The effect of HLA-C matching on acute renal transplant rejection

Christoph Frohn,1, Lutz Fricke2, Jan-Christoph Puchta3 and Holger Kirchner1

1 Institute of Immunology and Transfusion Medicine, and 2 Department of Internal Medicine, University of Lübeck School of Medicine, Lübeck and 3 Institute for Mathematics, University of Freiburg, Freiburg, Germany



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The acute immunological rejection and long time survival of kidney allografts are correlated with the human leukocyte antigen (HLA) match status between donor and recipient. HLA-A, -B and -DR have all turned out to be relevant HLA loci in several studies. The role of HLA-C has not been studied before now.

Methods. In 104 consecutive patient/donor pairs from our transplantation unit, we retrospectively analysed whether acute graft rejection is influenced by HLA-C match status between donor and recipient. For typing HLA-C alleles, we used an allele-specific PCR protocol in combination with serology.

Results. By analysing groups of donor/recipient pairs with a homogenous distribution of HLA-B mismatches in order to exclude an effect of the linkage disequilibrium between HLA-B/C, HLA-C mismatch turned out to be significantly correlated with acute transplant rejection in pairs with one additional mismatch on the B locus (P=0.004). Additional parameters that may hypothetically influence acute rejection episodes (HLA-A or DR mismatch, time of cold and warm ischaemia, previous transplantations, pre-existing HLA antibodies) were also analysed but cannot explain this finding.

Conclusion. HLA-C matching of all kidney donors and recipients seems to be an option to reduce the probability of acute rejection episodes. Further studies of greater patient cohorts analysing organ rejection and organ survival are warranted.

Keywords: graft rejection; HLA-C matching; kidney transplantation; transplantation immunology



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The transplantation of kidney allografts has become a standard therapy for end-stage renal disease. The acute rejection of the graft by the host's immune system remains an unsolved problem in this context. Immunosuppressive drugs help to prevent and manage acute rejection episodes in many situations. However, aside from other harmful side effects, they can also induce infections and cancer in a large number of patients [1]. In conclusion, selecting grafts that are immunologically compatible with the recipient is still recommended despite the fact that new and improved immunosuppressive regimes have been developed.

Allografts rejection is a complex phenomenon, which is mediated by different immune mechanisms. A pivotal role for host T-cells is generally accepted. These cells are triggered mainly by foreign human leukocyte antigen (HLA) molecules. Therefore matching has to be performed with respect to the HLA molecules when donor/recipient pairs are selected in the clinical transplantation setting. This matching significantly improves the outcome of transplantation, although other factors such as gender, race, time of ischaemia and cytomegalovirus (CMV) status also contribute to graft survival [25].

Current allocation protocols for cadaveric kidneys match for the HLA-A, -B and -DR loci. HLA-C is also typed by some transplantation centres, but no active attempts are made to avoid mismatches concerning this locus. From the immunological point of view, HLA-C may be just as important as other loci, because it does induce an antibody response and presents peptides similar to the HLA-A, -B and -DR molecules [6]. In the context of bone marrow transplantation, elevated levels of cytotoxic lymphocyte precursors could be shown in recipient pairs that were HLA-C mismatched but otherwise completely matched [7]. An impact of HLA-C matching on the probability of clinical manifest graft versus host reactions has also been demonstrated [8,9]. Concerning kidney transplantation, two case reports of acute kidney rejections that were probably due to a HLA-C mismatch have been published [10,11].

Despite the above mentioned facts, the potential role of HLA-C in solid organ transplantation has not yet been examined systematically. For this reason, 106 donor recipient pairs from our transplantation unit were retrospectively analysed with respect to histologically verified acute rejection episodes, match status for the HLA-A, -B, -C and -DR loci, and other variables such as the duration of cold and warm ischaemia. The aim of the study was to determine whether HLA-C is an independent factor for acute rejection.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
HLA match status and occurrence of acute rejection episodes were retrospectively examined by analysing 106 consecutive cadaveric kidney donor/recipient pairs from our renal transplantation unit from 1995 to 1997. The mean follow-up time was 42 months. One pair was excluded because of severe perioperative complications (cardiac infarction), which led to exclusion from the standard immunosuppressive treatment, and one patient died intraoperatively. Eighty-five out of 104 patients (81%) were transplanted for the first time. Adequate follow-up data (clinical course at least 1 year after transplantation and results of renal biopsies if performed) was available for all patients.

