Does minor histocompatibility antigen HA-1 disparity affect the occurrence of graft-versus-host disease in tunisian recipients of hematopoietic stem cells?

CLINICAL SCIENCE

Does minor histocompatibility antigen HA-1 disparity affect the occurrence of graft-versus-host disease in tunisian recipients of hematopoietic stem cells?

Mohamed Hichem Sellami^I; Lamia Torjemane^II; Alejandro Espadas de Arias^III; Houda Kaabi^I; Saloua Ladeb^II; Francesca Poli^III; Tarek Ben Othmane^II; Slama Hmida^I

^IImmunogenetic Applied to Cell Therapy research unit, Immunohematology and HLA-Typing Department, National Blood Transfusion Centre of Tunis, Tunisia

^IIDepartment of Hematology, National Bone Marrow Transplantation Centre of Tunis, Tunisia

^IIIDepartment of Regenerative Medicine, Ospedale Maggiore Policlinico di Milano, Italy. E-mail to: [e-mail]. Tel.: [phone]

ABSTRACT

INTRODUCTION: Minor histocompatibility antigen HA-1 (MiHAg-HA-1) disparity between a patient and his or her human leukocyte antigen (HLA) genoidentical donor has been widely associated with an increased risk of graft-versus-host disease following allogeneic hematopoietic stem cell transplantation.

OBJECTIVE: To examine the effect of HA-1 disparity on the incidence of both acute and chronic graft-versus-host disease in Tunisian recipients of hematopoietic stem cells.

METHODS: A total of 60 patients and their 60 respective sibling hematopoietic stem cell donors were enrolled in this study. All patients prophylactically received cyclosporine A and/or methotrexate for graft-versus-host disease. An HA-1 genotyping assay was performed with the SSP-PCR method, and HLA-A*0201- and/or HLA-A*0206-positive samples were identified using the Luminex HLA typing method.

RESULTS: The Luminex HLA typing assay showed that 54 patients were positive for either the HLA-A*0201 or HLA-A*0206 alleles. Among these cases, six pairs were mismatched for MiHAg-HA-1. Both acute and chronic graft-versus-host disease occurred in four mismatched patients (Fisher's p-values were 0.044 and 0.170, respectively). A univariate logistic regression model analysis showed that only acute graft-versus-host disease may be affected by recipient MiHAg-HA-1 disparity (p: 0.041, OR: 6.727), while chronic graft-versus-host disease correlates with both age and recipient/donor sex mismatch (p: 0.014, OR: 8.556 and p: 0.033, OR: 8.664, respectively).

CONCLUSION: Our findings support previously reported data suggesting a significant association between HA-1 disparity and the risk of acute graft-versus-host disease following hematopoietic stem cell transplantation.

Keywords: Hematopoietic stem cell transplantation; Graft-versus-host disease; Minor histocompatibility antigens; HA-1; HLA-A; Tunisian population.

INTRODUCTION

Graft-versus-host disease (GVHD) is the major cause of mortality and morbidity following an allogeneic hematopoietic stem cell transplantation (HSCT).^1-4

This complication has been widely linked to immunogenetic causes, such as a disparity between the recipient and his or her human leukocyte antigen (HLA) genoidentical donor for certain non-HLA antigens, called minor histocompatibility antigens (MiHAgs).^2,4-6 These antigens are endogenously synthesized polymorphic products that can be recognized by alloreactive T cells only in the context of major histocompatibility complex molecules (HLA).^6-9 The importance of MiHAgs, encoded by sex-linked or autosomal loci, in the occurrence of GVHD has been demonstrated previously.^2-4,6

Disparity in a limited number of immunodominant MiHAgs may be sufficient to cause acute GVHD, but the available data do not permit definitive conclusions.^5,10-16 Donor/recipient disparity for HA-1, the most extensively studied MiHAg and whose expression is limited to hematopoietic cells, was initially correlated with acute GVHD in studies involving small numbers of patients.^5,12-14 In analyses of larger cohorts, however, this disparity was not significantly associated with the development of acute GVHD.^17-20 Thus, additional studies in other populations of the same area of research are needed to confirm the previous result and define the effect of HA-1 disparity on GVHD occurrence following HSCT.

