HLA matching in hematopoietic cell transplantation.

Progress in hematopoietic cell transplantation has been greatly facilitated by our increasing knowledge of the HLA system, as well as by improved therapies for achieving sustained engraftment, preventing graft-versus-host disease, and protecting the patient from infection. Disparity for HLA genes can cause graft rejection and graft-versus-host disease and decrease survival in patients receiving grafts from both related and unrelated donors. The presence of patient alloantibodies against donor antigens demonstrated by a positive crossmatch is a strong risk factor for graft rejection. The availability of matched donors for patients lacking a genotypically HLA-matched sibling has been greatly improved by the establishment of international registries of HLA-typed volunteer donors. The development of accurate and reproducible high-resolution DNA-based typing methods has significantly improved the prospects for identifying unrelated donors who are well matched with the patient for HLA. The use of these methods to optimize donor selection will improve both donor identification and the success of unrelated donor transplants.

Effect of HLA mismatches on the outcome of hematopoietic transplants.

Hematopoietic cell transplantation from unrelated volunteer donors for the treatment of hematological malignancy can be optimized by complete and precise matching for HLA class I and II alleles between the donor and recipient. Survival is improved when the donor and recipient are matched for HLA-A, -B, -C, -DRB, -DQB1 and -DPB1 alleles. The risks of clinically severe graft-versus-host disease, graft failure and mortality are increased in the presence of multilocus mismatching. These findings demonstrate that HLA allelic differences are biologically relevant in human transplantation.

Optimizing outcome after unrelated marrow transplantation by comprehensive matching of HLA class I and II alleles in the donor and recipient.

In unrelated marrow transplantation, the benefit of matching class II HLA-DRB1 and DQB1 alleles of the donor and recipient is well documented. Little is known about the clinical relevance of matching for class I HLA-A, B, and C alleles. We used DNA-amplification methods to identify the HLA-A, B, and C alleles of 300 patients and their donors. The incidence of graft failure was correlated with multiple class I mismatching in the donor. The risk of grades III-IV acute graft-versus-host disease was highest with class II mismatching in the recipient. Mismatching for a single class I or class II allele had no effect on survival, but mortality was increased by mismatching for more than one class I allele and by simultaneous mismatching for class I and class II alleles. We conclude that matching HLA class I and class II alleles of the donor and recipient can improve outcome after unrelated marrow transplantation.

Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors.

Disparity for HLA-A or HLA-B antigens increases the risk of marrow graft rejection, but the relevance of HLA-C is unknown because typing methods have not been sufficiently accurate for clinical use. We designed a matched case-control study and employed DNA sequencing methods to evaluate the role of HLA-C disparity in 21 patients who experienced graft failure (cases) following transplantation with unmanipulated marrow from either HLA-A, B serologically matched, DRB1 matched (n = 14) or single locus mismatched (n = 7) unrelated donors. For each case, two patients who successfully engrafted were selected as controls based on similarity for factors known or suspected to influence engraftment. The estimated odds ratio (OR) of graft failure for an HLA-C mismatch relative to match (univariable model) was 5.2 (95% CI, 1.4, 19; P = .01). Serologically undetectable HLA-A or HLA-B allele disparity was also associated with graft failure. The association between HLA-C disparity and graft failure remained significant even after accounting for the contribution of HLA-A and/or HLA-B allele disparity (OR 4.0; 95% CI, 1.1, 15; likelihood ratio test P = .03). These results show that HLA-C functions as a transplantation antigen and that HLA-A and HLA-B allele mismatches are biologically important. Molecular-based methods for pretransplant assessment of class I compatibility should be implemented for the selection of unrelated marrow donors.

Definition of HLA-DQ as a transplantation antigen.

