In comparison with HLA-matched sibling bone marrow transplants, unrelated donor transplants are associated with increased graft-versus-host disease and graft failure. This is likely in part due to HLA incompatibilities not identified by current matching strategies. High resolution DNA-based typing methods for HLA class II loci have improved donor selection and treatment outcome in unrelated donor bone marrow transplantation. By using DNA-based typing methods for HLA-A and -B on a cohort of 100 potential bone marrow donor/patient pairs, we find that serological typing for HLA class I is limited in its ability to identify incompatibilities in unrelated pairs. Furthermore, the incompatibilities identified are associated with the presence at high frequency of alloreactive cytotoxic T-lymphocyte precursors. DNA typing also indicates that HLA-C mismatches are common in HLA-A and -B serologically matched pairs. Such mismatches appear to be significantly less immunogenic with respect to cytotoxic T-lymphocyte recognition, but are expected to influence natural killer cell activity. Thus, improved resolution of HLA class I shows many previously undisclosed mismatches that appear to be immunologically functional. Use of high resolution typing methods in routine matching is expected to improve unrelated donor selection and transplant outcome.

ALLOGENEIC BONE MARROW transplantation (BMT) involves a unique immunological assault on the recipient. HLA differences between individuals can lead to the expansion of high frequency alloreactive T cells, often leading to severe graft-versus-host disease (GVHD) in the allogeneic BMT setting.1 An HLA genotypically identical sibling is therefore the donor of choice. However, because such a donor is available to only about 30% of patients, alternative donors, such as partially HLA-matched related and phenotypically HLA matched unrelated donors, are increasingly used. Although single HLA differences may be tolerated in related donor transplants, especially in younger recipients,2 such donors are rare, and increased disparity is associated with greatly reduced transplant success.3Consequently, HLA-matched unrelated donor transplants are often used in the treatment of a range of hematological disorders.

HLA matching is complex involving at least three highly polymorphic loci, HLA-A, -B, and -DRB1, encoding more than 400 alleles.4 In siblings, this complexity is reduced, because matching simply involves distinguishing the inherited from noninherited haplotypes. However, unrelated donor/patient pairs must be matched for each antigen and/or allele and, due to the extensive polymorphism of the HLA system, the chance of unrelated individuals being HLA identical is remote.5 It is only with the establishment of large international registers of HLA-typed individuals (currently >4.6 million potential donors have been recruited worldwide) that identification of a suitable unrelated donor is practical. Compared with HLA genotypically identical sibling transplants, HLA phenotypically matched unrelated donor transplants are commonly associated with increased risk of GVHD posttransplant6 and failure to engraft. It is likely that the increase in such posttransplant complications is at least in part due to the presence of HLA alloantigens not discriminated in the current matching procedure.

HLA typing for DRB1 and DQB1 is now performed using DNA-based methods allowing the routine identification of most Caucasoid alleles. However, until recently, matching for HLA-A and -B has been dependent on serological techniques. Serological resolution was thought to be adequate, because, in contrast to the typing of HLA-DR and -DQ serology, serological typing for HLA-A and -B identifies a greater number of antigens and a high level of heterozygosity at each locus. Despite this, there are limitations in the use of HLA class I serological data for matching, as cellular,7biochemical,8 and sequencing strategies show.9Although the importance of such differences in BMT remains to be fully defined, case studies have implicated alloresponses due to a single amino acid difference in HLA-B44 subtypes in both graft rejection10 and GVHD.11 

Although matching at HLA-A and -B loci is known to be important, the impact of HLA-C on transplantation is unclear. HLA-C is the third classical class I locus to be described, but, due to its low level of cell surface expression and reduced polymorphism, has been considered as less immunologically relevant.12 However, HLA-C–restricted virus-specific,13tumor-specific,14 and allospecific cytotoxic T lymphocytes (CTLs)15 are found, indicating that HLA-C has a role to play in a range of T-cell immune responses.

Interest in HLA-C and transplantation has been stimulated by recent exposition of its role in modulating natural killer (NK) cell activity. Interaction of self-MHC with receptors on a subset of NK cells is crucial for the protection from NK cell-mediated lysis.16In humans, this interaction has been mapped to a dimorphic epitope at residues 77 and 80 on the α1 domain of HLA-C, each epitope interacting with a distinct killer inhibitory receptor (KIR).17 Those cells expressing HLA-C molecules with a motif shared with HLA-Cw*0303 (Ser77/Asn 80) are protected from lysis by NK clones expressing KIR2DL2/3, whereas expression of a motif shared with HLA-Cw*0401 (Asn77/Lys80) inhibits lysis by NK clones expressing KIR2DL1.18 Alloreactive NK cells can be generated against stimulators expressing the opposite motif to that of the responder.19 The implications for BMT may be seen in mouse experiments in which NK cells of an irradiated F1 hybrid are able to reject parental bone marrow20 that lacks expression of self-MHC class I molecules.21 Recent reports suggest increased likelihood of graft failure in HLA-C–mismatched BMT,22 23 which may be analogous to the mouse hybrid resistance model.

The frequency of such serologically undetected mismatches and their impact on transplantation have yet to be fully assessed. In a previous report, we described that high CTL precursor (CTLp) frequencies were common in HLA-A, -B serologically and biochemically matched unrelated pairs, suggesting that undetected mismatches are common.24We have applied high resolution HLA class I typing techniques to study the true level of HLA compatibility in conventionally matched unrelated patient/donor pairs. Comparison of CTLp frequencies and DNA typing information may allow the assessment of the functional significance of such mismatches in allorecognition.

Patients and donors.

Seventy-six patients and 100 potential unrelated bone marrow donors were included in this study. Donors were selected as the best available matches for patients on the basis of matching for HLA-A and -B by serology and DRB1 identity by DNA-based typing. All patient/potential donor pairs were also analyzed for alloreactive CTLp frequency in the graft-versus-host direction.