Immunosuppressive treatment
Patients receiving their first transplant were treated initially with 7.5 mg/kg cyclosporin; 2 mg/kg azathioprine, and 500, 250, 125 and 20 mg methylprednisolone. Patients receiving a second transplant were also treated with antithymocyte globulin (Thymoglobulin®, Merieux, France). Doses were adjusted according to T-cell counts and graft function. Rejection episodes were treated initially with methylprednisolone, and additional antibody-based therapy was performed if necessary (17/35 cases). The rejection therapy avoided acute graft loss in 30/48 cases.

Histocompatibility studies
In all individuals, complete HLA class-I typing was performed using a complement-mediated cytotoxicity assay with 174 test sera according to National Institutes of Health (NIH) standards on EDTA-anticoagulated blood. To ascertain HLA-C type, frozen material was analysed using a commercially available PCR kit, which discriminates 18 currently known broad specificities (HLA-C low resolution SSP, Dynal, Sweden). Furthermore, a self-designed, group-specific PCR protocol was also used that discriminated two alternative amino acids at positions 77 and 80 of the HLA-C locus (for details see ref. 12) to ascertain the results obtained by allele-specific PCR. HLA-DR type was also ascertained by genotyping in all cases at the time of transplantation (Dynal DR high resolution PCR kit). Due to the retrospective design of the study, no frozen material from 34 donor/recipient pairs was available to perform HLA-C genotyping. Sixteen had two different HLA-C alleles; it was therefore not possible that a serologically undetectable allele was overlooked in these cases (see below). Considering this fact and the very low discrepancy between serology typing and genotyping for HLA-C in our laboratory (19 out of 318 genotyped alleles were mistyped by serology, whereby 18 mistypings have been the result of the non-existence of a serological equivalent for these alleles [12]), these patients were included in the study. The 18 resting pairs, for which DNA was not available and in which a second undetectable allele was possible, were analysed separately. Mismatches were always defined on the serological split level, thus HLA-C/-DR subsplit discrepancies were not regarded a mismatch, but HLA-A or -B split discrepancies were.

Rejection
If a rejection episode was clinically suspected a biopsy was performed immediately. Histological evaluation was performed by the local pathologist according to the Banff scheme [13]; rejection episodes were considered if they occurred during the first year after transplantation. To exclude CMV infection as the cause of graft function loss, pp65 early antigen was tested on lymphocyte smears counting 400 000 cells [14].

The following parameters were also initially considered in the analysis: mismatches on the HLA-A, -B or -DR loci; time of ischaemia; age and gender of donor/recipient; pre-existence of cytotoxic antibodies; and underlying diagnosis leading to renal failure. All patients stayed in hospital until blood creatinine was stabilized to a constant level. The creatinine level at the day of discharge was also documented as an indicator of graft function.

Statistics
First, a univariate analysis was performed for all considered variables. Apart from HLA-C, only HLA-B had any influence on acute rejection probability, although this influence was not significant. An independent effect of HLA-C matching was then examined in subgroups of donor/recipient pairs in which HLA-B was distributed homogeneously. Evaluation of all resulting cross tables was performed by Fisher's exact test using the SPSS software program (SPSS, Chicago, IL, USA). The influence of the time of cold and warm ischaemia was calculated by the Wilcoxon ranked sums test. A graft survival analysis was also performed by calculating a Kaplan–Meier blot and log rank statistics (2 degrees of freedom) using SPSS software.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
HLA typing
Genotyping for HLA-C revealed unambiguous results in all cases. Differences between serotyping and genotyping were observed in 18/140 (13%) genotyped individuals and were always due to alleles for which no serological equivalent exists (most frequently HLA-C *12xx). Thus serotyping for HLA-C never indicated alleles that did not actually exist but sometimes overlooked alleles, especially those with no serological equivalent. As expected, HLA-A/-B mismatch was strongly correlated with HLA-C mismatch (P<0.001) as a consequence of linkage disequilibrium.

