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Keywords:

  • minor histocompatibility antigen;
  • HA-8;
  • graft-versus-host disease

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Summary.  We recently identified a new minor histocompatibility antigen, termed HA-8, which is presented by human leucocyte antigen (HLA)-A*0201 or HLA-A*0202 and expressed ubiquitously among tissues. A retrospective analysis of 577 Caucasian patients with HLA-A*0201 or A*0202 who had received a haematopoietic stem cell transplant from a human leucocyte antigen (HLA)-identical sibling was conducted to determine whether HA-8 disparity correlated with clinical outcome. HA-8 disparity was detected in 72 recipients, and grades II–IV graft-versus-host disease (GVHD) occurred in 46 (64%), compared with 251 (50%) of the 503 patients without HA-8 disparity. After adjusting for known risk factors for acute GVHD, this difference was statistically significant (odds ratio, 1·8; 95% confidence interval, 1·0–3·1; P = 0·04). However, the hazards of clinical extensive chronic GVHD, overall mortality and recurrent malignancy were not statistically significantly different between the two groups. These data suggest that the increased risk of acute GVHD associated with recipient HA-8 disparity was not sufficient to change other clinical outcomes.

Minor histocompatibility antigens (mHAs) are major histocompatiblity complex (MHC)-associated peptides that originate from polymorphisms in the genome and trigger T-cell responses that mediate graft-versus-host disease (GVHD) and graft-versus-tumour effects (Goulmy, 1997; Warren et al, 1998a). Although GVHD is a major cause of mortality and morbidity after haematopoietic cell transplantation (HCT) (Ferrara & Deeg, 1991; Sullivan, 1994), the importance of individual mHAs in the occurrence of GVHD in humans has been controversial (Goulmy et al, 1996; Tseng et al, 1999; Lin et al, 2001). It has been suggested that disparity for a limited number of immunodominant mHAs may be sufficient to cause acute GVHD (Behar et al, 1996; Goulmy et al, 1996; Martin, 1997; Tseng et al, 1999; Gallardo et al, 2001; Lin et al, 2001; Socie et al, 2001), but the available data do not permit definitive conclusions in this regard. Donor/recipient disparity in HA-1, the most extensively studied mHA whose expression is limited to haematopoietic cells, was initially correlated with acute GVHD in studies of small numbers of patients (Goulmy et al, 1996; Tseng et al, 1999; Gallardo et al, 2001; Socie et al, 2001). However, in our subsequent analysis of a large patient population, recipient HA-1 disparity was not found to be significantly associated with the development of acute GVHD (Lin et al, 2001).

Using a biochemical approach, we recently identified a new HLA-A*0201-restricted mHA, termed HA-8, that is encoded by the KIAA0020 gene (Brickner et al, 2001). The HA-8 peptide that is presented by HLA-A*0201 was recognized by a CD8+ cytotoxic T lymphocyte (CTL) clone (designated SKH-13 CTL), which was derived from the peripheral blood lymphocytes of a male patient who received a bone marrow transplant from his HLA-identical sister for chronic myelogenous leukaemia (Warren et al, 1998b). The molecular basis of the HA-8 mHA was found to result from a polymorphism at nt 864 of the KIAA0020 gene, that encodes either arginine (HA-8R) or proline (HA-8P) at position 1 of a 9-mer antigenic peptide. Only the HA-8R peptide can be translocated into the endoplasmic reticulum and presented on the cell surface, leading to recognition by HA-8-specific SKH-13 CTL. A synthetic HLA-A2 tetramer incorporating the HA-8R peptide detected reactive T cells (0·27% of CD8+ cells) on day 35 post-transplant peripheral blood mononuclear cells from which the clone was isolated, demonstrating that CTL clones specific for HA-8R were expanded in vivo (unpublished observation).

