03a,cAbbreviation: DSA, donor-specific antibody.*Renal transplant biopsy showed presence of capillaritis, polymorphonuclear leukocytes (PMNs), and positive C4d staining in >50% of the peritubular capillaries suggestive of AMR despite the lack of DSA in serum.**Not all recipients had urine protein/creatinine ratio available; thus, the total number of patients is different than that of the entire group.a P<0.05 for no DSA vs. DQ only.b P<0.05 for no DSA vs. nonDQ.c P<0.05 for no DSA vs. DQ+nonDQ.d P<0.05 for DQ only vs. DQ+nonDQ.e P<0.05 for DQ only vs. nonDQ.f P 50% of the peritubular capillaries suggestive of AMR despite the lack of DSA in serum. **Not all recipients had urine protein/creatinine ratio available; thus, the total number of patients is different than that of the entire group. a P<0.05 for no DSA vs. DQ only. b P<0.05 for no DSA vs. nonDQ. c P<0.05 for no DSA vs. DQ+nonDQ. d P<0.05 for DQ only vs. DQ+nonDQ. e P<0.05 for DQ only vs. nonDQ. f P 4000 MFI) DSA had a trend toward more AMR (3/19) compared with those with a weak DSA (0/14; P=0.06), as well as more graft loss (2/19 vs. 0/14, respectively; P=0.13). Renal function as demonstrated by serum creatinine and degree of proteinuria is shown in Table 2. Only patients with nonDQ antibodies exhibited higher creatinines early post transplant (6 months); however, at most recent follow-up, all patients with DSA, regardless of type, had significantly higher creatinines than no DSA patients (P<0.001). Even in patients with DQ DSA-only, the most recent serum creatinine was significantly higher than patients without DSA (1.6 vs. 1.3mg/dl, respectively; P=0.02). The degree of proteinuria was assessed by a spot urine protein-to-creatinine ratio and considered clinically significant when greater than 0.5. Again, an early difference was seen in the nonDQ group; however, by most recent follow-up, patients in both DQ-only and DQ+nonDQ groups demonstrated a higher rate of proteinuria compared with the no DSA group. For the entire cohort, uncensored graft survival, death-censored graft survival, and patient survival at 3 years post transplant were 92, 96, and 94%, respectively. Uncensored graft survival was significantly worse in the DQ+nonDQ group (52%) compared with all other groups (92–94%; P=0.0004). However, there was no difference in graft survival between the remaining three groups. Excluding patient death, nine graft losses occurred in the cohort. As shown in Table 3, there were no differences in causes of graft loss between the groups. Of the patients with DSA, all graft losses occurred in patients with MFI in the moderate, strong, or very strong range with none occurring in those with weak DSA (Figure 3b).Table 3Causes of graft loss by DSA group (excluding death)No DSA (n=3)BKN, CAN, unknownDQ only (n=1)RejectionNonDQ (n=1)BKNDQ+nonDQ (n=4)BKN, rejection (2), unknownAbbreviations: BKN, BK nephropathy; CAN, chronic allograft nephropathy. Open table in a new tab Abbreviations: BKN, BK nephropathy; CAN, chronic allograft nephropathy. Increasing evidence highlights the significant impact of DSA against HLA -A, -B, and -DR antigens in kidney transplantation; however, the importance of DQ DSA alone is not well described. Similar to other reports, we have noticed a predominance of DQ DSA through the use of routine post-transplant monitoring. Our results suggest that DQ antibodies are the most common de novo DSA detected post transplant, and that these antibodies may be associated with a detrimental effect in terms of rejection and graft dysfunction. Furthermore, while we observed no difference in graft survival in patients with DQ antibodies alone, DQ antibodies detected in conjunction with other class I and class II antibodies were associated with significantly reduced graft survival. Importantly, AMR and graft losses did not occur in patients with low levels of DQ-only antibodies. Here, we report an overall prevalence of de novo DSA of 18%, while the rate reported in the literature is highly variable ranging from 4 to 27%.2.Zhang Q. Liang L.W. Gjertson D.W. et al.Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction.Transplantation. 2005; 79: 591Crossref PubMed Scopus (85) Google Scholar, 4.Cooper J.E. Gralla J. Cagle L. et al.Inferior kidney allograft outcomes in patients with de novo donor-specific antibodies are due to acute rejection episodes.Transplantation. 2011; 91: 1103Crossref PubMed Scopus (97) Google Scholar, 11.Hourmant M. Cesbron-Gautier A. Terasaki P.I. et al.Frequency and clinical implications of development of donor-specific and non-donor-specific HLA antibodies after kidney transplantation.J Am Soc Nephrol. 2005; 16: 2804Crossref PubMed Scopus (257) Google Scholar, 12.Cardarelli F. Pascual M. Tolkoff-Rubin N. et al.Prevalence and significance of anti-HLA and donor-specific antibodies long-term after renal transplantation.Transpl Int. 2005; 18: 532Crossref PubMed Scopus (85) Google Scholar In studies that prospectively monitor for de novo DSA, this prevalence appears to consistently range from 22–27%.2.Zhang Q. Liang L.W. Gjertson D.W. et al.Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction.Transplantation. 2005; 79: 591Crossref PubMed Scopus (85) Google Scholar, 4.Cooper J.E. Gralla J. Cagle L. et al.Inferior kidney allograft outcomes in patients with de novo donor-specific antibodies are due to acute rejection episodes.Transplantation. 2011; 91: 1103Crossref PubMed Scopus (97) Google Scholar, 7.Piazza A. Poggi E. Borrelli L. et al.Impact of donor-specific antibodies on chronic rejection occurrence and graft loss in renal transplantation: posttransplant analysis using flow cytometric techniques.Transplantation. 2001; 71: 1106Crossref PubMed Scopus (123) Google Scholar Until recently, most studies reporting on the impact of class II antibodies focus primarily on outcomes related to DR. Through the use of solid phase antibody detection and identification methods, improved reporting and better characterization of HLA-directed antibodies, including DQ antibodies, have been attained before and after transplant. Of the studies published that report the percentage of de novo DSA directed against DQ, the rate reported varies from 33 to 90%.11.Hourmant M. Cesbron-Gautier A. Terasaki P.I. et al.Frequency and clinical implications of development of donor-specific and non-donor-specific HLA antibodies after kidney transplantation.J Am Soc Nephrol. 2005; 16: 2804Crossref PubMed Scopus (257) Google Scholar, 13.Iniotaki-Theodoraki A. Boletis J. Trigas G.C. et al.Humoral immune reactivity against human leukocyte antigen (HLA)-DQ graft molecules in the early posttransplantation period.Transplantation. 2003; 75: 1201Crossref Scopus (27) Google Scholar, 14.Ozawa M. Rebellato L.M. Terasaki P. et al.Longitudinal testing of 266 renal allograft patients for HLA and MICA antibodies: Greenville experience.Clin Transpl. 2006; 20: 265Crossref Scopus (19) Google Scholar This variation may be attributed to differences in sample collection times, assays used to detect antibodies, and the MFI cutoff considered significant. In the cohort of patients reported herein, 77% developed a DQ DSA. The reason for the high rate of DQ DSA remains unknown, even recent data indicate that HLA-DR mismatches may be more immunogenic than DQ. This may be in part due to the fact that DR, but not DQ, matching is taken into consideration in the current UNOS allocation system. In this report, we found that the majority of DSA occur within 6 months post transplant, with no difference in time to DSA development between all groups. Using a similar routine prospective DSA monitoring protocol, Cooper et al. found that 91% of DSA were detected within 6 months of transplant, while Zhang et al. reported that 63% of patients developed DSA within 1 month after transplant with the remaining 37% developing within 6 months post transplant.2.Zhang Q. Liang L.W. Gjertson D.W. et al.Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction.Transplantation. 