Molecular Typing Methods for Minor Histocompatibility Antigens

  • Eric Spierings
Part of the Methods in Molecular Biology book series (MIMB, volume 1109)


Minor histocompatibility (H) antigen mismatching leads to clinically relevant alloimmune reactivity. Depending on the tissue expression pattern of the involved minor H antigens, the immune response may either cause graft-versus-host disease and a graft-versus-tumor effect or lead to only a graft-versus-leukemia effect. Thus, identification of recipient–donor pairs with minor H antigen mismatches has clinical importance. This chapter describes molecular typing methods for molecular typing of minor H antigens.

Key words

Minor histocompatibility antigens Transplantation Stem cells Solid organ Graft-versus-host Graft-versus-tumor Graft rejection Genotyping 



Talitha de Hoop and Walter van Ginkel, University Medical Center Utrecht, are acknowledged for developing and validating the SBT protocols; Jos Pool and Jos Drabbels, Leiden University Medical Center, for developing and validating the uniform PCR-SSP protocols; and Erik Rozemuller, GenDx BV, Utrecht, for the implementation of automated allele assignment for minor H antigens after SBT in SBTEngine©.


  1. 1.
    Goulmy E (1996) Human minor histocompatibility antigens. Curr Opin Immunol 8:75–81PubMedCrossRefGoogle Scholar
  2. 2.
    Hambach L, Goulmy E (2005) Immunotherapy of cancer through targeting of minor histocompatibility antigens. Curr Opin Immunol 17:202–210PubMedCrossRefGoogle Scholar
  3. 3.
    Cai J, Lee J, Jankowska-Gan E, Derks R et al (2004) Minor H antigen HA-1-specific regulator and effector CD8+ T cells, and HA-1 microchimerism, in allograft tolerance. J Exp Med 199:1017–1023PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Verdijk RM, Kloosterman A, Pool J et al (2004) Pregnancy induces minor histocompatibility antigen-specific cytotoxic T cells: implications for stem cell transplantation and immunotherapy. Blood 103:1961–1964PubMedCrossRefGoogle Scholar
  5. 5.
    Mommaas B, Stegehuis-Kamp JA, Van Halteren AG et al (2004) Cord blood comprises antigen-experienced T cells specific for maternal minor histocompatibility antigen HA-1. Blood 105:1823–1827PubMedCrossRefGoogle Scholar
  6. 6.
    Van Halteren AG, Jankowska-Gan E, Joosten A et al (2009) Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members. Blood 114: 2263–2272PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Nielsen HS, Wu F, Aghai Z et al (2010) H-Y antibody titers are increased in unexplained secondary recurrent miscarriage patients and associated with low male: female ratio in subsequent live births. Hum Reprod 25: 2745–2752PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Mutis T, Goulmy E (2002) Hematopoietic system-specific antigens as targets for cellular immunotherapy of hematological malignancies. Semin Hematol 39:23–31PubMedCrossRefGoogle Scholar
  9. 9.
    Klein CA, Wilke M, Pool J et al (2002) The hematopoietic system-specific minor histocompatibility antigen HA-1 shows aberrant expression in epithelial cancer cells. J Exp Med 196: 359–368PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Fujii N, Hiraki A, Ikeda K et al (2002) Expression of minor histocompatibility antigen, HA-1, in solid tumor cells. Transplantation 73:1137–1141PubMedCrossRefGoogle Scholar
  11. 11.
    Mutis T, Brand R, Gallardo D, van Biezen A, Niederwieser D, Goulmy E (2010) Graft-versus-host driven graft-versus-leukemia effect of minor histocompatibility antigen HA-1 in chronic myeloid leukemia patients. Leukemia 24:1388–1392PubMedCrossRefGoogle Scholar
  12. 12.
    Goulmy E, Termijtelen A, Bradley BA, van Rood JJ (1976) Alloimmunity to human H-Y. Lancet 2:1206PubMedCrossRefGoogle Scholar
  13. 13.
    Voogt PJ, Fibbe WE, Marijt WA et al (1990) Rejection of bone-marrow graft by recipient-derived cytotoxic T lymphocytes against minor histocompatibility antigens. Lancet 335: 131–134PubMedCrossRefGoogle Scholar
  14. 14.
    Wilke M, Dolstra H, Maas F et al (2003) Quantification of the HA-1 gene product at the RNA level; relevance for immunotherapy of hematological malignancies. Hematol J 4: 315–320PubMedCrossRefGoogle Scholar
  15. 15.
    Dolstra H, Fredrix H, Maas F et al (1999) A human minor histocompatibility antigen specific for B cell acute lymphoblastic leukemia. J Exp Med 189:301–308PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Goulmy E (1997) Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy. Immunol Rev 157:125–140PubMedCrossRefGoogle Scholar
  17. 17.
    Dickinson AM, Wang XN, Sviland L et al (2002) In situ dissection of the graft-versus-host activities of cytotoxic T cells specific for minor histocompatibility antigens. Nat Med 8: 410–414PubMedCrossRefGoogle Scholar