Initial HLA typing for donor selection.

HLA-A and -B typing was performed by the standard complement-dependent microcytotoxicity test, using a combination of local and commercial (Biotest, Germany) serological typing trays. HLA-DR and -DQ types were assigned initially using commercial serological typing trays (Biotest). DNA from 97 of 100 pairs was then typed by a combination of polymerase chain reaction–sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction–sequence-specific primers (PCR-SSP) for DRB1 and similarly 95 of 100 for DQB1, as previously described.25 Where fully HLA compatible unrelated donors were not found on international volunteer registers, donors with minor HLA mismatches were considered. Minor mismatches were defined as either an HLA-A or -B subtype of a serologically related group as defined by the World Health Organization (WHO) nomenclature committee or mismatched at the molecular level for serologically defined HLA-DR1-14 specificities. Three pairs were serologically mismatched for HLA-A and 9 pairs for HLA-B, and 14 pairs were originally mismatched at the molecular level for HLA-DRB1. In total, mismatches were detected between 23 pairs at HLA-A, -B, and/or -DRB1.

Limiting dilution analysis.

Limiting dilution analysis was performed in the graft-versus-host direction as described by Kaminski et al26 to determine the frequency of patient-specific donor CTL precursors. A high CTLp frequency was taken to be greater than 1:105 peripheral blood mononuclear cells (PBMCs), because this correlates with poor transplant outcome.27 Low CTLp frequency was taken as being between 1:105 and 5:106, with CTLp frequencies less than this being regarded as negative.

DNA typing for HLA-A, -B, and -C.

To identify the presence of serologically undetected incompatibilities, a combination of DNA-based typing methods were used. HLA-A and -B were typed by previously described PCR-SSOP methods28,29 to confirm serotypes. Locus-specific oligotyping enabled the subtyping of most common HLA-A and -B serotypes to varying levels of resolution. The resolution offered by these techniques was dependent on the combination of alleles present, because heterozygosity often hindered allele-specific assignment. B locus oligotyping resolution was improved by using 3′ primers D1 and D2 separately where appropriate to amplify and analyze each allele individually.29 In total, the B locus alleles were amplified separately in 30% of pairs.

The polymorphisms within the serological specificities HLA-A2, -B35, and -B44 were further defined using a combination of group-specific amplification and oligotyping as described previously.30,31HLA-C typing was performed using either PCR-SSP32 or PCR-SSOP33 as described.

Reference strand-mediated conformation analysis (RSCA) matching of patient/donor pairs.

Because PCR-SSOP typing resolution is limited both by the number of probes used and ambiguities caused by heterozygosity, a conformation-dependent technique has been used on selected pairs. RSCA has recently been described as a method of identifying HLA alleles on the basis of mobility of a heteroduplex formed between the sense strand of a reference allele and the antisense strand of the unknown allele.34 

Briefly, HLA-A, -B, or -C amplification of a fragment containing exon 2, intron 2, and exon 3 was performed using locus-specific primers described previously.35 Reference strand amplification was performed from cell line DNA homozygous for the appropriate HLA allele using the same primers except with the sense primer labeled at the 5′ end with the Cy5 fluorochrome (Pharmacia Biotech, Uppsala, Sweden). Amplified reference and sample products were mixed at a 1:3 ratio and hybridized as previously described.34 Duplexes were separated by polyacrylamide gel electrophoresis (PAGE) using an ALFexpress automated sequencer (Pharmacia Biotech), and the mobility of the fluorescent duplex bands was analyzed using Fragment manager software (Pharmacia Biotech). By comparison with the mobilities of known heteroduplexes, it was possible to assign allelic specificities. Reference alleles used were as previously described,36 with the exception of HLA-B*1501 (amplified from DNA from IHW 9072 cell line) for confirmation of matching HLA-B62 seropositive donor/patient pairs.

Identification of serologically undetected HLA-A and -B incompatibilities.

To determine the level of HLA-A and -B undetected mismatching in serologically matched patient/donor pairs, samples were analyzed using higher resolution DNA-based methods. Patient/donor pairs were initially fully matched for HLA-A in 97% of cases (Fig 1). DNA typing of the patient/donor pairs confirmed the mismatches identified by serology. Two further patient/donor pairs were found to be incompatible using oligotyping for HLA-A, one indicating an HLA-A*02 subtype mismatch and the other an HLA-A*0301 versus -A*0302 mismatch (Table1). Specific subtyping confirmed the HLA-A*02 incompatibility as HLA-A*0201 versus -A*0205 and that 55 further HLA-A2 seropositive pairs were matched at the subtype level. That 91 of 92 HLA-A2 seropositive individuals were encoded by HLA-A*0201 reflects the dominance of this subtype in North European Caucasoids.37 RSCA matching indicated two further mismatches at A locus, an HLA-A*30 subtype mismatch and an HLA-A*03 heterozygote donor (A*0301, *03v) and HLA-A*0301 homozygous patient.

Fig. 1.

Level of matching achieved by conventional typing methods. Patients and potential donors were typed by serological methods for HLA-A and -B and DNA-based methods for HLA-DRB1 and -DQB1. Limiting dilution analysis was performed in the graft-versus-host direction to assess the frequency of host specific CTL precursors. Pairs with a high CTLp frequency (>1:105 PBMC) were judged to be mismatched at the cellular level.

Fig. 1.

Level of matching achieved by conventional typing methods. Patients and potential donors were typed by serological methods for HLA-A and -B and DNA-based methods for HLA-DRB1 and -DQB1. Limiting dilution analysis was performed in the graft-versus-host direction to assess the frequency of host specific CTL precursors. Pairs with a high CTLp frequency (>1:105 PBMC) were judged to be mismatched at the cellular level.

Close modal
Table 1.