The molecular characterization of novel mHAs has enabled studies of the role of individual determinants in GVHD. Recent studies in an in situ model of GVHD have suggested that CTLs specific for mHAs encoded by H-Y genes which are ubiquitously expressed, but not those specific for haematopoietic cell-specific genes such as HA-1, are targets of GVHD (Dickinson et al, 2002). The KIAA0020 gene encoding HA-8 is ubiquitously expressed on both haematopoietic and non-haematopoietic tissues (http://www.kazusa.or.jp/huge/gfpage/KIAA0020) and cells derived from these tissues are targets for HA-8 specific CTL in vitro. Thus, we investigated whether donor/recipient disparity of HA-8 is associated with an increased risk of GVHD in recipients of HLA-identical sibling HCT by genotyping the HA-8 locus in DNA samples from 577 HCT recipients.

Patient selection.  The study was approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center (FHCRC). The patient characteristics are summarized in Table I. DNA samples were extracted from HLA-A2-positive Caucasian patient and donor pairs (n = 584) who underwent a marrow or peripheral blood stem cell transplant from an HLA-identical sibling at the FHCRC (Seattle, WA, USA) between 1981 and 1998. This is almost the same patient population that was used in our prior analysis of HA-1 disparity and GVHD (Lin et al, 2001). All patients received methotrexate and cyclosporine for GVHD prophylaxis as previously described (Storb et al, 1986). Patients were assessed for acute and chronic GVHD according to previously published criteria (Glucksberg et al, 1974 and Loughran & Sullivan, 1990, respectively).

Table I.  Patient characteristics.
 HA-8 compatible (n = 505)HA-8 incompatible (n = 72)
  1. PBSC, peripheral blood stem cell.

  2. *Patients with haematological malignancy in relapse, accelerated phase or blast phase at the time of transplantation.

Median patient age [years (range)]35·6 (0·8–67·8)36·0 (0·8–61·9)
Disease status [n (%)]
 Advanced*158 (31)24 (33)
 Less advanced347 (69)48 (67)
Source of stem cells [n (%)]
 Bone marrow464 (92)63 (87·5)
 PBSC41 (8)9 (12·5)
Transplant year [n (%)]
 Before 1991271 (54)28 (39)
 1991 or later234 (46)44 (61)
Total body irradiation [n (%)]  
 None201 (40)30 (42)
 12 Gy160 (32)24 (33)
 >12 Gy144 (28)18 (25)
Patient/donor gender [n (%)]
 Female/female102 (20)13 (18)
 Female/male105 (21)18 (25)
 Male/female126 (25)17 (24)
 Male/male172 (34)24 (33)

Genotyping of HA-8 polymorphisms and HLA-A2.  Genotyping was performed blind to the recipient's GVHD status. Polymerase chain reaction (PCR) conditions and primers used for genotyping at the HA-8 locus have been described elsewhere (Brickner et al, 2001). Briefly, the antisense primer (5′-TCTAACACTTTGTCCCAGAATT-3′) was designed to produce an EcoR I site at its 3′ end when the primer anneals to the HA-8R allele. The PCR product for the HA-8P allele is resistant to EcoR I digestion. The sense primer used was 5′-GGATATACAGCAGAGCTTTC-3′. HA-8 was considered incompatible in the GVHD direction if the donor was HA-8P homozygous and the patient was either HA-8R homozygous or heterozygous (i.e. HA-8R/HA-8R or HA-8P/HA-8R).

The genotype of HLA-A2 in HA-8 incompatible pairs was confirmed as previously described (Tseng et al, 1999). Briefly, DNA samples were amplified by PCR using HLA-A2-specific 5′ primer A2F2M13R (5′-caggaaacagctatgaccTCTCAGCCACTCCTCGTCCCCAGGCTCT-3′) and HLA-A-specific 3′ primer AR1M13F (5′-tgtaaaacgacggccagtCGGGAGATCTACAGGCGATCAG-3′). The lower case letters indicate tagged M13 reverse and forward primer sequences, respectively. The amplified products were confirmed by size in agarose, purified (QIAquick PCR Purification kit; Qiagen, Valencia, CA, USA) and subjected to sequencing using M13 forward or reverse primer with the ABI Prism Big Dye Terminator cycle sequencing kit and an ABI 373A automated fluorescent sequencer system (Applied Biosystems, Foster City, CA, USA).