The minor histocompatibility antigen HA-1 was initially identified by a cytotoxic T cells clone that was recovered from a marrow transplant recipient with GVHD.²¹ This antigen is a nonapeptide that is part of a protein encoded by the HMHA1 gene that maps to chromosome 19p13.3.²² This gene has two allelic versions differing at positions 500 (rs3764653) and 504 (rs1801284) of the cDNA sequence, resulting in a single amino acid polymorphism.²³ The HMHA1^H allele encodes histidine (HA-1^H) at position 3 of the peptide (VLHDDLLEA), whereas the HMHA1^R allele encodes arginine (HA-1^R) at the same position. The HLA-A*0201 and HLA-A*0206 molecules have high affinities for the HA-1^H peptide, and the generated antigen/MHC complex is recognized by HA-1-specific cytotoxic T cells.^23,24 Unlike this peptide, the HA-1^R peptide does not generate a detectable immune response.²³ HA-1-positive individuals can be either homozygous or heterozygous carriers of the HA-1^H peptide. Thus, an HA-1 mismatch is defined by the presence of the HA-1^H allele in the host and not the donor.

Tunisia is a small North African country characterized by a mixed origin and a high rate of consanguinity.^25,26 Recently, we performed a population genetic study, focusing on HMHA-1 gene polymorphisms in Tunisians.²⁷ In this study, we found a noticeable probability that a Tunisian recipient of HSCs may be positive for HA-1/HLA-A*0201. Consequently, we examined the effect of HA-1 mismatch on the occurrence of GVHD. To achieve this purpose, a cohort of HSC recipients and their HLA-identical siblings was selected to conduct this retrospective study.

MATERIALS AND METHODS

To participate in this study, informed consent was obtained from all participants and/or their families according to a protocol approved by the Ethical Committee for Scientific and Medical Research of the National Blood Transfusion Center of Tunis (Tunisia).

Patient selection

Because HA-1 antigen presentation is restricted to the HLA-A*0201 and HLA-A*0206 molecules, we selected only HLA-A2-positive samples to perform this study. The cohort comprised 120 individuals: 60 patients and their 60 respective sibling hematopoietic stem cell donors. All subjects were Tunisian. The patients underwent HLA-matched HSCT in the National Bone Marrow Transplantation Centre of Tunis between 2000 and 2009. All patients prophylactically received cyclosporine A and/or methotrexate for GVHD and had grades 0-I or II-IV acute GVHD. See Table 1 for other clinical characteristics.

DNA samples

EDTA-treated blood samples were collected from all individuals prior to HSCT. DNA was purified from peripheral blood leukocytes using a DNA Blood Mini Kit (Qiagen, Leiden, the Netherlands) according to the manufacturer's instructions.

GVHD grading

The diagnosis and grading of acute and chronic GVHD were defined according to Seattle Transplant Team criteria.²⁸

HA-1 genotyping

HA-1 genotyping was performed using a previously described allele-specific PCR method.²² This SSP-PCR method uses two sets of primers: set 1 has a forward common primer and two allele-specific reverse primers, and set 2 has a reverse common primer and two allele-specific forward primers. The allele-specific primers target the polymorphic region that encodes the HA-1^H or HA-1^R allele. All primers were synthesized as oligonucleotides (Invitrogen Corporation, CA, USA).

PCR amplification was carried out in a final volume of 10 μL containing 200 μM of each dNTP, 0.2 μM of each primer (which detects a specific allele), 0.2 μM of control primers (which detect a sequence from the HGH allele), and 1.5 mM MgCl₂ in 2 μL of 5X Green Buffer (Promega Corporation, USA). Each PCR was performed using 150 ng of genomic DNA and 0.4 units of Taq polymerase (GoTaq^TM DNA Polymerase, Promega Corporation, USA).