Frequency of Mismatching Is Serotype Dependent

HLA Serotype
A2*,A3*,A30B35*,B39B44*,B51
Donor/patient pairs  56  24  4  15  8  25  4  
Mismatches detected  1  2  1  6  3  4  2  
Subtypes found 2  3  3  4  2  2  
HLA Serotype
A2*,A3*,A30B35*,B39B44*,B51
Donor/patient pairs  56  24  4  15  8  25  4  
Mismatches detected  1  2  1  6  3  4  2  
Subtypes found 2  3  3  4  2  2  
*

Mismatch detected by PCR-SSOP.

Mismatch detected by RSCA.

Original serological testing showed that 91% of patient/donor pairs were matched for HLA-B. A greater level of mismatching was found for HLA-B using DNA-based typing techniques. Discrepancies may occur between serological techniques due to the cross-reactivity of alloantisera, limitations in the alloantisera used, or the lack of expression of an allele. HLA-B SSOP found 3 samples to be misassigned by serology. In one case, HLA-B58 was missed and in another the same antigen was misassigned HLA-B57. In both cases, HLA-B62 was the second serotype expressed and errors in serological typing were likely due to serological cross-reactivity for HLA-B15 and -B17 groups. In the third, an HLA-B38 was not originally identified by serology. However, retyping by serology confirmed the presence of HLA-B38 as being expressed and not a null allele.

By using group-specific oligotyping methods,30,31 we identified heterogeneity within the HLA-B35 and -B44 serotypes. Four HLA-B*35 subtypes were identified at varying frequencies within the 27 individuals tested (Table 1). Importantly, 40% of the HLA-B35 seropositive patient/donor pairs tested were mismatched at the subtype level. HLA-B*44 subtyping indicated two common subtypes, HLA-B*4402 and -B*4403, in a 3:2 ratio. Despite the presence of these two common subtypes, only 4 of 25 (16%) of HLA-B44 seropositive pairs were mismatched at the subtype level. This low percentage is likely due to the HLA-B*44 subtypes commonly segregating on different haplotypes38 and matching for other loci fortuitously results in frequent HLA-B*44 subtype compatibility.

Oligotyping was limited in its resolution by the number of probes used and the heterozygosity exhibited by most samples. Two PCR primer mixes were used for HLA-B SSOP, which enabled the separate typing of each allele in a proportion of allelic combinations. However, RSCA was useful in identifying polymorphisms not detected by oligotyping and also in confirming homogeneity in potentially heterogeneous serotypes. Three HLA-B*51 subtypes and two commonly occuring HLA-B*39 subtypes were identified by this technique (Table 1) as well as those HLA-B*35 and -B*44 subtypes identified by SSOP. In contrast, only one subtype was detected in 45 HLA-B7 and 40 HLA-B8 seropositive individuals. Furthermore, HLA-B62, a serotype shown to be encoded by many distinct alleles,4 was identified as B*1501 in all 16 cases tested.

After molecular typing methods were applied, mismatching for HLA-A and -B increased to 7% and 27% of pairs, respectively. In total, those matched for HLA-A and -B decreased from 89% as initially detected by serology to 70% of pairs (Fig 2). The higher level of HLA-B mismatching reflects the limitations in serological resolution at this locus.

Fig. 2.

HLA matching for bone marrow donor selection. DNA based typing (▪) showed an increased level of mismatching for HLA-A and -B over that defined by serology (░). Although no HLA-C serology was performed for original typing, DNA-based typing indicated a high level of incompatibility.

Fig. 2.

HLA matching for bone marrow donor selection. DNA based typing (▪) showed an increased level of mismatching for HLA-A and -B over that defined by serology (░). Although no HLA-C serology was performed for original typing, DNA-based typing indicated a high level of incompatibility.

Close modal
Molecular HLA-C typing.

Unlike HLA-A and -B, no account was taken of HLA-C match status in the selection of donors for final stage testing. However, because HLA-B and HLA-C are in strong linkage disequilibrium, some level of matching was expected. To measure the level of matching at HLA-C and so determine its impact on the match status of patient donor pairs, PCR-SSP and PCR-SSOP typing methods were used. DNA-based typing identified HLA-C mismatching in 33 of 84 pairs tested (39%), a higher level than that seen for HLA-A or -B (Fig 2), with 8 pairs mismatched for both HLA-C alleles. Importantly, 24 of the 33 (73%) HLA-C mismatched pairs had further HLA-A and/or -B incompatibilities identified at the molecular level, indicating that HLA-C is a useful marker for other mismatches on the haplotype. The utility of HLA-C type as an indicator of HLA-B compatibility was allele dependent. HLA-B*4402 and B*4403 were predominantly associated with restricted HLA-C locus alleles, Cw*0501 and Cw*1601, respectively. In contrast, HLA-B*5101 was associated with a wide range of HLA-C alleles, as reported previously.15 39Thus, mismatching at HLA-C was not predictive of further mismatches for all haplotypes. Overall, 22 of 25 potential donor/patient pairs mismatched at HLA-B were also mismatched at HLA-C.

Mismatching for the HLA-C encoded motifs that interact with KIRs, thus influencing NK cell allorecognition, was next assessed. Each individual was classified as being positive for the Asn77 and Lys80 (group 1) and/or Ser77 and Asn80 (group 2) HLA-C molecules.18 We then calculated whether the HLA-C mismatched donor/patient pairs were also mismatched for this motif. Twenty-two of the 33 (67%) HLA-C mismatched pairs were also mismatched at the level of KIR binding motif. However, no mismatched pairs were homozygous for opposite KIR binding motifs, and so NK cell allorecognition would be expected to be unidirectional. Nine of the 22 mismatches may be expected to influence allorecognition in the graft-versus-host direction and 13 in the host-versus-graft direction.

Cellular recognition.