Statistical analysis.  The association of HA-8 disparity with the probability of acute GVHD was examined using univariate and multivariable logistic regression models. Cox regression was used to examine the association of HA-8 disparity with the hazards of clinical extensive chronic GVHD, relapse and overall mortality. Regression models for each of these endpoints were fit from among non-HA-8 variables, and HA-8 disparity was then added to each of these models. The models without HA-8 were then compared with the associated model containing HA-8 via the likelihood ratio test. Estimates of the probability of survival were calculated using the method of Kaplan and Meier (1958), and cumulative incidence estimates (Gooley et al, 1999) were used to summarize the probability of GVHD and relapse. Death without GVHD and death without relapse were regarded as a competing risk for GVHD and relapse, respectively. P < 0·05 were considered to be statistically significant. All reported P-values were two-sided, and those associated with regression models were derived from the Wald test. No adjustments were made for multiple comparisons.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Among the 584 donor/recipient pairs, genotyping detected 79 HA-8 incompatible cases (13·5%) in the GVHD direction (i.e. the immunogenic HA-8R allele was expressed in the recipient and the donor was homozygous for non-immunogenic HA-8P allele). HLA-A*0201 or A*0202 was present in 72 of the 79 donors. As the CTL specific for HA-8R recognized only target cells expressing either HLA-A*0201 or A*0202 (data not shown), patients with other HLA-A2 subtypes (n = 7) were excluded, and a total of 577 pairs were further analysed. The HA-8 incompatible and HA-8 compatible groups were similar in risk factors for GVHD identified previously (Weisdorf et al, 1991; Nash et al, 1992) although parity data for female donors were not completely available (data not shown).

A total of 575 recipients were evaluable for acute GVHD. Forty-six (64%) of the 72 patients with HA-8 disparity developed grades II–IV acute GVHD, compared with 251 (50%) of the 503 patients without HA-8 disparity. Recipient HA-8 disparity was associated with a statistically significantly increased probability of grades II–IV GVHD [Table II, odds ratio, 1·8; 95% confidence interval (CI), 1·0 to 3·1; P = 0·04] in a multivariate regression model that adjusted for stage of disease, age, source of stem cells, total body irradiation (TBI) dose, HA-1 disparity and transplantation before or after 1991, which corresponded to a change in the physicians responsible for assigning the grade of acute GVHD at the FHCRC. The distribution of organ stages for acute GVHD was similar among patients with and without HA-8 disparity (data not shown).

Table II.  Multivariable regression models testing the correlation between HA-8 disparity and outcomes following haematopoietic cell transplantation (HCT).
EndpointHA-8 disparityOdds/hazard ratio* (95% CI)P-value (LRT P-value)
Absent (%)Present (%)
  1. GVHD, graft-versus-host disease; LRT, likelihood ratio test.

  2. *Odds ratio for acute GVHD, hazard ratio for other endpoints.

  3. †Adjusted for HA-1 disparity, stage of disease, age, source of stem cells, GVHD grader, TBI dose.

  4. ‡Adjusted for stage of disease, age, source of stem cells.

  5. §Adjusted for stage of disease, age, source of stem cells, TBI dose.