2005; 79: 591Crossref PubMed Scopus (85) Google Scholar,4.Cooper J.E. Gralla J. Cagle L. et al.Inferior kidney allograft outcomes in patients with de novo donor-specific antibodies are due to acute rejection episodes.Transplantation. 2011; 91: 1103Crossref PubMed Scopus (97) Google Scholar We did notice a slight delay in detection in the DQ+nonDQ DSA group, with a median time of detection around 11 months compared with the other groups at 6 months. This may be the result of a higher rate of detection ‘for cause’ as opposed to predetermined screening time points, specifically for episodes of graft dysfunction. Consequently, this group had a higher rate of AMR, in many cases as a result of noncompliance. We found an increased incidence of AR in each of the DSA groups compared with the no DSA group. The rate of AR in the DQ-only patients compared with the no DSA patients was approximately double. The difference lacked statistical significance, likely a result of the small sample size; however, the trend may be clinically relevant. In contrast to an earlier report, the incidence of AMR in the DQ-only group was significantly higher than in the no DSA group.13.Iniotaki-Theodoraki A. Boletis J. Trigas G.C. et al.Humoral immune reactivity against human leukocyte antigen (HLA)-DQ graft molecules in the early posttransplantation period.Transplantation. 2003; 75: 1201Crossref Scopus (27) Google Scholar Furthermore, recent data indicate that DQ antibodies are common during periods of AMR and is suggestive of the DQ antigen being upregulated by cells as part of an inflammatory response.15.Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2011; 89: 277Crossref Scopus (153) Google Scholar Controversy exists with regards to whether inferior renal outcomes are due to de novo DSA themselves or to previous rejection episodes in patients with DSA.4.Cooper J.E. Gralla J. Cagle L. et al.Inferior kidney allograft outcomes in patients with de novo donor-specific antibodies are due to acute rejection episodes.Transplantation. 2011; 91: 1103Crossref PubMed Scopus (97) Google Scholar In the current study, while we noted an increased incidence of AR in the DQ group, we did not find a difference in graft survival at 3 years when including patients with AR possibly due to the relatively short-term follow-up in our cohort, or to possible interventions made soon after DSA detection. However, patients expressing DQ antibodies in conjunction with HLA-A, -B, or -DR antibodies did show inferior graft survival suggesting a cumulative effect of the number, types, and strength of antibodies present. Overall, we feel that even in the absence of AR, the presence of de novo DSA likely represents a proportion of patients who are generally under-immunosuppressed and are thus at risk for subsequent acute or chronic rejection. This is supported by studies demonstrating an independent effect of DSA on allograft survival3.Worthington J.E. Martin S. Husseini A.L. et al.Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome.Transplantation. 2003; 75: 1034Crossref PubMed Scopus (260) Google Scholar, 8.Lee P.C. Zhu L. Terasaki P.I. et al.HLA-specific antibodies developed in the first year posttransplant are predictive of chronic rejection and renal graft loss.Transplantation. 2009; 88: 568Crossref PubMed Scopus (140) Google Scholar, 16.Mao Q. Teraskai P.I. Cai J. et al.Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study.Am J Transplant. 2007; 7: 864Crossref PubMed Scopus (127) Google Scholar, 17.Meier-Kriesche H.U. Ojo A.O. Hanson J.A. et al.Increased impact of acute rejection on chronic allograft failure in recent era.Transplantation. 2000; 70: 1098Crossref PubMed Scopus (179) Google Scholar, 18.Waaga A.M. Gasser M. Laskowski I. et al.Mechanisms of chronic rejection.Curr Opin Immunol. 2000; 12: 517Crossref PubMed Scopus (66) Google Scholar and several studies demonstrating the presence of DSA, including DQ antibodies in grafts lost owing to chronic rejection.3.Worthington J.E. Martin S. Husseini A.L. et al.Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome.Transplantation. 