Role of Human Leukocyte Antigen, Donor-Specific Antibodies, and Their Impact in Renal Transplantation

Role of Human Leukocyte Antigen, Donor-Specific Antibodies, and Their Impact in Renal Transplantation U. Mathuram Thiyagarajan, A. Bagul, J. Frost, T. Horsburgh, and M.L. Nicholson ABSTRACT Introduction. The clinical significance of the presence of antibody against human leukocyte antigen (HLAb) and donor-specific antibodies (DSAb) prior to renal transplantation remains unclear. This study was done to assess the impact of HLAb and DSAb on graft function, rejection episodes, and graft survival in renal transplantation. Methods. The Luminex (Luminex, Austin, Texas, United States) is a solid-phase assay using micro-spheres and it is more sensitive at detecting human leukocyte antigen (HLA) antibodies than conventional tests. This retrospective analysis involved 141 consecutive renal transplant recipients between May 2007 and 2009 and with a minimum of 2 years of follow-up. Results. Luminex was positive for HLA class I in 35 and negative in 106; similarly class II positivity was noted in 23 and negative in 118. The DSAb were positive in 33 and negative in 108 recipients. The HLA class I, class II, and DSA-positive groups showed no difference in renal function assessed by estimated glomerular filtration rate (eGFR) at 2 years (52 ⫾ 29 vs 52 ⫾ 22; 56 ⫾ 29 vs 51 ⫾ 29; 48 ⫾ 18 vs 53 ⫾ 19; P ⫽ not significant [NS]). But rejection episodes at 1 year were significantly high in HLA class I and DSAb-positive group (17/35 vs 27/106; P ⫽ .019 and 16/33 vs 29/108; P ⫽ .035). The rejection episodes in the HLA class II–positive group did not show any difference when compared with the negative group (9/23 vs 40/118; P ⫽ .63). Graft survival was not affected by positivity to any of these antibodies at 2 years. Conclusion. Having HLA class I, class II, and DSAb does not have any influence on early and intermediate graft function. The HLA class I and DSAb positivity increases rejection episodes within 1 year in renal transplantation. Graft survival was not affected by class I, class II, and DSAb at 2 years. HE association between a positive crossmatch and a poor graft survival has been identified since 1969,1 thus mandating a negative crossmatch prior to renal transplantation. The recent implementation of Luminex technology (Luminex, Austin, Texas, United States) using an array of microspheres coated with individual human leukocyte antigen (HLA) has added a new dimension in renal transplantation. This is a more sensitive method for detecting HLA antibodies (HLAb) and has been used as a surrogate for the pretransplantation crossmatch. These can be further divided into donor-specific antibodies (DSAb) and nonspecific HLAb. The presence of HLAb and its clinical importance in renal transplantation remains controversial. Some studies found that the presence of HLAb increases the chance of acute rejection,2–5 whereas others could not

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replicate this finding.6 – 8 It is imperative to note that in the United Kingdom, 23% of patients awaiting their first renal transplantation and 52% of patients awaiting retransplantation are sensitized to HLAb.9 This study was performed to assess the impact of having HLAb and DSAb prior to the

From the Department of Infection, Immunity & Inflammation, Transplant Surgery Group, University Hospitals of Leicester, Leicester General Hospital, Leicester, United Kingdom. Address reprint requests to Umasankar Mathuram Thiyagarajan, MBBS, MS, MRCS, Department of Infection, Immunity & Inflammation, Transplant Surgery Group, University Hospitals of Leicester, Leicester General Hospital, Leicester, LE5 4PW, United Kingdom.

Crown Copyright © 2012 Published by Elseiver Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 44, 1231–1235 (2012)

0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2011.10.054 1231

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transplantation and their impact on early graft function, rejection episodes, and graft survival.