It has been reported that HLA-A and -B mismatches detected by serology and at the DNA level are recognized in vitro by high frequency CTL.24 40 In total, 86% of all donor/patient pairs mismatched for HLA-A or -B in the graft-versus-host direction fell into the high CTLp frequency group. Although the range of CTLp frequencies was broad, there was no evidence that mismatches detected by DNA typing were less immunogenic than those typed by serological methods, suggesting they may be as functionally relevant. In contrast to HLA-A and -B mismatches, 40% of HLA-C incompatible pairs fell into the low or negative CTLp frequency group. This suggests that HLA-C alloantigens may be less immunogenic than HLA-A or -B. To further investigate this, the donor/patient pairs were divided into those with only HLA-C mismatches and those with HLA-C plus other HLA class I incompatibilities. Mean CTLp frequencies differed significantly between both groups (P = .0004), with 82% of pairs with only HLA-C mismatches being in the low or negative CTLp frequency group (Fig 3). Only four donor/patient pairs were identified with HLA-A or -B mismatches in the graft-versus-host direction without HLA-C incompatibility. For these four pairs, a mean CTLp frequency of 1:30,000 was obtained (range, 1:23,000 to 1:36,000).

Fig. 3.

The effect of HLA-C mismatching on CTLp frequency. CTLp frequencies of 30 pairs with patient HLA-C locus incompatibilities are shown above. Mean CTLp frequencies differed significantly between those with only HLA-C mismatches (1:316,500) and those with further HLA class I mismatches (1:47,110). This indicates that HLA-C alloreactive T cells are found at a lower frequency than those at HLA-A and -B. The limit of sensitivity of this assay is 1 patient-specific donor CTLp/5 × 105 or higher. Mean CTLp frequencies differed significantly between groups according to the Mann-Whitney test.

Fig. 3.

The effect of HLA-C mismatching on CTLp frequency. CTLp frequencies of 30 pairs with patient HLA-C locus incompatibilities are shown above. Mean CTLp frequencies differed significantly between those with only HLA-C mismatches (1:316,500) and those with further HLA class I mismatches (1:47,110). This indicates that HLA-C alloreactive T cells are found at a lower frequency than those at HLA-A and -B. The limit of sensitivity of this assay is 1 patient-specific donor CTLp/5 × 105 or higher. Mean CTLp frequencies differed significantly between groups according to the Mann-Whitney test.

Close modal

Previous studies have indicated undetected HLA class I mismatches to be responsible for high CTLp frequencies. Despite using DNA-based HLA-A, -B, and -C matching for serologically matched pairs, 24 of 47 pairs (51%) with a high CTLp frequency had no discernible mismatch at HLA class I (Fig 4). Furthermore, only 2 DRB1 and 4 DQB1 mismatches were detected in the 24 pairs in the high CTLp and no HLA class I mismatch group.

Fig. 4.

DNA-based typing shows many HLA class I mismatches in the high CTLp frequency group not identified using serological methods. (▪) Matched pairs; (░) those with detected HLA class I mismatches. Forty-seven donors had high (>1:105) patient-specific CTLp frequencies. Only 8 (17%) of these had HLA class I mismatches detected by serological typing, reflecting the low overall number of HLA class I serological mismatched pairs. The proportion of HLA class I mismatched pairs in the high CTLp frequency group increased to 23 (49%) after DNA-based typing, with 21 pairs mismatched at the HLA-B and/or -A locus. However, 24 pairs (51%) with high CTLp frequencies appear to be compatible for HLA class I.

Fig. 4.

DNA-based typing shows many HLA class I mismatches in the high CTLp frequency group not identified using serological methods. (▪) Matched pairs; (░) those with detected HLA class I mismatches. Forty-seven donors had high (>1:105) patient-specific CTLp frequencies. Only 8 (17%) of these had HLA class I mismatches detected by serological typing, reflecting the low overall number of HLA class I serological mismatched pairs. The proportion of HLA class I mismatched pairs in the high CTLp frequency group increased to 23 (49%) after DNA-based typing, with 21 pairs mismatched at the HLA-B and/or -A locus. However, 24 pairs (51%) with high CTLp frequencies appear to be compatible for HLA class I.

Close modal
Overall level of matching.

Donors were selected on the basis of being closely matched for HLA-A and -B by serological methods and -DRB1 (and DQB1) by molecular methods. Using molecular typing methods for HLA-A and -B and including HLA-C typing data, the level of matching was seen to be greatly reduced. Of 89 pairs (89 donors for 76 patients) studied, 63% were originally regarded as fully matched. After DNA-based typing, this figure was reduced to 46%. However, single detected mismatches indicate mismatched haplotypes and increase the chances of there being incompatibilities at other loci. By using high resolution class I typing, we have found 43% of donor patient pairs with two or more mismatches and 17% with three or more (Fig5).

Fig. 5.

Multiple mismatches are shown by high resolution HLA class I typing. (▪) Original matching level; (░) the matching level obtained after DNA typing for HLA class I. After serological testing for HLA-A, -B and DNA typing for HLA-DRB1, -DQB1, 92% of pairs fell in the 0 or 1 mismatch group. This was reduced to 69% after DNA typing of HLA-A, -B, and -C. The increase in pairs with 3 or more mismatches increased from 2% to 16%, reflecting the association of HLA subtypes on different haplotypes.

Fig. 5.

Multiple mismatches are shown by high resolution HLA class I typing. (▪) Original matching level; (░) the matching level obtained after DNA typing for HLA class I. After serological testing for HLA-A, -B and DNA typing for HLA-DRB1, -DQB1, 92% of pairs fell in the 0 or 1 mismatch group. This was reduced to 69% after DNA typing of HLA-A, -B, and -C. The increase in pairs with 3 or more mismatches increased from 2% to 16%, reflecting the association of HLA subtypes on different haplotypes.

