Pretransplant Donor‐Specific HLA Class‐I and ‐II Antibodies Are Associated With an Increased Risk for Kidney Graft Failure

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q‐fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG‐DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q‐fixing DSA. Patients with both class‐I plus ‐II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q‐fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan‐IgG SAB assay are generally related to C1q‐fixation. We conclude that the presence of class‐I plus ‐II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q‐fixing IgG‐DSA could not be assessed due to their low prevalence.     

Alloantibody Levels and Acute Humoral Rejection Early After Positive Crossmatch Kidney Transplantation

We examined the course of donor ‐ specific alloantibody (DSA) levels early after transplant and their relationship with acute humoral rejection (AHR) in two groups of positive crossmatch (+XM) kidney transplant recipients: High DSA group—41 recipients with a baseline T‐ or B‐cell flow crossmatch (TFXM, BFXM) channel shift ≥300 (molecules of equivalent soluble fluorochrome units (MESF) of approximately 19 300) who underwent pretransplant plasmapheresis (PP), and Low DSA group—29 recipients with a baseline channel shift <300 who did not undergo PP. The incidence of AHR was 39% (16/41) in the High DSA group and 31% (9/29) in the Low DSA group. Overall, mean DSA levels decreased by day 4 posttransplant and remained low in patients who did not develop AHR. By day 10, DSA levels increased in patients developing AHR with 92% (23/25) of patients with a BFXM >359 (MESF of approximately 34 000) developing AHR. The BFXM and the total DSA measured by single antigen beads correlated well across a wide spectrum suggesting that either could be used for monitoring. We conclude that AHR is associated with the development of High DSA levels posttransplant and protocols aimed at maintaining DSA at lower levels may decrease the incidence of AHR.     

Class II Alloantibody and Mortality in Simultaneous Liver‐Kidney Transplantation

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver–kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody‐mediated kidney rejection. To further investigate the risk associated with donor‐specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre‐SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity ≥ 2,000 = positive). Post‐SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p = 0.006), liver allograft rejection (p = 0.002), patient death (p = 0.02), liver allograft loss (p = 0.02) and renal allograft loss (p = 0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR = 2.2; p = 0.043) and liver allograft loss (HR = 2.2; p = 0.044). These data warrant reconsideration of the approach to DSA in SLKT.     

Baseline Donor‐Specific Antibody Levels and Outcomes in Positive Crossmatch Kidney Transplantation

Renal transplant candidates with donor‐specific alloantibody (DSA) have increased risk of antibody‐mediated allograft injury. The goal of this study was to correlate the risk of antibody‐mediated rejection (AMR), transplant glomerulopathy (TG) and graft survival with the baseline DSA level (prior to initiation of pretransplant conditioning). These analyses include 119 positive crossmatch (+XM) compared to 70 negative crossmatch (?XM) transplants performed between April 2000 and July 2007. Using a combination of cell‐based crossmatch tests, DSA level was stratified into very high +XM, high +XM, low +XM and ?XM groups. In +XM transplants, increasing DSA level was associated with increased risk for AMR (HR = 1.76 [1.51, 2.07], p = 0.0001) but not TG (p = 0.18). We found an increased risk for both early and late allograft loss associated with very high DSA (HR = 7.71 [2.95, 20.1], p = 0.0001). Although lower DSA recipients commonly developed AMR and TG, allograft survival was similar to that of ?XM patients (p = 0.31). We conclude that the baseline DSA level correlates with risk of early and late alloantibody‐mediated allograft injury. With current protocols, very high baseline DSA patients have high rates of AMR and poor long‐term allograft survival highlighting the need for improved therapy for these candidates.     

Determination of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotypes

Kwok J, Chan GSW, Lam MF, Yan T, Tang L, Kwong KM, Chan KW, Chan TM. Determination of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotypes.
Clin Transplant 2010 DOI: 10.1111/j.1399‐0012.2010.01246.x
© 2010 John Wiley & Sons A/S. Abstract: Objective: To determine donor human leukocyte antigen (HLA) from renal allograft biopsies in transplant recipients whose donor HLA phenotype is not known. Methods: Renal allograft biopsies were obtained from seven renal transplant recipients when indicated for allograft dysfunction or proteinuria. DNA was extracted fresh from allograft specimens, and HLA typing was performed with polymerase chain reaction‐specific sequence primers (PCR‐SSP) and polymerase chain reaction‐sequence‐specific oligonucleotides (PCR‐SSO). Results: HLA typing of the seven renal allograft biopsies was composed of both recipient and donor HLA phenotypes, allowing the determination of the donor HLA and the degree of HLA mismatching. Conclusions: Deducing mismatched donor HLA antigens in renal allograft recipients enables detection of donor‐specific antibodies, and the management of humoral rejection, and enables more appropriate selection of a donor organ should future retransplantation be required.     