DETECTION OF HLAb AND DSAb

Sera from consecutive patients undergoing transplantation between May 2007 and May 2009 were tested for the presence of anti-HLA antibodies using the multiplex technology SPA. Class I (ie, HLA-A/B) and class II (ie, HLA-DR/DP/DQ) HLAb were tested using LabScreen LS1A01 and LS2A01 (OneLambda, Canoga Park, Calif, United States) commercialised in the United Kingdom by VHBIO (Gateshead, England, UK). Briefly, 8 ␮L of serum samples were incubated with HLA class I– coated and HLA class II– coated microspheres for 30 minutes in the dark under gentle agitation. The specimens were then washed 5 times before being incubated with anti-human immunoglobulin (Ig) G-conjugated phyco-erythrine in the same conditions as in the first incubation. The Labscan 100 flow analyzer (Luminex, Austin, Tex, United States) was used for beads and data acquisition. Data were then exported to HLA Visual software (OneLambda) for analysis. The cut-off level was nominally defined as a baseline normalized ⬎500 mean fluorescence intensity units (MFI). The presence of DSA was assigned by comparing the various HLA specificities proposed by the software analysis with the HLA typing of the donor for all transplant recipients. The highest reacting “self” bead was identified, and any beads with double the normalized value of this bead were considered as positive. To define positivity, the median MFI value of the 3 negative controls was subtracted from the MFI value of the capture bead. This adjusted MFI was then compared with the adjusted MFI of the positive control bead. A ratio of ⱖ5% and/or an adjusted MFI value of ⱖ1000 for the capture bead were considered to be positive. The presence of DSA was assigned by comparing the various HLA specificities proposed by the software analysis with the HLA typing of the donor for all transplant recipients. Luminex results did not influence transplant management because this was a retrospective analysis.

PATIENTS AND METHODS The analysis was carried out on retrospectively collected data of consecutive renal recipients between May 2007 and 2009 and with a minimum follow-up of 2 year following transplantation. Data collection and assessment by the investigator were made prior to the knowledge of HLA antibody status of the recipients. A total of 141 patients were then grouped according to HLA class I, class II, and DSAb; HLA class I positivity was noted in 35, whereas 106 were negative. Similarly, HLA class II antibody was positive in 23 and negative in 118. The DSAb was positive in 33, whereas it was negative in 108 patients. The demographics between the 2 groups showed no difference in age and gender (Table 1) .

Immunosuppression Protocol In our center, we use a tacrolimus, mycophenolate mofetil, and prednisolone immunosuppressive regimen. Among the 141 recipients, 22 of them were switched to rapamycin at 6 months following transplantation due to their participation in another trial. All of our recipients received interleukin (IL)-2 receptor monoclonal antibody (Simulect, Novartis Pharmaceuticals, Surrey, United Kingdom) preoperatively and on the 4th postoperative day while methylprednisolone 0.5 g plus CoAmoxiclav 1.2 g was given at induction. Seven of 33 recipients received 3 doses of 0.5 g methylprednisolone because of high DSAb titer. All recipients were followed up carefully and their serum tacrolimus level maintained at 6 –10 ng/mL in the first 3 months thereafter between 4 and 8 ng/mL; for rapamycin the therapeutic level ranged between 8 and 15 ng/mL. ATG (Thymoglobulin®, Genzyme, Cambridge, Mass) was given postoperatively to 5 patients for delayed graft function (as per our unit protocol).

Surveillance Biopsy and Treatment of Rejection All the recipients in our study had surveillance biopsy at 1, 3, 6, and 12 months after transplantation. Transplant renal biopsy was done using a 16-gauge core needle; the tissue specimen was looked at under light microscopy, immunoflorescence, and immunohistochemistry (SV40 antigen). The antibody-mediated rejection was diagnosed based on presence of peri-capillary C4d and up to date Banff classification was used for grading of rejection. Banff 1A, 1B type of rejection was treated by 3 doses of 0.5 g of methylprednisolone on consecutive days. The Banff 2A, 2B types of rejection were treated in the same way as mentioned plus the mycophenolate mofetil dose was increased to full dose. All steroid rejections were treated using anti-thymocyte globulin (Thymoglobulin, Genzyme,

Table 1. Demography Parameter

HLA I–Positive

HLA I–Negative

HLA II–Positive

HLA II–Negative

DSAb-Positive

DSAb-Negative

Donor average age (y) Donor median age (y) Donor M:F Recipient age (y) Recipient median age (y) Recipient M:F HLA A⫹B Mismatch (Mean ⫾ SD) HLA DR mismatch (Mean ⫾ SD)