Grades II-IV acute GVHD†251/505 (50)46/72 (64)1·8 (1·0–3·1)0·04 (0·04)
Clinical extensive chronic GVHD‡181/505 (36)31/72 (43)1·3 (0·9–1·9)0·17 (0·18)
Relapse§137/505 (27)19/72 (27)0·8 (0·5–1·3)0·33 (0·31)
Mortality§257/505 (51)31/72 (43)0·8 (0·5–1·1)0·19 (0·18)

Analysis of individual mHA loci may not be sufficient to determine the contribution to GVHD as the development of T-cell responses to a single mHA may be influenced by a variety of factors. We considered the possibility that a combination of disparities at both HA-1 and HA-8 loci might confer a higher risk of severe acute GVHD. However, we only identified five recipients that were incompatible at both loci with their donors (Table III). Two of the three patients who had sex-matched donors did not develop any acute GVHD. The two remaining patients had additional H-Y disparity and both developed grade II acute GVHD.

Table III.  Summary of five patients receiving HA-1 and HA-8 double disparate transplant.
UPNSex donor/patientDisease statusAcute GVHD gradeChronic GVHDSurvival (days)
  1. GVHD, graft-versus-host disease; UPN, unique patient number.

  2. *Not evaluable because of early death.

4278M/MAdvanced0NE*50
4591F/MAdvancedIIYes>4349
7572F/MLess advancedIINE*30
12106F/FLess advancedIIINE*41
13315F/FLess advanced0No>831

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Previous reports in small numbers of patients have suggested a correlation between recipient HA-1 disparity and acute GVHD (Goulmy et al, 1996; Gallardo et al, 2001; Socie et al, 2001). However, in a prior analysis of almost the same patient population used in this study, recipient HA-1 disparity was not found to be statistically significantly associated with the development of acute GVHD (Lin et al, 2001). Although our results suggest that HA-8 disparity is more likely to be associated with the occurrence of acute GVHD, both studies were limited by the relatively low frequency of patients with mHA disparity.

Multiple disparities in immunogenic mHAs have been suggested to augment the occurrence of GVHD (Martin, 1997). In our study, two patients receiving HA-1, HA-8 and H-Y disparate transplant developed grade II acute GVHD, but three patients receiving both HA-1 and HA-8 disparate transplant did not. As the number of cases that were disparate for multiple molecularly characterized mHAs was limited, it is too early to draw any conclusion. In addition, other factors such as polymorphisms in genes that encode cytokines or cytokine receptors (Cavet et al, 1999; Socie et al, 2001; Nordlander et al, 2002) may be critical in determining the outcome of mismatches at mHA loci. Recipient antigen-presenting cells play a key role in the induction of mHA-specific responses of donor T cells, and cytokine gene polymorphisms, bacterial or viral infections that may influence the maturation, function or activity of host antigen-presenting cells could also have an impact on the development of acute GVHD following allogeneic HCT (Holler, 2002). Further studies that consider both mHA disparity, cytokine gene polymorphisms, and other factors will need to be conducted to elucidate their relative role in the development of GVHD and HCT outcome.

Although HA-8 incompatibility showed a statistically significant correlation with acute GVHD, we observed no such association with chronic GVHD, leukaemia relapse or overall survival (Table II). As a result of the low frequency of HA-8 disparity, and the corresponding low number of events among HA-8 disparate patients, there was limited power to detect statistically significant associations. Thus, our results cannot exclude a possible association of HA-8 disparity with these outcomes. A very large multicentre study would be necessary to assess the impact of single or multiple mHAs disparity and other polymorphisms on important clinical endpoints.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Supported by grants from the National Institutes of Health CA18029 (S. R. R. M. -T. L., J. A. H.), AI44134 (V. H. E.), CA18221 (P. J. M.), HL36444 (P. J. M.), AI33484 (J. A. H.), AI49213 (P. J. M., J. A. H., T. G.); a Lilly Clinical Investigator Award from the Damon Runyon Cancer Research Foundation (E. H. W.); Kirby Foundation Postdoctoral Fellow of the American Cancer Society (A. G. B.); Research on Human Genome, Tissue Engineering Food Biotechnology (Y. A., T. T.); Second Term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health, Labour, and Welfare, Japan (T. T.); and Grant-in-Aid for Scientific Research (Y. A.).

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  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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