Recent studies have demonstrated the importance of recipient HLA-DRB1 allele disparity in the development of acute graft-versus-host disease (GVHD) after unrelated donor marrow transplantation. The role of HLA-DQB1 allele disparity in this clinical setting is unknown. To elucidate the biological importance of HLA-DQB1, we conducted a retrospective analysis of 449 HLA-A, -B, and -DR serologically matched unrelated donor transplants. Molecular typing of HLA-DRB1 and HLA-DQB1 alleles revealed 335 DRB1 and DQB1 matched pairs; 41 DRB1 matched and DQB1 mismatched pairs; 48 DRB1 mismatched and DQB1 matched pairs; and 25 DRB1 and DQB1 mismatched pairs. The conditional probabilities of grades III-IV acute GVHD were 0.42, 0.61, 0.55, and 0.71, respectively. The relative risk of acute GVHD associated with a single locus HLA-DQB1 mismatch was 1.8 (1.1, 2.7; P = 0.01), and the risk associated with any HLA-DQB1 and/or HLA-DRB1 mismatch was 1.6 (1.2, 2.2; P = 0.003). These results provide evidence that HLA-DQ is a transplant antigen and suggest that evaluation of both HLA-DQB1 and HLA-DRB1 is necessary in selecting potential donors.

Six new DR52-associated DRB1 alleles, three of DR8, two of DR11, and one of DR6, reflect a variety of mechanisms which generate polymorphism in the MHC.

We have sequenced DNA from six new DR52-associated DRB1 alleles initially detected by PCR/SSOP analysis. Three DR8 associated alleles differed from previously known alleles by single nucleotide substitutions. DRB1*0807 and DRB1*0811 both vary from DRB1*08021 at codon 57 resulting in two different amino acids at this residue. DRB1*0807 was identified in samples of Brazilian origin while *0811 was identified among samples from the Tlingit Native American population of Southeast Alaska. DRB1*0814, identified in a family of Chinese origin, differed from DRB1*08032 at codon 12 at both the nucleotide and the amino acid level. In addition, two alleles of DR11, DRB1*1113 and *1119, were each detected in Caucasian individuals. DRB1*1113 differs from other DR11 alleles at codons 37, 67, 70 and 74, while DRB1*1119 differs from *1101 by a single nucleotide substitution at codon 67. Finally, DRB1*1418 was detected in a sample from an Asian or Pacific Islander and shares sequences with several other DR52-associated DRB1 alleles. These six DRB1 alleles appear to have been generated by either gene conversion events, DRB1*1113 and *1418, or by point mutations, DRB1*0814, *0807, *0811 and *1119, although the single nucleotide substitutions found in the latter three alleles are also present in at least one other DRB1 allele and, therefore, could have been the product of gene conversions.

Evaluation of the mixed lymphocyte culture (MLC) assay as a method for selecting unrelated donors for marrow transplantation.

The utility of the MLC assay as a test of HLA-D region matching and predictor of graft-versus-host disease (GvHD) was evaluated in 435 patients receiving marrow grafts from unrelated donors. Donors and recipients were phenotyped for HLA-A, B and DR antigens by serology, tested in MLC, and retrospectively genotyped for DRB1, B3, B4, B5, DQB1 and DPB1 alleles by PCR/SSOP. Of the 244 HLA-A, B, DR-identical donor-recipient pairs with valuable MLC and DRB1 typing results available, 208 were matched for HLA-A, B and DRB1, while 36 were matched for HLA-A and B and mismatched for a DRB1 allele. Donor anti-recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from 7.2 to 100%, with a median of 4.0%. A comparison of reactivity in MLC between pairs matched versus mismatched for DRB1 alleles showed a significant overlap in the distribution of RRs. Using optimally-defined RR cutoffs of 4 and 16%, no correlation between MLC results and risk of developing clinically significant grades III-IV GvHD (p=0.6 and 0.5, respectively) was found when the contribution of DRB1 mismatch was accounted for. Matching for DRB1 alleles, in contrast, was a better predictor of clinically significant GvHD, with DRB1-matched transplant recipients less likely to develop grades III-IV GvHD than DRB1-mismatched recipients (p=0.14). Among the 208 patients and donors matched for DRB1 alleles, the MLC, although reactive (RR > 4.0%) in 45% of cases, did not predict GvHD. Overall, these results underscore the limitations in using the MLC to predict DRB1 matching or risk of clinically significant GvHD among patients receiving unrelated marrow grafts. The availability of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing provides a method for donor matching that is rapid, precise and superior to the MLC for predicting clinically relevant outcome.