Close modal

Unrelated donor BMT has only been made practical by the establishment of large volunteer registries and implementation of HLA typing techniques with sufficient resolution for matching. Although HLA-matched siblings can be identified by a combination of a mixed lymphocyte reaction (MLR) assay and serological typing, this combination of tests has not proven effective in predicting GVHD in the unrelated donor setting.41 DNA-based typing methods have therefore been developed to allow accurate matching of HLA class II loci and are now used routinely for selection of matched unrelated donors, with improved transplant results.42 In contrast, HLA-A and -B specificities have until recently only been defined using classical serological methods. We have implemented DNA typing methods for HLA-A, -B, and -C to study the deficiencies of current HLA class I typing in accurately matching unrelated pairs. It is only by identifying the precise level of matching in BMT that the importance of serologically undefined differences can be assessed.

By using DNA-based typing for HLA class I, we have been able to identify many more serologically undetected mismatches in HLA-B than HLA-A. It appears that most HLA-A serotypes in our population are encoded for by one dominant allele as indicated by HLA-A2 subtyping (Table 1). In contrast, several HLA-B serotypes were encoded by multiple alleles. Although the incompatibilities identified by DNA-based typing involved molecular differences of as little as one amino acid, these are likely to be in positions on the HLA molecule expected to interact with bound peptide and/or T-cell receptor.43,44 Such small differences have been shown to generate vigorous alloreactive CTL responses in the BMT setting leading to severe complications.10 11 To further emphasize the functional relevance of these mismatches, high CTLp frequencies were detected in 86% of HLA-A, -B mismatched pairs.

We have confirmed that HLA-C incompatibilities are frequent in serologically HLA-A, -B matched unrelated pairs.15,45However, due to the distribution of polymorphic residues within HLA-C antigens and their reduced cell surface expression, it has been suggested that HLA-C may not be as immunologically relevant as the other classical class I loci.46 It has previously been suggested that HLA-C incompatibilities do correlate with high CTLp frequency,47 although in that study HLA-A and -B typings had not been determined using high resolution methods. In contrast, we have demonstrated that, whereas HLA-A and -B mismatches correlate with a high CTLp frequency, this was not so for HLA-C mismatching. By using high resolution matching techniques, we show that, of all the C locus mismatched pairs with high CTLp frequency, 89% had other class I differences. Furthermore, 82% of those with no other HLA class I mismatch were found to have a low or negative CTLp frequency (Fig 4). This supports the view that HLA-C may be of less immunological relevance than HLA-A and -B with respect to CTL surveillance.

Whereas HLA-C incompatibilities may not be a major target for alloreactive CTLs, certain mismatches will result in differential expression of the motifs responsible for modulating alloreactive NK cell activity. Mismatching for the HLA-C encoded NK resistance motifs was seen in 67% of those mismatched at this locus and 26% of all pairs studied. Because HLA phenotype appears to play an important role in defining the KIR repertoire of an individual,19,48potentially alloreactive NK cells are likely to be present in a significant proportion of unrelated donor transplants and their possible impact should be considered. A phenomenon in the mouse known as hybrid resistance in which bone marrow from an MHC homozygous parent is rejected by its F1 hybrid offspring has been shown to be mediated by NK cells.20 It is now known that the lack of self-MHC molecules on murine donor bone marrow cells leaves them vulnerable to recipient NK-mediated lysis.21 An analogous situation has been demonstrated in vitro in the human, with HLA-C playing a key role in the protection from NK lysis.49 

The detection of high CTLp frequency has been hypothesized to be a useful indicator of HLA class I incompatibilities not identified by serological methods.24,50,51 However, the range of CTLp frequencies for the same HLA mismatch may be large between unrelated individuals (Breur-Vriesendorp et al52 and unpublished data). Whether the absolute frequency or the qualitative properties of alloreactive CTLs calculated in vitro is important is not clear. Evidence suggests the CTLs involved in GVHD are not the same as those generated in vitro.51 Despite the use of high resolution HLA matching techniques, a significant number of pairs in the high CTLp frequency group (51%) had no detected mismatches in the GVHD direction. In such cases, full HLA-A, -B, and -C sequencing would be useful in confirming patient/donor HLA matching. A possible explanation for such CTL activity is that the peptide repertoire varies sufficiently between unrelated individuals to induce a strong alloreactive CTL response in vitro. In fact, serological reagents have been used to distinguish HLA-B antigens in the absence of allelic differences.53 54 CTLp frequency is often used as a criterion for selection of a suitable unrelated donor. It will therefore be important to assess the functional significance of high CTLp frequencies in the absence of HLA incompatibilities.

The term minor HLA mismatch is commonly used in reference to an HLA-A, -B mismatch of the same serological cross-reactive group or HLA class II allele encoding a product of the same serological group. Matching for HLA-B serological splits has been shown to be of little benefit in renal transplantation.55 However, evidence suggests that these incompatibilities are of major importance to the outcome of allogeneic BMT. Serologically undetected HLA class I mismatches are recognized efficiently by alloreactive CTL correlating with poor transplant outcome.27,40,56 57 

By improving the resolution of HLA class I matching, the number of perfectly matched pairs has significantly decreased. Furthermore, many pairs were found to have multiple mismatches, which has been shown to increase the risk of posttransplant complications. Thus, the problem created by high resolution typing of HLA loci is a reduction in the number of matched donors that can be provided. However, this study has demonstrated that many of the HLA mismatches identified are on haplotypes frequent in the Caucasoid population. Therefore, the likelihood increases that several unrelated donors will be matched at the serological level at HLA class I. The development of techniques such as RSCA will allow the convenient, rapid, and accurate screening of a large number of such potential donors. By using such a strategy, HLA matching levels should improve. However, there will remain a significant number of patients without fully HLA matched donors and we need to assess carefully what level of HLA mismatching can be acceptable for a beneficial outcome to BMT. Retrospective analyses of unrelated donor transplants using high resolution typing have been performed by several groups in an attempt to identify those mismatches best tolerated,58 59 and indications that mismatched transplants can be successful, especially for younger patients, are encouraging. The hope is that such studies will allow the rational selection of the most appropriate mismatched bone marrow donor.