43 14–64 19:16 47 25–71 7:28 1.91 ⫾ 1.05

46 13–71 47:59 46 25–66 30:76 2.11 ⫾ 0.94

45 13–64 8:15 43 28–65 8:15 1.47 ⫾ 1.04

45 14–71 58:60 49 21–71 76:42 2.17 ⫾ 1.05

44 14–64 18:15 48 25–71 7:26 1.93 ⫾ 1.05

46 13–71 48:60 46 21–66 32:76 2.18 ⫾ 1.02

0.85 ⫾ 0.69

0.93 ⫾ 0.70

0.73 ⫾ 0.68

0.94 ⫾ 0.69

0.84 ⫾ 0.68

0.85 ⫾ 0.69

Abbreviations: M, male; F, female.

ROLE OF HLA

Fig 1.

Episodes of rejection in the study population.

Cambridge, Mass, United States) 1.5 g/kg for 10 days and dose intervals were decided based on daily CD3 lymphocyte counts.

Parameters and Statistical Analysis First, we assessed the renal allograft function using an estimated glomerular filtration rate (eGFR mL/min) at 3, 6, 12, and 24 months following transplantation. The episodes of biopsy-proven acute rejection (BPAR) were from our dedicated unit’s database, and the eGFR were from the proton software system (Clinical Computing Limited, London, United Kingdom); they were assessed as a secondary end points. Data were recorded in an Excel spread sheet (Microsoft, Redmond, Wa, United States) and GraphPad Instat version 3.06 (GraphPad Software, Inc, San Diego, Calif, United States). Descriptive statistics for continuous variables were recorded as mean ⫾ SD or median (range) according to whether or not they were normally distributed. Normality testing was performed using the Kolmogorov-Smirnov test. Comparison between groups was performed with the Student t test for parametric variables and the Mann-Whitney test was used for nonparametric variables. Categorical variables were analysed using the Fisher exact test. Statistical significance was defined as P value ⬍ .05.

RESULTS Allograft Function

When assessing the graft function between HLA class I–positive versus–negative group, there was no difference found at 3 (mean eGFR 59 ⫾ 17 vs 58 ⫾ 17), 6 (mean eGFR 48 ⫾ 15 vs 50 ⫾ 15), 12 (mean eGFR 53 ⫾ 15 vs 52 ⫾ 15), and 24 months (mean eGFR 52 ⫾ 29 vs 51 ⫾ 30). Similarly no difference was observed between the HLA class II–positive versus –negative group at 3 (mean eGFR 56 ⫾ 18 vs 59 ⫾ 17), 6 (mean eGFR 50 ⫾ 15 vs 51 ⫾ 15), 12 (mean eGFR 50 ⫾ 15 vs 52 ⫾ 15), and 24 months (mean eGFR 55 ⫾ 29 vs 51 ⫾ 29). The graft function in the DSAb-positive group also did not differ from a negative group at 3 (58 ⫾ 17 vs 59 ⫾ 17), 6 (47 ⫾ 15 vs 51 ⫾ 15), 12 (52 ⫾ 15 vs 52 ⫾ 15), and 24 months (48 ⫾ 18 vs 53 ⫾ 19).

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of rejection episodes were noted in the DSAb-positive group (17/33 vs 32/108; P ⫽ .035; Fig 1). But the HLA class II–positive group did not show any difference in rejection episodes compared with the negative group (9/23 vs 40/118; P ⫽ .63). The graft survival at 2 years in the HLA class I–positive versus–negative group was not different (2/35 vs 3/106; P ⫽ .59). Similar results were also seen in the HLA class II (21/23 vs 4/118; P ⫽ 1.0) and DSAb-positive (1/33 vs 4/108; P ⫽ 1.0) versus-negative groups (Fig 2). Among the 5 grafts lost, 2 of them were because of noncompliance with medication; 2 patients lost their graft due to recurrent urinary tract infection sepsis; and the fifth patient by unknown cause possibly early chronic allograft nephropathy. None of these 5 graft losses were related to immunological reasons. DISCUSSION