The amplification protocol was 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 61°C for 40 s, and elongation at 72°C for 30 s; the final elongation step was 72°C for 5 min (Gene Amp PCR System 9600, Perkin Elmer, CT, USA). The PCR products were visualized on agarose gels (1.5%) with UV illumination and photodocumented (UViTech^TM, Germany).

HLA-A*0201/HLA-A*0206 allele typing

Because HA-1 antigen presentation is restricted to HLA-A*0201 and HLA-A*0206 molecules, only carriers of these alleles were selected for the statistical analysis. All patients and donors were previously typed as HLA-A2-positive using standard clinical laboratory techniques with serological or molecular testing methods (One Lambda, USA and Innogenetics, Belgium). HLA-A*0201 and HLA-A*0206 allele typing was initially performed with Luminex^TM technology (One Lambda Corporation, USA) and was confirmed in some cases with the SSP-PCR method (One Lambda Corporation, USA).

Statistical analysis

The association between HA-1 mismatch and the incidence of either acute or chronic GVHD was evaluated using Fisher's exact test. The odds ratios for univariate and multivariate logistic regression models were used to analyze the correlations between risk factors and the probabilities of acute and chronic GVHD. All statistical analyses were performed with SPSS software (v. 17.0).

RESULTS

Among 60 HLA-A2-positive patient/donor pairs, the Luminex^TM molecular genotyping assay showed that 53 cases were positive for the HLA-A*0201 allele, and 1 pair carried the HLA-A*0206 variant. The remaining six cases expressed the HLA-A*0205 allele and were excluded from the statistical analysis. Thus, 54 pairs were carriers of the specific HLA molecules needed for HA-1 peptide presentation. When typed for this MiHAg, only six pairs were mismatched (11.11%) vs. forty-eight identical pairs (88.89%). Acute GVHD of grades II-IV occurred in only four HA-1 mismatched recipients vs. eleven matched pairs (Table 2). Like the majority of cases, these four patients had received CSA and MTX as prophylaxis for GVHD. When treated in a univariate logistic regression model, HA-1 disparity appeared to be correlated with an increased risk of acute GVHD (p: 0.041, OR: 6.727) but showed only a trend toward significance in the multivariate logistic regression analysis. Furthermore, acute GVHD was not influenced by factors such as sex mismatch, conditioning and patient age. All results are summarized in Tables 2 and 3.

vs. only 4 mismatched pairs. When we compared its incidence between HA-1-matched and mismatched recipients, we found no significant difference. This finding was determined from a statistical analysis using Fisher's exact test and verified with univariate and multivariate logistic regression models (

Tables 2 and

3). Interestingly, chronic GVHD may be significantly promoted by both donor/recipient sex mismatch and patient age (adulthood). A total of 82.4% of patients who developed chronic GVHD were sex mismatched with their siblings, and 94.11% of patients who developed this outcome were adults. None of the other risk factors, including conditioning and malignancy, were significant in the paradigm of chronic GVHD incidence.