The authors thank I. Anthony Dodi and Steven G.E. Marsh for critical review of the manuscript and the staff of the Anthony Nolan Tissue-typing Laboratories for excellent technical assistance.

Supported by The Anthony Nolan Bone Marrow Trust. J.R.A. is a recipient of a fellowship from the Consejo Nacional de Ciencia y Tecnologia, Mexico and Overseas Research Students Awards (CVCP) U.K.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Kaminski
E
Hows
J
Man
S
Brookes
P
Mackinnon
S
Hughes
T
Avakian
O
Goldman
JM
Batchelor
JR
Prediction of graft versus host disease by frequency analysis of cytotoxic T cells after unrelated donor bone marrow transplantation.
Transplantation
48
1989
608
2
Beatty
PG
Anasetti
C
Hansen
JA
Longton
GM
Sanders
JE
Martin
PJ
Mickelson
EM
Choo
SY
Petersdorf
EW
Pepe
MS
Appelbaum
FR
Bearman
SI
Buckner
CD
Clift
RA
Peterson
FB
Singer
J
Stewart
PS
Storb
RF
Sullivan
KM
Tesler
MC
Witherspoon
RP
Thomas
ED
Marrow transplantation from unrelated donors for treatment of hematologic malignancies: Effect of mismatching for one HLA locus.
Blood
81
1993
249
3
Beatty
PG
Clift
RA
Mickelson
EM
Nisperos
BB
Flournoy
N
Martin
PJ
Sanders
JE
Stewart
P
Buckner
CD
Storb
R
Thomas
ED
Marrow transplantation from related donors other than HLA-identical siblings.
N Engl J Med
313
1985
765
4
Bodmer
JG
Marsh
SG
Albert
ED
Bodmer
WF
Bontrop
RE
Charron
D
Dupont
B
Erlich
HA
Fauchet
R
Mach
B
Mayr
WR
Parham
P
Sasazuki
T
Schreuder
GM
Strominger
JL
Svejgaard
A
Terasaki
PI
Nomenclature for factors of the HLA System, 1996.
Hum Immunol
53
1997
98
5
Speiser
DE
Tiercy
JM
Rufer
N
Chapuis
B
Morell
A
Kern
M
Gmur
J
Gratwohl
A
Roosnek
E
Jeannet
M
Relation between the resolution of HLA-typing and the chance of finding an unrelated bone marrow donor.
Bone Marrow Transplant
13
1994
805
6
Beatty
PG
Hansen
JA
Longton
GM
Thomas
ED
Sanders
JE
Martin
PJ
Bearman
SI
Anasetti
C
Petersdorf
EW
Mickelson
EM
Pepe
MS
Appelbaum
FR
Buckner
CD
Clift
RA
Petersen
FB
Stewart
PS
Storb
RF
Sullivan
KM
Tesler
MC
Witherspoon
RP
Marrow transplantation from HLA-matched unrelated donors for treatment of hematologic malignancies.
Transplantation
51
1991
443
7
Brenner
MB
McLean
J
Yang
SY
van der Poel
JJ
Pious
D
Strominger
JL
Clonal T lymphocyte recognition of the fine structure of the HLA-A2 molecule.
J Immunol
135
1985
384
8
Ragupathi
G
Cereb
N
Yang
SY
The relative distribution of B35 alleles and their IEF isotypes in a HLA-B35-positive population.
Tissue Antigens
46
1995
24
9
Fleischhauer
K
Kernan
NA
Dupont
B
Yang
SY
The two major subtypes of HLA-B44 differ for a single amino acid in codon 156.
Tissue Antigens
37
1991
133
10
Fleischhauer
K
Kernan
NA
O’Reilly
RJ
Dupont
B
Yang
SY
Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44.
N Engl J Med
323
1990
1818
11
Keever
CA
Leong
N
Cunningham
I
Copelan
EA
Avalos
BR
Klein
J
Kapoor
N
Adams
PW
Orosz
CG
Tutschka
PJ
Baxter-Lowe
LA
HLA-B44-directed cytotoxic T cells associated with acute graft-versus-host disease following unrelated bone marrow transplantation.
Bone Marrow Transplant
14
1994
137
12
Lawlor
AD
Warren
E
Ward
FE
Parham
P
Comparison of class I MHC alleles in humans and apes.
Immunol Rev
113
1990
147
13
Chen
BP
Lam
V
Kraus
EE
DeMars
R
Sondel
PM
Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules.
Hum Immunol
26
1989
137
14
van der Bruggen
P
Szikora
JP
Boel
P
Wildmann
C
Somville
M
Sensi
M
Boon
T
Autologous cytolytic T lymphocytes recognize a MAGE-1 nonapeptide on melanomas expressing HLA-Cw*1601.
Eur J Immunol
24
1994
2134
15
Grundschober
C
Rufer
N
Sanchez-Mazas
A
Madrigal
A
Jeannet
M
Roosnek
E
Tiercy
JM
Molecular characterization of HLA-C incompatibilities in HLA-ABDR-matched unrelated bone marrow donor-recipient pairs. Sequence of two new Cw alleles (Cw*02023 and Cw*0707) and recognition by cytotoxic T lymphocytes.
Tissue Antigens
49
1997
612
16
Ljunggren
H-S
Karre
K
In search of the ‘missing self’: MHC molecules and NK cell recognition.
Immunol Today
11
1990
237
17
Moretta
A
Vitale
M
Bottino
C
Orengo
AM
Morelli
L
Augugliaro
R
Barbaresi
M
Ciccone
E
Moretta
L
P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities.
J Exp Med
178
1993
597
18
Ciccone
E
Pende
D
Viale
O
Than
A
Di Donato
C
Orengo
AM
Biassoni
R
Verdiani
S
Amoroso
A
Moretta
A
Moretta
L
Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: Expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2).
J Exp Med