Class II HLA Epitope Matching—A Strategy to Minimize De Novo Donor‐Specific Antibody Development and Improve Outcomes

De novo donor‐specific antibody (dnDSA) develops in 15–25% of renal transplant recipients within 5 years of transplantation and is associated with 40% lower graft survival at 10 years. HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA‐DR/DQ/DP conformational epitopes for 286 donor–recipient pairs. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus‐specific epitope mismatches were more numerous in patients who developed HLA‐DR dnDSA alone (21.4 vs. 13.2, p < 0.02) or HLA‐DQ dnDSA alone (27.5 vs. 17.3, p < 0.001). An optimal threshold for epitope mismatches (10 for HLA‐DR, 17 for HLA‐DQ) was defined that was associated with minimal development of Class II dnDSA. Applying these thresholds, zero and 2.7% of patients developed dnDSA against HLA‐DR and HLA‐DQ, respectively, after a median of 6.9 years. Epitope specificity analysis revealed that 3 HLA‐DR and 3 HLA‐DQ epitopes were independent multivariate predictors of Class II dnDSA. HLA‐DR and DQ epitope matching outperforms traditional low‐resolution antigen‐based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long‐term graft outcome.     

Clinical impact of the baseline donor‐specific anti‐human leukocyte antigen antibody measured by Luminex single antigen assay in living donor kidney transplant recipients after desensitization therapy

The aim of this study is to investigate the clinical impact of donor‐specific anti‐HLA‐antibody (HLA‐DSA) baseline levels, measured using the Luminex single antigen assay (LSA), in living donor kidney transplantation (LDKT). Total 129 cases of LDKT were divided into four groups according to baseline mean fluorescence intensity (MFI) HLA‐DSA values: Strong (n = 6), >10 000; Moderate (n = 8), 5 000–10 000; Weak (n = 11), 1 000–5 000, Negative (n = 104), <1 000. Pretransplant desensitization (DSZ) was performed to decrease the MFI to weak or negative values before KT. Clinical outcomes in the four groups were compared. After DSZ, HLA‐DSA decreased to weak or negative levels in all patients; Acute rejections developed more frequently in strong group [5/6 (83.3%)] compared with other three groups (P < 0.05), and especially acute antibody‐mediated rejection (AAMR) developed almost exclusively in strong group [4/6 (66.7%)]. Strong HLA‐DSA levels at baseline were more predictive of AAMR than either type of XM (complement‐dependent lymphocytotoxicity or flow cytometry) in ROC analysis. Allograft function in this group showed significant deterioration during follow‐up compared with the other groups. In conclusion, strong HLA‐DSA levels at baseline are associated with worse allograft outcome even after successful desensitization; therefore, strict monitoring and strong maintenance immunosuppression may be required in such patients.     

Rethinking the advantage of zero‐HLA mismatches in unrelated living donor kidney transplantation: implications on kidney paired donation

The OPTN/UNOS Kidney Paired Donation (KPD) Pilot Program allocates priority to zero‐HLA mismatches. However, in unrelated living donor kidney transplants (LDKT)—the same donor source in KPD—no study has shown whether zero‐HLA mismatches provide any advantage over >0 HLA mismatches. We hypothesize that zero‐HLA mismatches among unrelated LDKT do not benefit graft survival. This retrospective SRTR database study analyzed LDKT recipients from 1987 to 2012. Among unrelated LDKT, subjects with zero‐HLA mismatches were compared to a 1:1–5 matched (by donor age ±1 year and year of transplantation) control cohort with >0 HLA mismatches. The primary endpoint was death‐censored graft survival. Among 32,654 unrelated LDKT recipients, 83 had zero‐HLA mismatches and were matched to 407 controls with >0 HLA mismatches. Kaplan–Meier analyses for death‐censored graft and patient survival showed no difference between study and control cohorts. In multivariate marginal Cox models, zero‐HLA mismatches saw no benefit with death‐censored graft survival (HR = 1.46, 95% CI 0.78–2.73) or patient survival (HR = 1.43, 95% CI 0.68–3.01). Our data suggest that in unrelated LDKT, zero‐HLA mismatches may not offer any survival advantage. Therefore, particular study of zero‐HLA mismatching is needed to validate its place in the OPTN/UNOS KPD Pilot Program allocation algorithm.     