Rejection-univariate analysis (Table 1Go)
Time of warm and cold ischaemia, number of previous transplantations and gender match had no demonstrable influence on acute rejection episodes. HLA-B mismatch was weakly correlated with the rejection probability (P=0.1). HLA-A and -DR had no influence on rejection, and the same was observed for all other considered variables. The strongest influence on acute rejection as determined by univariate analysis was exerted by HLA-C (P=0.002), whereby the cut-off point was between 0 or 1 and 2 mismatches.


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Table 1. Univariate analysis of the influence of all initially considered parameters on acute transplant rejection

 

Analysis of the independent influence of HLA-C (Table 2Go)
The primary goal of our study was to examine an independent effect of HLA-C mismatch on graft rejection. To exclude the HLA-B mismatch effect from this analysis, we had to consider separate subgroups of patients with a homogenous HLA-B match status. Within the subgroup of patients showing one HLA-B mismatch (Table 2bGo), HLA-C was found to exert a significant influence (P=0.004, or P=0.022 if only pairs with HLA-C determination on a genomic level were considered; see subjects and methods section) on the probability of acute rejection episodes. In contrast, no influence of C mismatch was observed in the subgroup of patients with no additional B mismatches (Table 2aGo). In summary, the HLA-C mismatch status is an independent factor in acute rejection, at least when paired with additional B mismatches.


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Table 2. Analysis of an independent HLA-C effect on transplant rejection excluding the effect of HLA-B mismatch by focusing the analysis on pairs without mismatch at this locus (a) and with one mismatch (b)

 

Graft survival
To prove whether or not the observed influence of the HLA-C match status also affects the graft survival, even in the small cohort, we performed a Kaplan–Meier analysis (Figure 1aGo and bGo). We established that there was a tendency towards better graft survival in the HLA-C-matched pairs. This effect was again more pronounced in pairs with one additional B mismatch, nearly reaching significance when pairs with no and two HLA-C mismatches were compared in this subgroup (P=0.055).



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Fig. 1. Kaplan–Meier analysis of the cumulative graft survival in different HLA-C match constellations. (a) All patients. (b) Patients with exactly one mismatch on the B locus thereby ruling out the possibility that different B-mismatches in the considered groups are responsible for observed survival differences.

 

Creatinine
The creatinine level at the time of discharge was not influenced by either HLA-A/-B or HLA-C mismatching.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We retrospectively analysed factors that might influence the probability of acute renal transplant rejection, as confirmed by histopathological diagnosis in 106 consecutive donor/recipient pairs. Despite the limited number of cases, univariate analysis revealed a statistically significant correlation with rejection probability for HLA-C and a trend for HLA-B. By subgroup analysis the independent contribution of the C locus could be established. The C effect could not be demonstrated in those pairs without additional B mismatches. Apart from problems due to the relatively small observation cohort, immunological reasons may also exist, presumably a form of ‘hyper-additive’ effect of the stimulation of B- and C-specific T-cell receptors which may explain the latter observation. All other variables, including HLA-A and -DR mismatch, revealed no correlations.