176
1992
963
19
Colonna
M
Brooks
EG
Falco
M
Ferrara
GB
Strominger
JL
Generation of allospecific natural killer cells by stimulation across a polymorphism of HLA-C.
Science
260
1993
1121
20
Bennett
M
Biology and genetics of hybrid resistance.
Adv Immunol
41
1987
333
21
Yu
YYL
George
T
Dorfman
JR
Roland
J
Kumar
V
Bennet
M
The role of Ly49A and 5E6(Ly49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts.
Immunity
4
1996
67
22
Bishara
A
Amar
A
Brautbar
C
Condiotti
R
Lazarovitz
V
Nagler
A
The putative role of HLA-C recognition in graft versus host disease (GVHD) and graft rejection after unrelated bone marrow transplantation (BMT).
Exp Hematol
23
1995
1667
23
Petersdorf
EW
Longton
GM
Anasetti
C
Mickelson
EM
McKinney
SK
Smith
AG
Martin
PJ
Hansen
JA
Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors.
Blood
89
1997
1818
24
O’Shea
J
Madrigal
A
Davey
N
Brookes
P
Scott
I
Firman
H
Lechler
R
Goldman
J
Batchelor
R
Measurement of cytotoxic T lymphocyte precursor frequencies reveals cryptic HLA class I mismatches in the context of unrelated donor bone marrow transplantation.
Transplantation
64
1997
1353
25
Jordan
F
McWhinnie
AJ
Turner
S
Gavira
N
Calvert
AA
Cleaver
SA
Holman
RH
Goldman
JM
Madrigal
JA
Comparison of HLA-DRB1 typing by DNA-RFLP, PCR-SSO and PCR-SSP methods and their application in providing matched unrelated donors for bone marrow transplantation.
Tissue Antigens
45
1995
103
26
Kaminski
E
Hows
J
Goldman
J
Batchelor
R
Optimising a limiting dilution culture system for quantitating frequencies of alloreactive cytotoxic T lymphocyte precursors.
Cell Immunol
137
1991
88
27
Spencer
A
Brookes
PA
Kaminski
E
Hows
JM
Szydlo
RM
van Rhee
F
Goldman
JM
Batchelor
JR
Cytotoxic T lymphocyte precursor frequency analyses in bone marrow transplantation with volunteer unrelated donors. Value in donor selection.
Transplantation
59
1995
1302
28
Oh
SH
Fleischhauer
K
Yang
SY
Isoelectric focusing subtypes of HLA-A can be defined by oligonucleotide typing.
Tissue Antigens
41
1993
135
29
Middleton
D
Williams
F
Cullen
C
Mallon
E
Modification of an HLA-B PCR-SSOP typing system leading to improved allele determination.
Tissue Antigens
45
1995
232
30
Tiercy
JM
Djavad
N
Rufer
N
Speiser
DE
Jeannet
M
Roosnek
E
Oligotyping of HLA-A2, -A3, and -B44 subtypes. Detection of subtype incompatibilities between patients and their serologically matched unrelated bone marrow donors.
Hum Immunol
41
1994
207
31
Gauchat-Feiss
D
Rufer
N
Speiser
D
Jeannet
M
Roosnek
E
Tiercy
JM
Heterogeneity of HLA-B35. Oligotyping and direct sequencing for B35 subtypes reveals a high mismatching rate in B35 serologically compatible kidney and bone marrow donor/recipient pairs.
Transplantation
60
1995
869
32
Bunce
M
Barnardo
MC
Welsh
KI
Improvements in HLA-C typing using sequence-specific primers (PCR-SSP) including definition of HLA-Cw9 and Cw10 and a new allele HLA-”Cw7/8v”.
Tissue Antigens
44
1994
200
33
Kennedy
LJ
Poulton
KV
Dyer
PA
Ollier
WE
Thomson
W
Definition of HLA-C alleles using sequence-specific oligonucleotide probes (PCR-SSOP).
Tissue Antigens
46
1995
187
34
Argüello
RJ
Little
A-M
Pay
AL
Gallardo
D
Rojas
I
Marsh
SGE
Goldman
JM
Madrigal
JA
Mutation detection and typing of polymorphic loci through double-strand conformation analysis.
Nat Genet
18
1998
192
35
Cereb
N
Maye
P
Lee
S
Kong
Y
Yang
SY
Locus-specific amplification of HLA class I genes from genomic DNA: locus-specific sequences in the first and third introns of HLA-A, -B, and -C alleles.
Tissue Antigens
45
1995
1
36
Argüello
J
Little
A-M
Bohan
E
Gallardo
D
O’Shea
J
Dodi
I
Goldman
JM
Madrigal
JA
A high resolution HLA class I and class II matching method for bone marrow donor selection.
Bone Marrow Transplant
22
1998
527
37
Krausa
P
Brywka
M
III
Savage
D
Hui
KM
Bunce
M
Ngai
JLF
Teo
DLT
Ong
YW
Barouch
D
Allsop
CEM
Hill
AVS
McMichael
AJ
Bodmer
JG
Browning
MJ
Genetic polymorphism within HLA-A*02: significant allelic variation revealed in different populations.
Tissue Antigens
45
1995
223
38
Petersdorf
EW
Setoda
T
Smith
AG
Hansen
JA
Analysis of HLA-B*44 alleles encoded on extended HLA haplotypes by direct automated sequencing.
Tissue Antigens
44
1994
211
39
Bunce
M
Barnardo
MC
Procter
J
Marsh
SG
Vilches
C
Welsh
KI
High resolution HLA-C typing by PCR-SSP: Identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology [corrected and republished article originally printed in Tissue Antigens 48:680, 1996].