DISCUSSION

GVHD is one of the major complications of allogeneic HSCT, even in an HLA genoidentity context.^1-4 Disparity between a recipient and his or her HLA-identical sibling for certain minor histocompatibility antigens may be sufficient to cause GVHD following HSCT.^2,4-6 Based on these data, we investigated the effect of recipient MiHAg-HA-1 disparity on GVHD occurrence in a Tunisian series of HSC allografts. After screening an HLA-A*0201/HLA-A*0206-positive cohort, we performed HA-1 typing for each recipient/donor pair. The correlation analysis showed that acute GVHD may be affected by HA-1 mismatch because Fisher's p-value for this relationship was 0.044. This result was confirmed by the univariate logistic regression analysis, but in the multivariate regression model, we noticed only a trend toward an increased probability of Acute GVHD of grades II-IV. The same multivariate analysis showed no significant association between risk factors such as patient age, conditioning or sex mismatch and the incidence of acute GVHD. As expected, our data support previously reported findings correlating acute GVHD occurrence with recipient HA-1 disparity,^5,12-14 but a definitive conclusion cannot be deduced. This fact may be explained by the prophylactic protocol received by patients enrolled in each study. Thus, we believe that the actual effect of HA-1 disparity cannot be evaluated in subjects who receive CSA and MTX to prevent GVHD occurrence. Likewise, differences in the methods used for sample selection or GVHD grading could affect the level of significance. In our opinion, to assess this relationship more accurately, we need to investigate the effect of HA-1 disparity in a universal context, particularly in patients who have not received GVHD prophylaxis. We believe that this procedure may be feasible if it is performed first in murine models and then in human patients.

Our study showed that chronic GVHD may be affected by both donor/recipient sex mismatch and adulthood but not by HA-1 disparity. Statistically, 82.4% of patients who developed chronic GVHD were sex mismatched with their HLA-identical siblings (recipient sex-ratio: 1.33). Furthermore, patient age appeared to be a critical player in chronic GVHD onset because 94.11% of those patients who developed chronic GVHD were adults. Both sex mismatch and adulthood have been described as favorable conditions for the occurrence of these effects, especially through T cells alloreactivity against Y-encoded antigens when the recipient/donor pairs have different genders.^5,29-31 Thus, our findings are in agreement with those findings reported by Gallardo et al.; in their large pool of patients, they found no association between chronic GVHD and HA-1 disparity.¹³

On the other hand, chronic GVHD may be affected by acute GVHD status. This outcome occurred in 53% of patients who developed acute GVHD (data not shown). This fact may be explained by the presence of T cells that are primed by chronic antigen stimulation due to the presence of ubiquitous MiHAgs.³ Our findings support previous data qualifying acute GVHD as the main risk factor for the development of chronic GVHD,³² but future studies are needed to investigate the role of regulatory T cells, B cells and other immune cells. In this regard, recent data have suggested a critical role for humoral immunity in the chronic GVHD paradigm because there is a strong correlation between this outcome and both increased numbers of B cells with altered Toll-like Receptor 9 (TLR9) responses and the presence of antibodies against some minor histocompatibility antigens.^30,32-33

In conclusion, this study showed that acute GVHD but not chronic GVHD may be affected by HA-1 disparity in Tunisian HSC recipients. Consequently, we believe that HA-1 typing may be useful in cases where there is more than one HSC donor, which occurs frequently in Tunisian families (data not shown). Overall, we believe that our results are significant and can encourage further study in this area of cell therapy in Tunisia because this study is the first to report the role of immunogenetic markers in the occurrence of GVHD. It will be important, however, to expand the patient cohort and investigate the effects of HA-1 disparity on other endpoints, including leukemia relapse, disease-free survival and overall survival.

ACKNOWLEDGMENTS

We would like to thank the technicians from the National Blood Transfusion Center and the National Bone Marrow Transplantation Centre of Tunis for their valuable technical assistance. We are also grateful to our Italian colleagues from the Department of Regenerative Medicine, Organ and Tissue Transplantation Immunology, Ospedale Maggiore Policlinico di Milano and from Lagitre s.r.l, Italy. This work was supported by the Tunisian Ministry for Higher Education, Scientific Research and Technology and the Tunisian Ministry of Public Health.

ELECTRONIC DATABASE INFORMATION

Accession numbers and URLs for data in this article are as follows:

> GenBank: http://www.ncbi.nlm.nih.gov/GenBank/ (HA-1 Gene ID: 23526)

> EnsemblGene: http://www.ensembl.org/ (HA-1 Ensembl Gene ID: ENSG00000180448).