Many studies in large patient cohorts have shown a positive effect of HLA matching on renal allograft survival time [2,3,15]. The undesirable nature of the longer allocation procedure was more than compensated for by the benefits of matching [3]. The occurrence of acute rejection episodes as confirmed by histopathological diagnosis was not analysed in any of these studies. The impact of HLA matching on acute rejection episodes of renal allografts has been examined specifically in a few smaller studies [1619]. We used this parameter as the primary readout to monitor matching effects for two reasons: (i) acute rejection is an a priori immunological phenomenon and thus well suited for monitoring the effects of HLA matching; (ii) ~50% of all cases of graft function loss are actually caused by immunological rejection, despite the fact that immunosuppressive regimes are given in such cases [20]. The data presented in other reports [16,17,19] are consistent with our data to the extent that a correlation was observed between HLA mismatch and the incidence of rejection episodes. However, with respect to the individual HLA loci involved, results are contradictory. In the studies cited, the HLA-DR locus has the greatest influence, followed by HLA-B. In contrast, our data do not demonstrate any influence of HLA-DR match—not even a tendency—on rejection probability. One explanation for this discrepancy might be our local matching policy, which places high priority on matching for DR (60% no mismatch; 34% one mismatch; 6% two mismatches), thus the number of DR-mismatched donor/recipient pairs is probably too small to result in a statistically significant clinical DR-effect.

Our most remarkable finding was the significant correlation between HLA-C mismatching and rejection. This factor has never been considered in clinical practice and has never before been analysed in the context of solid organ transplantation. This may in part be due to the fact that serological typing has been considered to be less reliable [21] for the HLA-C locus than for other HLA loci. Today, PCR kits are commercially available which provide clear results in most cases [12]. Moreover, the discrepancy between serotyping and genotyping does not exceed 7.5% if sufficient HLA-C sera are used in the standard NIH cytotoxicity test [12]. Another reason for the lack of interest in HLA-C may be that its expression on cell surfaces is quite low [22]. However, HLA-C-presented antigens are recognized by T cells [7,23]. In the context of bone marrow transplantation, the influence of HLA-C has now been examined where HLA-C mismatch turns out to be a stronger predictor of graft versus host disease than HLA-A or -B mismatching [9].

The reciprocal influence of the B and C loci needs special consideration. The B and C loci are in close proximity to one another on chromosome 6; therefore the linkage disequilibrium is very strong. This phenomenon is also reflected by our data, which show a highly significant correlation between HLA-B and -C match status. As we were not able to perform genotyping for HLA-B for logistical reasons, one might argue that HLA-C may only be an indicator for additional B mismatches which have been overlooked by serological typing. However, the problems of serotyping are much less important for HLA-B alleles than reported for HLA-DR alleles [24]. Typing errors concerning broad specificities or splits rarely occur when typing HLA-B serologically. Subsplit mismatches may appear if serotyping is replaced by genotyping (e.g. *4402/*4403; *3901/*3906) but have never been reported to exert an influence on rejection. In our cohort, even mismatch on the split level did not exert a significant influence (Table 1Go). Moreover, our subgroup analysis revealed the clearest correlation between C mismatch and rejection rate in the subgroup with additional mismatch on the B locus. Thus, the considered pairs are a priori B mismatched, even on a serological level. HLA-B genotyping would not have changed the HLA-B match status if it had been performed in the subgroup upon which our conclusions are based because it is extremely unlikely that additional genotyping for HLA-B will turn a mismatch into a match (whereby changes in the opposite direction will occur).

Another question of practical importance is the frequency with which different allocation decisions result if allocation procedures are changed and HLA-C is considered with high priority. An analysis of our data demonstrates that in spite of the linkage disequilibrium, matching for B does not necessarily implicate matching for C. In our cohort, 13 pairs had C mismatching on haplotypes that were matched for B. This demonstrates clearly that additional matching for C can influence the allocation decision in a relevant proportion of cases that are already B matched.

In conclusion, HLA-C matching of all kidney donors and recipients may result in a better transplantation outcome. We strongly support the idea that clinical prospective studies should be performed in which rejection episodes and graft survival should be assessed in larger patient collectives.



   Acknowledgments
 
The authors would like to thank Petra Koritke for excellent technical assistance, Lothar Rink for helpful scientific discussions and Julian Keogh for critically reading the manuscript.



   Notes
 
Correspondence and offprint requests to: Dr C. Frohn, Institute of Immunology and Transfusion Medicine, Ratzeburger Allee 160, D-23538 Lübeck, Germany. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 30.11.99
Revision received 26. 9.00.