In our study, the presence of HLA class I, class II, and DSAb were not associated with a negative impact on graft function and survival. But having HLA class I and DSAb led to a higher incidence of BPAR within 1 year. Having HLAb prior to transplantation was initially considered as a contraindication; this also has been shown to have a negative impact on the graft, although it is still a matter of debate. Some studies did support that having HLAb can cause high rejection episodes and lead to early graft failure,1,3,10 –12 whereas others showed that having HLAb did not have any effect on the immediate- and medium-term graft function as well as on rejection episodes.6,7,13,14 In the modern era of immunosuppression, we have performed this retrospective study to answer the same question. First, a detailed analysis of these studies showed a significant difference in immunosuppression protocol and, hence, probably the dilemma of difference in the graft function and rejection episodes. Phelan et al7 in live donor renal transplants used anti-thymocyte globulin (Thymoglobulin, Genzyme Corp, Cambridge, Mass, United States) in HLA mismatched recipients in addition to the tacrolimus, mycophenolate mofetil, and prednisolone therapy. Second, there was a great difference in the timing and the frequencies of transplant renal biopsies; most studies did

BPAR and Graft Survival

There is a high incidence of episodes of BPAR within 1 year between HLA class I–positive versus HLA class I–negative (20/35 vs 29/106; P ⫽ .002; Fig 1) Similarly, high incidences

Fig 2. Graft survival at 2 years in the DSAb-positive vs the -negative group.

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not perform surveillance biopsy.6,7,15 Among the studies that had performed surveillance biopsy, the timing and frequencies of biopsy were different.3 To the best of our knowledge, this is the only study that used a standard immunosuppression protocol on recipients with HLAb/ HLA mismatch. We have practiced expectant management and performed surveillance transplant biopsies at 1, 3, 6, and 12 months after transplantation. All of the biopsies reported herein as positive were clinically relevant and had been treated according to Banff grading. When assessing the graft function, the mean eGFR at 12 months in HLA class I antibody–positive and –negative groups was 56 ⫾ 14.98 and 51 ⫾ 14.98, respectively. For HLA class II antibody, the positive group had an eGFR of 54 ⫾ 14.98 and the negative group had 52 ⫾ 14.98. Thus, no significant difference in eGFR was noted in all groups at 12 months. Similar results were observed at 2 years as well. Our study also clearly showed that having HLA class I, and class II antibodies does not have any influence on graft function within 2 year after live donor transplantation. By performing surveillance biopsy, we understand that most subclinical and histologically proven rejection would have been reported. But despite this, the rejection rate was not significantly different between the HLA class II–positive and –negative groups. The impact of DSAb on rejection is very obvious in our study, which correlates well with the previously published literature.3 Bielmann et al3 had shown similar incidences of rejection (4 among 9 patients, 44%) in patients with DSAb but all recipients who were positive for DSAb were treated with ATG (ATG Fresenius, Fresenius Medical Care, Switzerland) and IgG. Herein, the protocol biopsy was performed only at 3 and 6 months after transplantation. The limitations of our study are that it was retrospective with a small number of patients with 2 years of follow-up. However, the positive points are that it included standard immunosuppression and a regular surveillance biopsy for 1 year following transplantation.

PERFECT HLA MATCH: A FRIEND OR A FOE?

As we know, evidence does support both sides of the argument; the question remains. Is there a need for a perfect HLA match? Well, most transplantation surgeons say why not? But there are some concerns about the risk of having a perfect HLA match in living related renal transplantation, particularly when the cause of renal failure is secondary to glomerulonephritis. Glassock et al14 reported that there was a high incidence (65%) of recurrent glomerulonephritis after transplantation between identical twins, suggesting that the genetic similarities between donor and recipient might increase the risk of recurrent disease. A similar high incidence of recurrent glomerulonephritis in recipients of a HLA-identical living related donor graft has recently been reported in other studies.16 In an early

THIYAGARAJAN, BAGUL, FROST ET AL

work by Berger et al,17 5 of 7 HLA-identical siblings (ages, 10 –32 years) had recurrent IgA nephropathy. Ponticelli et al also reported that “at least 35% of IgA Nephropathy patients after transplantation had [non-protocol] biopsy-proven recurrence, and younger patients were more prone to the risk of recurrence and it is more frequent with zero-HLA mismatched living donors.”18,19 In summary, graft function and graft survival appear to be multifactoral and the presence of HLA class I, class II, and DSAb does not appear to have any significant effect on them. HLA class I and DSAb increased the risk of BPAR within 1 year after transplantation. This retrospective study needs to be further assessed and its finding supported by a multicenter, international randomized control trial with long-term follow-up to accurately assess the impact of HLA and DSAb on the graft.