Received for publication on July 02, 2010

First review completed on July 26, 2010

Accepted for publication on August 09, 2010

• 1. Bay JO, Cabrespine A, Peffault de Latour R. Role of monoclonal antibodies in the treatment of acute graft versus host disease. Bulletin du Cancer. 2007;94:33-41.
• 2. Sun Y, Tawara I, Toubai T, Reddy P. Pathophysiology of Acute Graft-vs-Host Disease: Recent Advances. Translational research. 2007;150: 197-214.
• 3. Toubai T, Sun Y, Reddy P. GVHD pathophysiology: is acute different from chronic? Best Practice & Research Clinical Haematology. 2008; 21:101-17.
• 4. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet. 2009;373:1550-61.
• 5. Goulmy E, Schipper R, Pool J, Blokland E, Falkenburg JHF, Vossen J, et al. Mismatch of minor histocompatibility antigen between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation. New England Journal of Medicine. 1996;334:281-5.
• 6. Hambach L, Spierings E, Goulmy E. Risk assessment in hematopoietic stem cell transplantation: Minor histocompatibility antigens. Best Practice & Research Clinical Haematology. 2007;20:171-87.
• 7. Simpson E, Scott D, James E, Lombardi G, Cwynarski K, Dazzin F, et al. Minor H antigens: genes and peptides. Transplant Immunology. 2002;10:115-23.
• 8. Falkenburg F, Van de Corput F, Marijt EWA, Willemze R. Minor histocompatibility antigens in human stem cell transplantation. Experimental Haematology. 2003;31:743-51.
• 9. Malarkannan S, Regunathan J, Timler AM. Minor Histocompatibility Antigens: Molecular targets for immunomodulation in tissue transplantation and tumor therapy. Clinical and Applied Immunology Reviews. 2005:5:95-109.
• 10. Behar E, Chao NJ, Hiraki DD, Krishnaswamy S, Brown BW, Zehnder JL, et al. Polymorphisms of adhesion molecule CD31 and its role in acute graft-versus-host disease. New England Journal of Medicine. 1996;334:286-91.
• 11. Martin PJ. How much benefit can be expected from matching for minor antigens in allogeneic marrow transplantation? Bone Marrow Transplantation. 1997;20:97-100.
• 12. Tseng LH, Lin MT, Hansen JA, Gooley T, Pei J, Smith AG, et al. Correlation between Disparity for the Minor Histocompatibility Antigen HA-1 and the Development of Acute Graft-Versus-Host Disease after Allogeneic Marrow Transplantation. Blood. 1999;94:2911-14.
• 13. Gallardo D, Aróstegui JI, Balas A, Torres A, Caballero D, Carreras E, et al. Disparity for the minor histocompatibility antigen HA-1 is associated with an increased risk of acute graft-versus-host disease (GvHD) but it does not affect chronic GvHD incidence, disease-free survival or overall survival after allogeneic human leucocyte antigen-identical sibling donor transplantation. British Journal of Haematology. 2001;114:931-6.
• 14. Socié G, Loiseau P, Tamouza R, Janin A, Busson M, Gluckman E. et al. Both genetic and clinical factors predict the development of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Transplantation. 2001;72:699-706.
• 15. Cavanagh G, Chapman CE, Carter V, Dickinson AM, Middleton PG. Donor CD31 genotype impacts on transplant complications after human leukocyte antigen-matched sibling allogeneic bone marrow transplantation. Transplantation. 2005;79:602-5.
• 16. El-Chennawi FA, Kamel HA, Mosaad YM, El-Sherbini SM, El-Billey NA. Impact of CD31 mismatches on the outcome of hematopoeitic stem cell transplant of HLA-identical sibling. Hematology. 2006;11: 227-34.