The significance of HLA-DRB1 matching on clinical outcome after HLA-A, B, DR identical unrelated donor marrow transplantation.

Despite matching for serologically defined HLA-A, B, DR antigens, acute graft-versus-host disease (GVHD) is a major complication contributing to increased morbidity and mortality in patients who undergo marrow transplantation from unrelated donors. The extent to which unrecognized mismatching for alleles that encode DR1-DR18 contribute to the increased risk of acute GVHD and overall survival is unknown. We analyzed 364 patients and their HLA-A, B, DR serologically matched donors to determine whether molecular typing of DRB1 alleles can allow more accurate donor/recipient matching and thereby improve clinical outcome after marrow transplantation. DRB1 alleles were typed by sequence-specific oligonucleotide probe hybridization methods. Selected alleles were confirmed by DNA sequencing. Of the 364 pairs, 305 were matched and 59 were mismatched for DRB1. The probability of moderate to severe acute GVHD was .48 for the matched and .70 for the mismatched patients. Compared with mismatched patients, the estimated relative risk (RR) of GVHD for matched patients was .58 (95% confidence interval [CI], .40 to .85). DRB1 matching decreased the risk of transplant-related mortality (RR, .66; 95% CI, .44 to .97) and was associated with decreased overall mortality (RR, .71; 95% CI, .51 to 1.0). Therefore, matching DRB1 alleles of the donor and recipient decreases the risk of acute GVHD and improves survival after unrelated marrow transplantation. These results indicate that prospective matching of patients and donors for DRB1 alleles is warranted.

Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: matching for transplants from related haploidentical donors.

The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-DR region by HTC typing. Donor antirecipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II-IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.

The MLC assay as a test for HLA-D region compatibility between patients and unrelated donors: results of a national marrow donor program involving multiple centers.

Mixed lymphocyte culture (MLC) assays from 763 patients and prospective unrelated marrow transplant donors identified through the National Marrow Donor Program (NMDP) were analyzed for their overall utility in measuring HLA-D region compatibility. The assays were performed at 31 different transplant centers (range, 1 to 197; median, 9 assays per center). A total of 325/763 (42.6%) of the tests were judged to be uninterpretable, either due to lack of sufficient control cells included in the assay (89 tests) or to insufficient reactivity by patient and/or donor cells (236 tests). Among the 438 tests that could be interpreted, HLA-Dw phenotyping with HLA-D homozygous cells was performed for a subset of 190. The relative response (RR) values from these 190 tests, however, were not clearly separable into distinct populations; i.e., RR values corresponding to Dw identity versus nonidentity between patient and donor could not be reliably discriminated. The predictive value of a nonreactive MLC for Dw identity was calculated to be 0.91 for RRs of < or = 20%, while the predictive value of a reactive MLC for Dw nonidentity was 0.35 for RRs of > 20%. These results, based on an analysis of data submitted from multiple transplant centers testing patients who had a variety of hematologic disorders, suggest that the MLC assay is a relatively imprecise method for determining HLA-D region compatibility between patient and prospective unrelated marrow donor.

Selective T-cell-receptor gene usage in allorecognition and graft-versus-host disease.