Donor‐Specific HLA Antibodies in a Cohort Comparing Everolimus With Cyclosporine After Kidney Transplantation

Donor‐specific HLA antibodies (DSA) have a negative impact on kidney graft survival. Therefore, we analyzed the occurrence of DSA and antibody‐mediated rejection (AMR) in patients from two prospective randomized trials in our center. At 3–4.5 months posttransplant 127 patients were randomized to continue cyclosporine or converted to everolimus therapy. The presence of DSA was prospectively assessed using Luminex assays. AMR was defined according to the Banff 2009 classification. Antibody screening was available in 126 patients with a median follow‐up of 1059 days. Seven out of 65 (10.8%) patients on cyclosporine developed DSA after a median of 991 days. In comparison, 14/61 patients (23.0%) randomized to everolimus developed DSA after 551 days (log‐rank: p = 0.048). Eight patients on everolimus compared to two patients on cyclosporine developed AMR (log‐rank: p = 0.036). Four of 10 patients with AMR—all in the everolimus group—lost their graft. A multivariate regression model revealed everolimus, >3 mismatches and living donor as significant risk factors for DSA. Acute rejection within the first year, >3 mismatches, everolimus and living donor were independent risk factors for AMR. This single center analysis demonstrates for the first time that everolimus‐based immunosuppression is associated with an increased risk for the development of DSA and AMR.     

Trends in Immune Cell Function Assay and Donor‐Specific HLA Antibodies in Kidney Transplantation: A 3‐Year Prospective Study

The immune cell function assay (ICFA) and de novo anti‐donor‐specific HLA antibodies (DSA) have been proposed as assays for immune monitoring in renal transplantation, but longitudinal studies examining the modification of both parameters over time and their relation with clinical events are lacking. We prospectively measured longitudinal changes in ICFA and DSA levels in 55 kidney transplant recipients over 3‐year follow‐up (534 visits) and analyzed their relation with the risk of developing acute rejections or infections. Seven patients (12.7%) developed biopsy‐proven acute rejection, and 20 (36.4%) developed viral infections. At 3 years posttransplant, 28% of the patients had developed de novo DSA. ICFA levels peaked at 1–2 months posttransplant (p = 0.005) and leveled off thereafter. They were not associated with the risk of acute rejections, viral infections or development of de novo DSA. Instead, the incidence of de novo DSA was higher in patients who previously had viral infections (adjusted‐odds ratio of de novo DSA associated with prior infections: 6.03 [95% CI, 1.64–22.06; p = 0.007]). Our prospective, longitudinal study does not support using ICFA to quantify the immune risk in kidney transplantation. Further studies are needed to confirm the relationship between viral infections and the subsequent development of de novo DSA.     

Evolution and Clinical Pathologic Correlations of De Novo Donor‐Specific HLA Antibody Post Kidney Transplant

The natural history for patients with de novo donor‐specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pretransplant DSA, with a mean follow‐up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA posttransplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years posttransplant. Independent predictors of dnDSA were HLA‐DRβ1 MM > 0 (OR 5.66, p < 0.006); and nonadherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p = 0.061). The median 10‐year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody‐mediated injury can occur and progress in patients with dnDSA in the absence of graft dysfunction and furthermore, nonadherence and cellular rejection contribute to dnDSA development and progression to graft loss.     

Extremely High Association Between Appearance of HLA Antibodies and Failure of Kidney Grafts in a Five-Year Longitudinal Study

Longitudinal studies were conducted over a five-year period for HLA antibodies on 493 sera tested from 54 kidney transplant patients. HLA single antigen beads were employed to establish donor specificity of the antibodies. Only 3 of 22 patients without antibodies rejected a graft in contrast to 17 out of 32 patients with posttransplant antibodies (p = 0.003). Using a serum creatinine value of 4.0 mg/dL as the cut-off for a failed graft, 4 of 22 patients without antibodies failed compared to 21 of 32 with antibodies (p = 0.0006). Among patients with donor-specific antibodies (DSA) 13 of 15 failed (p = 0.000004). Even among patients with non-donor specific antibodies (NDSA), 8 of 17 failed (p = 0.05). Among patients who could be identified as making de novo antibodies (since they developed antibodies while not having antibodies for more than six months after transplantation), 6 of 11 failed (p = 0.03). Sequential testing for HLA antibodies shows that antibodies appear prior to a rise in serum creatinine and subsequent graft failure. The very strong association between the production of HLA antibodies after transplantation and graft failure indicates the importance of monitoring for posttransplant HLA antibodies.     