• 17. Lin MT, Gooley T, Hansen JA, Tseng LH, Martin EG, Singleton K, et al. Absence of statistically significant correlation between disparity for the minor histocompatibility antigen HA-1 and outcome after allogenic hematopoietic cell transplantation [Letter]. Blood. 2001;98: 3172-3.
• 18. Tait BD, Maddison R, McCluskey J, Deayton S, Heatley S, Lester S, et al. Clinical relevance of the minor histocompatibility antigen HA-1 in allogeneic bone marrow transplantation between HLA identical siblings. Transplantation Proceedings. 2001;33:1760-1.
• 19. Nesci S, Bufû O, Iliescu A, Andreani M, Lucarelli G. Recipient mHag-HA1 disparity and aGVHD in thalassemic-transplanted patients. Bone Marrow Transplantation. 2003;31:575-8.
• 20. Spellman S, Warden MB, Haagenson M, Pietz BC, Goulmy E, Warren EH. Effects of Mismatching for Minor Histocompatibility Antigens on Clinical Outcomes in HLA-Matched, Unrelated Hematopoietic Stem Cell Transplants. Biology of Blood and Marrow Transplantation. 2009;15:856-63.
• 21. Goulmy E. Minor histocompatibility antigens in man and their role in transplantation, in Morris PJ, Tilney NL (eds): Transplantation Reviews 2. Philadelphia, PA, Saunders. 1988;p 29.
• 22. Wilke M, Pool J, Den Haan JMM, Goulmy E. Genomic identification of the minor histocompatibility antigen HA-1 locus by allele specific PCR. Tissue Antigens. 1998;52:312-7.
• 23. Tseng LH, Lin MT, Martin PJ, Pei J, Smith AG, Hansen JA. Definition of the gene encoding the minor histocompatibility antigen HA-1 and typing for HA-1 from genomic DNA. Tissue Antigens. 1998; 52:305.
• 24. Torikai H, Akatsuka Y, Miyauchi H, Terakura S, Onizuka M, Tsujimura K, et al. The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206. Bone Marrow Transplantation. 2007;40:165-74.
• 25. Hajjej A, Kâabi H, Sellami MH, Dridi A, Jeridi A, Hmida S. The contribution of HLA class I and II alleles and haplotypes to the investigation of the evolutionary history of Tunisians. Tissue Antigens. 2006;68:153-62.
• 26. Kerkeni E , Monastiri K, Seket B, Guediche MN, Ben Cheikh H. Interplay of Socio-economic Factors, Consanguinity, Fertility, and Offspring Mortality in Monastir, Tunisia. Croat Med J. 2007;48:701-7.
• 27. Sellami MH, Ben Ahmed A, Kaabi H, Jridi A, Dridi A, Hmida S. HA-1 and HA-2 minor histocompatibility antigens in Tunisians. Tissue Antigens. 2010;75:720-3.
• 28. Przepiorka D, Weisdorfm D, Martinm P, Klingemannm HG, Beattym P, Hows J, et al. Meeting report: consensus conference on acute GVHD grading. Bone Marrow Transplantation. 1995;15:825-8.
• 29. Eisner MD, August CS. Impact of donor and recipient characteristics on the development of acute and chronic graft-versus-host disease following pediatric bone marrow transplantation. Bone Marrow Transplantation. 1995;15:663-8.
• 30. Miklos DB, Kim HT, Miller KH, Guo L, Zorn E, Lee SJ, et al. Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission. Blood. 2005;105:2973-8.
• 31. Grathwohl A. Prognostic factors in chronic myeloid leukemia allografting. Seminar Hematol. 2003;40:13-21.
• 32. Lee SJ. New approaches for preventing and treating chronic graft-versus-host disease. Blood. 2005;105:4200-06.
• 33. She H, Gilman AL, Aslanian S, Shimizu H, Krailo, M, Chen Z, et al. Altered toll-like receptor 9 responses in circulating B cells at the onset of extensive chronic graft-versus-host disease. Biology of Blood and Marrow Transplantation. 2007;13:386-97.

Publication Dates

• Publication in this collection
  05 Jan 2011
• Date of issue
  2010

History

• Received
  02 July 2010
• Accepted
  26 July 2010