A CORRELATION BETWEEN HLA-C MATCHING AND DONOR... : Transplantation (original) (raw)
During the past decade, increasing use has been made of registries of HLA-typed volunteers willing to provide bone marrow for transplantation into recipients who lack an HLA-identical sibling donor. It was anticipated that grafts from unrelated donors would be attended by greater risks of severe graft-versus-host disease (GVHD*) than those encountered when using donor marrow from an HLA-identical sibling. The reason for this is that HLA-identical siblings have inherited the same HLA region of chromosome 6 from each parent and thus share precisely the same HLA haplotypes and are therefore HLA genotypically identical. In the case of unrelated, volunteer donors, the exactness of HLA match with the recipient depends on the resolution and accuracy of the techniques employed and the particular loci of the HLA region examined.
The clinical results of bone marrow transplantation (BMT) with grafts from unrelated, volunteer donors have been sufficiently promising to encourage wider use of this strategy. However, many questions about the level of HLA matching necessary for an acceptable clinical result remain to be answered. For example, is it critical to match donor and recipient for all class I and II HLA genes and how precise should this match be? To obtain a functional index of the immunological strength of antigenic incompatibilities, we have used limiting dilution assays to estimate the frequencies of donor lymphocytes with antirecipient alloreactivity (1, 2). The results show that high frequencies of precursors of alloreactive (donor versus recipient) cytotoxic T lymphocytes (CTLp) are associated with significantly higher risks of acute GVHD and death. In these studies, effects of mismatching at the HLA-C locus were not examined because of suspicions that HLA-C assignments based on serological tests were frequently incorrect and unreliable. Recently, we have developed a reliable DNA-based method using the polymerase chain reaction and sequence-specific primers (PCR-SSP) to amplify sequences of the HLA-C alleles (3, 4) and have now included this technique in the battery of HLA-matching tests performed on blood samples from potential donors and recipients before BMT. We show here that donor/recipient mismatching at HLA-C, a locus not previously considered of importance in BMT, is correlated with “high” CTLp frequencies that are not associated with a defined mismatch of HLA-A or HLA-B. The results demonstrate that HLA-C matching by this inexpensive and rapid method significantly improves the ability to identify donor/recipient pairs with high CTLp frequencies, a patient group with high risk of severe acute GVHD.
HLA compatibility was determined for 36 patient/unrelated, volunteer donor pairs being assessed for bone marrow grafts. Donors were selected from the Anthony Nolan Research Centre panel. Full details of the patients and HLA-A, -B, -DR, and -DQ typing methods are described elsewhere (1). In brief, 28 patients received BMT for chronic myeloid leukemia. Unrelated donors were selected on the basis of matching by conventional serological methods for HLA-A, -B, -DR, and -DQ polymorphisms, using antisera supplied by the United Kingdom Transplant Support Service Authority, supplemented by some locally derived reagents. Class I (HLA-A and -B) allele assignments were confirmed by one-dimensional isoelectric focusing (IEF) of 35S-methionine-labeled immunoprecipitates using the monoclonal antibody W6/32. Class II (HLA-DRB and -DQB) alleles were confirmed by analyses of DNA restriction fragment length polymorphisms and/or PCR-SSP methodology. The HLA-A, -B, -DR, and -DQ specificities of the donor/recipient pairs together with the CTLp frequencies are shown in Table 1.
CTLp frequencies of each donor antirecipient combination were estimated by limiting dilution analysis as described previously (5). Donor/recipient pairs were classified according to CTLp frequencies into two groups, referred to as the high frequency group (HF, >1 in 100×103) and the low frequency group (LF, <1 in 100×103).
High and low frequencies are defined using a cutoff value of 1 in 100×103. This figure was derived from early studies (2, 6) in which the range of CTLp frequencies was measured in three distinct cohorts of patients: HLA-identical siblings, matched unrelated pairs, and mismatched pairs. Sibling pair frequencies were consistently less than 1 in 100×103, while mismatched pairs all gave frequencies of >1 in 130×103. Matched unrelated pairs ranged from <1 in 500×103 to 1 in 10×103. The cutoff frequency was taken to be that between the upper limit of the sibling pairs and the lower limit of the mismatched group. Subsequent analyses of clinical posttransplant data have confirmed the validity of these criteria.