Clinical impact of preformed donor‐specific denatured class I HLA antibodies after kidney transplantation

Class I single‐antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA‐sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor‐specific antibodies (DSA) using two different assays: an acid‐treated SAFB assay (anti‐dHLA DSA) and the iBeads assays (SAFB+/iBeads‐ DSA). Eighty‐five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520–13 882]). Anti‐dHLA and SAFB+/iBeads‐ DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500–1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti‐dHLA DSA or only SAFB+/iBeads‐ DSA developed acute clinical antibody‐mediated rejection in the first‐year post‐transplantation, and their five‐yr death‐censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T‐cell flow cytometry cross‐match. Therefore, both anti‐dHLA DSA and SAFB+/iBeads‐ DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.     

Five‐Year Outcomes in Living Donor Kidney Transplants With a Positive Crossmatch

Renal transplant candidates with high levels of donor‐specific anti‐HLA antibodies have low transplantation rates and high mortality rates on dialysis. Using desensitization protocols, good short‐term outcomes are possible in “positive crossmatch kidney transplants (+XMKTx)”, but long‐term outcome data are lacking. The aim of the current study was to determine actual 5‐year graft outcomes of +XMKTx. We compared graft survival and the functional and histologic status of 102 +XMKTx to 204 ?XMKTx matched for age and sex. Actual 5‐year death‐censored graft survival was lower in the +XMKTx group (70.7% vs. 88.0%, p < 0.01) and chronic injury (glomerulopathy) was present in 54.5% of surviving grafts. Graft survival was higher in recipients with antibody against donor class I only compared with antibody against class II (either alone or in combination with class I) (85.3% vs. 62.6%, p = 0.05) and was similar to ?XMKTx (85.3 vs. 88.0%, p = 0.64). Renal function and proteinuria ranged across a wide spectrum in all groups reflecting the different histological findings at 5 years. We conclude that when compared to ?XMKTx, +XMKTx have inferior outcomes at 5 years, however, almost half of the surviving grafts do not have glomerulopathy and avoiding antibodies against donor class II may improve outcomes.     

Long‐term follow up for anti‐HLA donor specific antibodies postrenal transplantation: high immunogenicity of HLA class II graft molecules

Τhe clinical significance of de novo post‐transplant anti‐HLA donor‐specific antibodies (DSA) was evaluated using 4241 serum samples collected between 2000 and 2007 from 597 renal transplant recipients. Patients transplanted before December 1996 (n = 77) were included in the historic group and those transplanted thereafter (n = 520) were included in the study group. All recipients were negative for DSA before transplantation (Tx). Post‐Tx, de novo DSA were detected in 92/597 (15.4%) patients, while 196 had third party anti‐HLA antibodies (DSA‐negative). DSA were more frequent in the historic group (33.8%) compared with the study group (12.7%) (P < 0.001). Anti‐HLA class‐II DSA predominated in both groups (84.6% vs. 69.7%). Recipients of HLA class II‐incompatible grafts developed DSA more frequently than those receiving HLA class II‐compatible grafts (17.9% vs.7.9%, P = 0.003), directed mainly against HLA‐DQ graft molecules (64/446, 14.4%). DSA production was not different between presensitized and nonsensitized patients (P = 0.842). Graft survival was higher in patients without antibodies compared with DSA‐positive (log‐rank test, P = 0.002) and DSA‐negative patients (log‐rank test, P = 0.002). Univariate and multivariate analysis showed independent association for DSA class I (HR = 31.78), DSA class II (HR = 20.92) and non‐DSA (HR = 5.94) and graft failure. We conclude that HLA class II incompatible graft transplantations need careful monitoring and should be avoided in high immunological risk cases.     

Donor Origin of BK Virus in Renal Transplantation and Role of HLA C7 in Susceptibility to Sustained BK Viremia

In a previous study, we performed serial BK virus (BKV), polymerase chain reaction (PCR) and detected active BKV infection in 70 (35.4%) of 198 renal transplant recipients. In the current study, pre-transplant donor and recipient samples were analyzed for BKV antibody titer and HLA alleles. Donor antibody titer was inversely proportional to onset of viruria, p<0.001, directly proportional to duration of viruria, p=0.014 and directly proportional to peak urine viral titer p=0.005. Recipient pairs receiving kidneys from the same donor were concordant for BKV infection, p=0.017, and had matched sequences of segments of the NCCR and VP1 genes that tended to vary among recipients of kidneys from different donors. We did not see an association of HLA A, B, or DR, HLA allele mismatches or total HLA mismatches and BK infection. However, all 11 recipients with sustained BK viremia received kidneys from donors lacking HLA C7, and 10 recipients also lacked C7. These findings derive from the largest and most comprehensive prospective study of BKV infection in renal transplant recipients performed to date. Our data support donor origin for early BKV infection in kidney transplant recipients, and suggest that a specific HLA C locus may be associated with failure to control BKV infection.