Immune recognition of foreign HLA molecules is initiated by T cell recognition mediated by alloreactive T cell receptor (TCR) molecules. We analyzed the diversity of TCR expression in the clinical setting of allorecognition in a patient with acute graft-versus-host disease following bone marrow transplantation. Nearly 200 TCR transcripts from peripheral blood lymphocytes were cloned and sequenced at two time points during GVHD. HLA genes in the transplant donor and the recipient were mismatched for a very specific HLA-DR subtype: HLA-DRB1 genes in the donor (DR4/Dw4) and the recipient (DR4/Dw14) encode HLA molecules that differ at only two amino acids, providing a very restricted target for allorecognition. We also studied TCR genes from five T cell clones derived in vitro from mixed lymphocyte cultures between Dw4-positive responder and Dw14-positive stimulator cells. Comparisons of the derived TCR sequences implicate nonrandom patterns of TCR selection both in vivo and in vitro.

The role of HLA-DPB1 disparity in the development of acute graft-versus-host disease following unrelated donor marrow transplantation.

The role of HLA-DPB1 disparity in the development of acute graft-versus-host disease (GVHD) following unrelated donor (URD) marrow transplantation is unknown. We studied 129 patients who underwent marrow transplantation from HLA-A, -B, -DRB, and -DQB matched URDs to determine whether matching for HLA-DPB1 alleles significantly decreased the risk of developing acute GVHD. HLA-DPB1 alleles were determined by sequence-specific oligonucleotide hybridization and by the number of patient DPB1 alleles not shared by the donor scored. The Kaplan-Meier probability of developing grades II to IV acute GVHD was determined for patients incompatible for zero (group A), one (group B), or two (group C) DPB1 alleles. Of the 129 pairs, there was no recipient DPB1 incompatibility in 28 (22%), one DPB1 mismatch in 72 (56%), and two DPB1 mismatches in 29 (22%). The probability of grades II to IV acute GVHD was 0.69 (0.50, 0.86) for group A, 0.83 (0.73, 0.91) for group B, and 0.72 (0.56, 0.87) for group C (P = .63). These results indicate that matching patients and unrelated donors for HLA-A, -B, -DRB, and -DQB does not predict for matching at DPB1. However, recipient incompatibility for DPB1 alleles does not detectably influence the risk of acute GVHD. Therefore, HLA-DP disparity should not be used as an exclusion criterion for donor selection in unrelated marrow transplantation.

Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus.

One hundred twelve patients less than 36 years old received marrow grafts from unrelated donors as treatment for hematologic malignancy. Seventy donor/recipient pairs were phenotypically identical for HLA-A, -B, and -D, while 42 had a "minor" disparity at one HLA locus. There was an increase in the risk of acute graft-versus-host disease (GVHD) in patients receiving HLA-partially matched grafts compared with those receiving HLA-matched grafts (51% v 36% probability of grades III-IV acute GVHD). However, in this cohort of patients, there was no significant difference in survival (at 1.5 years, 46% v 51% for good-risk patients, 44% v 30% for poor-risk patients). This finding suggests that some degree of HLA disparity can be tolerated in young patients transplanted from unrelated donors for malignant disease, thus making transplantation an option available to larger numbers of patients.

Allogeneic marrow transplantation: the Seattle experience.

Allogeneic marrow transplantation from a normal donor provides potential life-saving therapy for a variety of inherited and acquired diseases of the hematopoietic and immune systems including malignancies. However, even with an HLA-identical sibling donor the success of allografts can be compromised by GvHD and associated complications such as opportunistic infection. Non-HLA minor histocompatibility determinants still represent significant barriers to safe and successful transplants in some cases. In the absence of an HLA-identical sibling, alternate donors, including HLA-matched unrelated volunteers and HLA partially matched relatives can provide opportunity for effective therapy, though with increased risk of graft rejection and GvHD. HLA matching appears to be a critical factor, with the incidence and severity of GvHD exceeding acceptable limits as the number of incompatible HLA antigens increases. Although the United States NMDP and the marrow donor registries in other countries that currently have more than 1.2 million HLA-typed donors have profoundly improved the likelihood of finding an HLA-matched unrelated donor for the majority of White patients, many Whites and most patients of other racial origin cannot at this time find a match. Effective and safe treatment for these patients will require improved methods for managing HLA-mismatched transplants.