2003; 75: 1034Crossref PubMed Scopus (260) Google Scholar,19.Campos E.F. Tedesco-Silva H. Machado P.G. et al.Post-transplant anti-HLA class II antibodies as risk factor for late kidney allograft failure.Am J Transplant. 2006; 6: 2316Crossref PubMed Scopus (114) Google Scholar All graft losses and AMRs in the DQ-only group occurred in patients with an MFI of at least 4000, similar to the results by Everly et al.1.Everly M.J. Everly J.J. Arend L.J. et al.Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss.Am J Transplant. 2009; 9: 1063Crossref PubMed Scopus (173) Google Scholar showing that in patients with AMR and DSA, those with higher DSA MFI's had worse outcomes, and of all patients with AMR and DSA only 4 of 22 (18%) had DSA 4000–8000 MFI, strong >8000–15,000 MFI, and very strong >15,000 MFI. In our system, 4000 MFI is the approximate lower limit of a positive B-cell flow crossmatch, while 8000 and greater MFI antibodies are associated with increasingly positive AHG cytotoxic crossmatches. Peak-MFI DSA was defined as the highest MFI of a single DSA in each individual recipient. Donor and recipient typings were done by molecular methods according to their respective manufacturers directions. All recipients and living donors were typed by a flow bead array using sequence-specific oligonucleotide probes (Labtype SSO, One Lambda) with positive hybridization detected by Luminex and data analyzed by Fusion software (One Lambda). All deceased donors were typed via sequence-specific primers (HLA-A/B/DR/DQ-SSP UniTray, Life Technologies/Invitrogen, Carlsbad, CA), results obtained by 2% agarose/ethidium bromide gel electrophoresis, and data analyzed by vendor supplied software. Renal biopsies were evaluated by light microscopy and immunofluoresce. Electron microscopic examination was performed when glomerular pathology was suspected. The histopathological features and the classification of rejection were reported according to Banff 2005 updates. We required the simultaneous presence of diffuse linear C4d deposition in the peritubular capillaries, and presence of DSA, and morphological evidence of graft injury to diagnose AMR. Severe intimal arteritis, glomerulitis, capillaritis, and fibrinoid vascular necrosis and thrombosis were features of AMR. Staining for C4d in the biopsies was performed on snap-frozen portion of the biopsy by indirect immunofluorescence technique using monoclonal antibody to C4d (Biogenesis, Kidlington, UK) at a dilution of 1:75. In the rare event that snap-frozen tissue of the kidney biopsy is not available, C4d staining was performed on a paraffin section using a polyclonal C4d antibody (1:50; American Research Products, Waltham, MA). Patients were categorized into one of the four following groups based on presence and type of DSA: no DSA (those without any detectable DSA); DQ-only (those with only a DQ DSA); nonDQ (those with DSA directed against an HLA-A, -B, and -DR, but without a DQ DSA), and DQ+nonDQ DSA (those with DSA against HLA-A, -B, and/or -DR, as well as against DQ). Baseline categorical and continuous variables were compared among groups using 2 and one-way analysis of variance tests, respectively. For Tables 1 and 2, outcomes were compared between individual groups using 2 and t-tests for categorical (gender, ethnicity, retransplant status, induction agent, and rejection) and continuous variables (age, PRA, creatinine, and time to rejection), respectively. Differences in immunosuppressant dosages and levels (Supplementary Table S1 online) were detected using analysis of variance testing, with subsequent inter-group comparisons made by t-tests. Graft survival, defined as freedom from the return to dialysis or patient death, was computed using the Kaplan–Meier method and compared using the log-rank test. We would like to thank the following for their help: Kevin M. Burns and Smaroula Dilioglou, Department of Pathology, The Methodist Hospital, Houston, TX, USA. Table S1. Immunosuppressive use by DSA Group. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
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