REFERENCES 1. Patel R, Terasaki P: Significance of the positive crossmatch test in kidney transplantation. N Engl J Med 280:735, 1969 2. Ishida H, Tanabe K, Furusawa M, et al: Evaluation of flow cytometric panel reactive antibody in renal transplant recipients examination of 238 cases of renal transplantation. Transpl Int 18:163, 2005 3. Bielmann D, Honger G, Lutz D, et al: Pretransplant risk assessment in renal allograft recipients using virtual crossmatching. Am J Transplant 7:626, 2007 4. Patel AM, Pancoska C, Mulgaonkar S, et al: Renal transplantation in patients with pre-transplant donor-specific antibodies and negative flow cytometry crossmatches. Am J Transplant 7:2371, 2007 5. Lefaucheur C, Suberbielle-Boissel C, Hill GS, et al: Clinical relevance of preformed HLA donorspecific antibodies in kidney transplantation. Am J Transplant 8:324, 2008 6. Billen EVA, Christiaans MHL, van den Berg-Loonen EM: Clinical relevance of Luminex donor-specific crossmatches: data from 165 renal transplants. Tissue Antigens 74:205, 2009 7. Phelan D, Thalachallour M, Ramachandran S, et al: Living donor renal transplantation in the presence of donor-specific human leukocyte antigen antibody detected by solid-phase assay. Human Immunol 70:584, 2009 8. Tuncera M, Gurkanb A, Erdoganb O, et al: Lack of impact of human leukocyte antigen matching in living donor kidney transplantation: experience at Akdeniz University. Transplant Proc 37:2969, 2005 9. Fuggle SV, Martin S: Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients. Transplantation 86:384, 2008 10. Cecka JM, Zhang Q, Reed EF: Preformed cytotoxic antibodies in potential allograft recipients: recent data. Human Immunol 66:343, 2005 11. Gibney EM, Cagle LR, Freed B, et al: Detection of donorspecific antibodies using HLA-coated microspheres: another tool for kidney transplant risk stratification. Nephrol Dial Transplant 21:2625, 2006 12. Susal C, Dohler B, Opelz G: Presensitized kidney graft recipients with HLA class I and II antibodies are at increased risk for graft failure: A Collaborative Transplant Study report. Human Immunol 70:569, 2009 13. Tuncera M, Gurkanb A, Erdoganb O, et al: Lack of impact of human leukocyte antigen matching in living donor kidney transplantation: experience at Akdeniz University. Transplant Proc 37:2969, 2005

ROLE OF HLA 14. Glassock RJ, Feldman D, Reynolds ES, et al: Human renal isografts: a clinical and pathologic analysis. Medicine 47:411, 1968 15. Aubert V, Venetz J-P, Pantaleo G, et al: Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant. Human Immunol 70:580, 2009 16. Andresdottir MB, Hoitsma AJ, Assmann KJ, et al: The impact of recurrent glomerulonephritis on graft survival in recipients of human histocompatibility leucocyte antigen-identical living related donor grafts. Transplantation 68:623, 1999

1235 17. Berger J, Yaneva H, Nabarra B, et al: Recurrence of mesangial deposition of IgA after renal transplantation. Kidney Int 7:232, 1975 18. Ponticelli C, Traversi L, Feliciani A, et al: Kidney transplantation in patients with IgA mesangial glomerulonephritis. Kidney Int 60:1948, 2001 19. McDonald SP, Russ GR: Recurrence of IgA nephropathy among renal allograft recipients from living donors is greater among those with zero HLA mismatches. Transplantation 82:759, 2006