Matching of patient/donor pairs for HLA-C was carried out retrospectively using PCR-SSP as developed by Bunce et al. (3, 4). Briefly, 20 PCR primer pairs were used to identify the 18 alleles/allele group shown in Table 2. All the known HLA-C alleles were considered. A 30-cycle PCR protocol was employed using an ammonium sulfate buffer system subsequent to treatment of the target DNA sample with heparinase II (7). The tests were performed and HLA-C alleles assigned before the sample code was broken.
The detectable specificities for serology, RFLP and SSP are shown in Table 2 and those for IEF are shown in Table 3.
The distribution of HLA-C mismatches in the donor/recipient pairs was significantly correlated with the CTLp frequencies. The contingency table (Table 4) shows the data.
Only 2 of the 17 pairs in the HF group were mismatched for HLA-A and/or -B; the mismatches were due to variants not detected by serological methods, and were identified by IEF. One of these pairs was also HLA-C mismatched. There were a further seven pairs without HLA-A or -B mismatches identified that demonstrated a donor antirecipient mismatch at the HLA-C locus.
In the LF group there were no donor antirecipient HLA-A or -B mismatches detected. Only one pair was mismatched for HLA-C in this group.
It has been demonstrated previously that unrelated patient/donor pairs generating high (>1 in 100,000) donor antirecipient CTLp frequencies suffer a significantly higher incidence of severe, acute GVHD and death following BMT (1, 8). However, some other groups have reported a failure to corroborate these findings (9, 10). Such discrepant observations may be attributable to a number of factors. First, in some cases, detailed information relating to the patient groups studied, such as pretransplant conditioning and patient homo/heterogeneity, have not been provided, either of which may critically affect results. Second, these patient cohorts are generally small, and may therefore be prone to statistical inaccuracies. In addition, definition of posttransplant complications including GVHD may vary between centers. Last, the CTLp assay itself has not been fully standardized between laboratories and no true consensus for optimal methodology has been established.
Here we present results that show that HLA-C incompatibility in unrelated donor/recipient pairs, in which no other class I HLA mismatch has been detected, is significantly associated with high CTLp frequencies, and the associated risk of severe, acute GVHD. The numbers are too small, however, to directly measure any significant association between C-locus incompatibilities and GVHD. Measurement of CTLp frequencies is a time-consuming and laborious procedure, limiting its widespread use for selection of unrelated donors. Alternative methods for the detection of clinically significant incompatibility are clearly desirable. With the introduction of this DNA-based method of HLA-C typing, our ability to identify mismatches correlating with high CTLp frequencies has been substantially improved. The results also indicate that it will be necessary to achieve a similar level of typing and matching at the other HLA class I loci to optimize selection of unrelated donors in BMT.
A number of class II incompatibilities were detected but these were evenly distributed throughout the cohort and were not statistically significant. This is in keeping with the immunological dogma that CTL are predominantly class I restricted. Further study of these class II-mismatched pairs using the recently developed HTLp assay might yield more information regarding the role of class II in the generation of GVHD. At this stage, HTLp assays have not been performed.
Recently, two studies have demonstrated a high degree of previously undetected mismatching at the HLA-C locus among unrelated donor/recipient pairs matched at all other loci (11, 12). Our findings support these data and, additionally, are the first to show a significant correlation between HLA-C matching and a proven marker for clinical outcome in the CTLp frequency. This is in agreement with the nonsignificant trend reported by Santamaria and co-workers (11), in which class I molecular mismatching was associated with an increased incidence of GVHD.
Theoretically, HLA-C incompatibilities could themselves be the targets of immunological attack. HLA-C molecules can be recognized by receptors of human CTL in both in vitro culture (13) and in vivo allograft rejection (14). Alternatively, HLA-C incompatibilities may act as “markers” for other, unidentified HLA incompatibilities in linkage disequilibrium. Clearly, it will be necessary to apply higher resolution HLA typing techniques to other class I loci (HLA-A and -B) than are currently employed, in order to address this question. Sequencing of the HLA genes in the HLA-C mismatched pairs will be necessary in order to unambiguously determine the importance of HLA-C polymorphisms as transplantation antigens in unrelated BMT.
Footnotes
Abbreviations: BMT, bone marrow transplantation; CTLp, cytotoxic T lymphocyte precursors; GVHD, graft-versus-host disease; HF, high CTLp frequency; IEF, isoelectric focusing; LF, low CTLp frequency; PCR, polymerase chain reaction; SSP, sequence-specific primers.
REFERENCES
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