Mixed leukocyte culture reactivity and graft-versus-host disease in HLA-identical marrow transplantation for leukemia.

The results of pretransplant mixed leukocyte culture (MLC) assays were compared to subsequent risk of graft-versus-host disease (GVHD) in 783 patients receiving marrow transplants from HLA genotypically identical sibling donors. The mean MLC response observed between 1303 normal HLA identical sibling pairs was 0.0 +/- 4.2% RR. The donor anti-recipient MLC reaction, an in vitro response that presumably might be relevant to GVHD, was significantly increased (greater than mean + 2 sd) in 83 (10.6%) of the cases, most often in patients in relapse at the time of testing. No association was found, however, between this increased donor anti-recipient MLC reactivity pretransplant and the incidence or severity of subsequent acute or chronic GVHD. These data suggest that the increased MLC responses sometimes observed between leukemia patients and their HLA identical sibling donors prior to marrow transplantation do not represent genetic differences capable of causing GVHD.

T-cell receptor V beta selectivity in T-cell clones alloreactive to HLA-Dw14.

The HLA-DR4 subtypes Dw14 and Dw4 are T-cell-defined allospecificities encoded by the DRB1*0404 and DRB1*0401 genes, respectively. Although these allelic subtypes differ in only two amino acids, allorecognition between Dw14 and Dw4-positive individuals is brisk. This provides an opportunity to analyze T-cell receptor (TCR) usage in a very limited and specifically targeted case, namely the Dw4 anti-Dw14 allogeneic T-cell response. The variable (V), diversity (D), and joining (J) region sequences of the TCR beta chain from two different Dw14-specific alloreactive T-cell clones derived from a Dw4 donor were examined. Clone EMO25 recognized the Dw14.1, Dw14.2, and Dw15 subtypes, which share a DRB1 polymorphism at codon 71 on a DR4 background, while clone EMO36 reacted with only the Dw14.1 subtype associated with polymorphisms at codons 71 and 86. TCR beta cDNA from each clone was amplified using an anchored polymerase chain reaction (PCR) and subsequently expanded with V beta- and C beta-specific primers for asymmetric PCR and direct DNA sequencing. Both clones were found to express the same TCR V beta 8.2 gene segment; however, they have several different residues within the V beta-D beta-J beta junctional regions. V beta 8 usage was also enriched in polyclonal cells obtained from mixed lymphocyte cultures performed between the Dw4 and Dw14 responder-stimulator combination from which EMO25 and EMO36 were derived.

T-cell clones identify three distinct epitopes associated with HLA-Dw14.

Alloreactive T-cell clones were used to study allodeterminants associated with the HLA-DR1, -DR4, and -DRw14 allelic families. Three clones derived by priming against the DR4,Dw14 alloantigen were tested against a panel of HLA-D homozygous B-cell lines and homozygous and heterozygous peripheral blood lymphocytes. Each clone was blocked by monoclonal antibodies specific for HLA-DR, but not HLA-DQ or -DP, molecules, and each showed a unique pattern of allorecognition when tested against the cell panel. Clone 14B appeared to recognize a specific sequence, termed L67-A74, comprised of amino acids in the third hypervariable region of the alpha-helix of the DR beta 1 molecule, and expressed on certain DR1-, DR4-, and DRw14-positive cells. Clone EMO25 recognized the same L67-A74 sequence, but only when expressed on DR4-positive cells, suggesting a role for residues in the first and second hypervariable regions of DR4-positive DR beta 1 molecules in T-cell recognition. Clone EM036 also recognized the L67-A74 sequence, but only when expressed on DR4,Dw14.1-positive cells, implicating residues at positions 57 and 86 of the alpha-helix in T-cell recognition. These results demonstrate the range of specific T-cell responses that are possible against alloepitopes expressed by a single class II allele (Dw14), and are an indication of the diverse regions of the class II molecule that can contribute to allorecognition sites.