Tissue Antigens
50
1997
100
40
Speiser
DE
Loliger
CC
Siren
MK
Jeannet
M
Pretransplant cytotoxic donor T-cell activity specific to patient HLA class I antigens correlating with mortality after unrelated BMT.
Br J Haematol
93
1996
935
41
Mickelson
EM
Longton
G
Anasetti
C
Petersdorf
E
Martin
P
Guthrie
LA
Hansen
JA
Evaluation of the mixed lymphocyte culture (MLC) assay as a method for selecting unrelated donors for marrow transplantation.
Tissue Antigens
47
1996
27
42
Petersdorf
EW
Longton
GM
Anasetti
C
Martin
PJ
Mickelson
EM
Smith
AG
Hansen
JA
The significance of HLA-DRB1 matching on clinical outcome after HLA-A, B, DR identical unrelated donor marrow transplantation.
Blood
86
1995
1606
43
Bjorkman
PJ
Saper
MA
Samraoui
B
Bennett
WS
Strominger
JL
Wiley
DC
The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.
Nature
329
1987
512
44
Bjorkman
PJ
Parham
P
Structure, function, and diversity of class I major histocompatibility complex molecules.
Annu Rev Biochem
59
1990
253
45
Santamaria
P
Reinsmoen
NL
Lindstrom
AL
Boyce-Jacino
MT
Barbosa
JJ
Faras
AJ
McGlave
PB
Rich
SS
Frequent HLA class I and DP sequence mismatches in serologically (HLA-A, HLA-B, HLA-DR) and molecularly (HLA-DRB1, HLA-DQA1, HLA-DQB1) HLA-identical unrelated bone marrow transplant pairs.
Blood
83
1994
280
(erratum 83:3834, 1994)
46
Lawlor
DA
Zemmour
J
Ennis
PD
Parham
P
Evolution of class-I MHC genes and proteins: From natural selection to thymic selection.
Annu Rev Immunol
8
1990
23
47
Barnardo
MC
Davey
NJ
Bunce
M
Brookes
PA
Lechler
RI
Welsh
KI
Batchelor
JR
A correlation between HLA-C matching and donor antirecipient CTL precursor frequency in bone marrow transplantation.
Transplantation
61
1996
1420
48
Valiante
NM
Uhrberg
M
Shilling
HG
Lienert-Weidenbach
K
Arnett
KL
D’Andrea
A
Phillips
JH
Parham
P
Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors.
Immunity
7
1997
739
49
Moretta
L
Ciccone
E
Mingari
MC
Biassoni
R
Moretta
A
Human natural killer cells: Origin, clonality specificity, and receptors.
Adv Immunol
55
1994
341
50
Rufer
N
Tiercy
JM
Speiser
DE
Helg
C
Gratwohl
A
Chapuis
B
Jeannet
M
Roosnek
E
Characterization of pretransplant CTL activity in “perfectly matched” unrelated bone marrow donor/recipient combinations.
Bone Marrow Transplant
11
1993
20
(suppl 1)
51
Rufer
N
Helg
C
Tiercy
JM
Barbey
C
Gratwohl
A
Chapuis
B
Jeannet
M
Roosnek
E
Recognition of major histoincompatibilities after transplantation with marrow from HLA closely matched donors.
Transplantation
63
1997
1833
52
Breur-Vriesendorp
BS
Vingerhoed
J
Schaasberg
WP
Ivanyi
P
Variations in the T-cell repertoire against HLA antigens in humans.
Hum Immunol
27
1990
1
53
Domena
JD
Arnett
KL
Marsh
SG
Bodmer
JG
Parham
P
Alloantibodies can discriminate three populations of HLA-B40 molecules encoded by B*4002.
Tissue Antigens
44
1994
57
54
Adams
EJ
Scott
I
Shah
A
Arnett
KL
Marsh
SG
Madrigal
JA
Parham
P
Homogeneity of allelic sequence for serological variants of HLA-B53.
Tissue Antigens
46
1995
330
55
Sanfilippo
F
Vaughn
WK
Light
JA
LeFor
WM
Lack of influence of donor-recipient differences in subtypic HLA-A, B antigens (splits) on the outcome of cadaver renal transplantation.
Transplantation
39
1985
151
56
Roosnek
E
Hogendijk
S
Zawadynski
S
Speiser
D
Tiercy
JM
Helg
C
Chapuis
B
Gratwohl
A
Gmur
J
Seger
R
Jeannet
M
The frequency of pretransplant donor cytotoxic T cell precursors with anti-host specificity predicts survival of patients transplanted with bone marrow from donors other than HLA-identical siblings.
Transplantation
56
1993
691
57
Keever-Taylor
CA
Passweg
J
Kawanishi
Y
Casper
J
Flomenberg
N
Baxter-Lowe
LA
Association of donor-derived host-reactive cytolytic and helper T cells with outcome following alternative donor T cell-depleted bone marrow transplantation.
Bone Marrow Transplant
19
1997
1001
58
Madrigal
JA
Arguello
R
Scott
I
Avakian
H
Molecular histocompatibility typing in unrelated donor bone marrow transplantation.
Blood Rev
11
1997
105
59
Hansen
JA
Petersdorf
E
Martin
PJ
Anasetti
C
Hematopoietic stem cell transplants from unrelated donors.
Immunol Rev
157
1997
141

Author notes

Address reprint requests to J. Alejandro Madrigal, MD, PhD, The Anthony Nolan Research Institute, The Royal Free Hospital, Pond Street, London, NW3 2QG, UK; e-mail: madrigal@rfhsm.ac.uk.

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