  18. 18.
    de Bueger MM, Bakker A, van Rood JJ, Van der Woude F, Goulmy E (1992) Tissue distribution of human minor histocompatibility antigens. Ubiquitous versus restricted tissue distribution indicates heterogeneity among human cytotoxic T lymphocyte-defined non-MHC antigens. J Immunol 149:1788–1794PubMedGoogle Scholar
  19. 19.
    Dolstra H, Fredrix H, Preijers F et al (1997) Recognition of a B cell leukemia-associated minor histocompatibility antigen by CTL. J Immunol 158:560–565PubMedGoogle Scholar
  20. 20.
    Wilke M, Pool J, den Haan JM, Goulmy E (1998) Genomic identification of the minor histocompatibility antigen HA-1 locus by allele-specific PCR. Tissue Antigens 52: 312–317PubMedCrossRefGoogle Scholar
  21. 21.
    Wilke M, Pool J, Goulmy E (2002) Allele specific PCR for the minor histocompatibility antigen HA-2. Tissue Antigens 59:304–307PubMedCrossRefGoogle Scholar
  22. 22.
    Spierings E, Brickner AG, Caldwell JA et al (2003) The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein. Blood 102:621–629PubMedCrossRefGoogle Scholar
  23. 23.
    den Haan JM, Sherman NE, Blokland E et al (1995) Identification of a graft versus host disease-associated human minor histocompatibility antigen. Science 268:1476–1480CrossRefGoogle Scholar
  24. 24.
    Spierings E, Drabbels J, Hendriks M et al (2006) A uniform genomic minor histocompatibility antigen typing methodology and database designed to facilitate clinical applications. PLoS ONE 1:e42PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Deng G (1988) A sensitive non-radioactive PCR-RFLP analysis for detecting point mutations at 12th codon of oncogene c-Ha-ras in DNAs of gastric-cancer. Nucleic Acids Res 16:6231PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Mercier B, Ferec C, Dufosse F, Huart JJ (1992) Improvement in HLA-DQB typing by PCR-RFLP—introduction of a constant restriction site in one of the primers for digestion control. Tissue Antigens 40:86–89PubMedCrossRefGoogle Scholar
  27. 27.
    Tseng LH, Lin MT, Martin PJ, Pei J, Smith AG, Hansen JA (1998) Definition of the gene encoding the minor histocompatibility antigen HA-1 and typing for HA-1 from genomic DNA. Tissue Antigens 52:305–311PubMedCrossRefGoogle Scholar
  28. 28.
    Pierce RA, Field ED, Mutis T et al (2001) The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein. J Immunol 167: 3223–3230PubMedCrossRefGoogle Scholar
  29. 29.
    Brickner AG, Warren EH, Caldwell JA et al (2001) The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing. J Exp Med 193: 195–206PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Lo YMD, Tein MSC, Lau TK et al (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 62: 768–775PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Murata M, Warren EH, Riddell SR (2003) A human minor histocompatibility antigen resulting from differential expression due to a gene deletion. J Exp Med 197:1279–1289PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994PubMedCrossRefGoogle Scholar
  33. 33.
    Akatsuka Y, Warren EH, Gooley TA et al (2003) Disparity for a newly identified minor histocompatibility antigen, HA-8, correlates with acute graft-versus-host disease after haematopoietic stem cell transplantation from an HLA-identical sibling. Br J Haematol 123: 671–675PubMedCrossRefGoogle Scholar
  34. 34.
    Fehse B, Chukhlovin A, Kuhlcke K et al (2001) Real-time quantitative Y chromosome-specific PCR (QYCS-PCR) for monitoring hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation. J Hematother Stem Cell Res 10:419–425PubMedCrossRefGoogle Scholar
  35. 35.
    Longo MC, Berninger MS, Hartley JL (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93:125–128PubMedCrossRefGoogle Scholar
  36. 36.
    Kreiter S, Wehler T, Landt O, Huber C, Derigs HG, Hess G (2000) Rapid identification of minor histocompatibility antigen HA-1 subtypes H and R using fluorescence-labeled oligonucleotides. Tissue Antigens 56:449–452PubMedCrossRefGoogle Scholar
  37. 37.
    Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–131, and 134–138PubMedGoogle Scholar
  38. 38.
    Arguello JR, Little AM, Pay AL et al (1998) Mutation detection and typing of polymorphic loci through double-strand conformation analysis. Nat Genet 18:192–194PubMedCrossRefGoogle Scholar
  39. 39.
    Arostegui JI, Gallardo D, Rodriguez-Luaces M et al (2000) Genomic typing of minor histocompatibility antigen HA-1 by reference strand mediated conformation analysis (RSCA). Tissue Antigens 56:69–76PubMedCrossRefGoogle Scholar
  40. 40.
    Kamei M, Nannya Y, Torikai H et al (2009) HapMap scanning of novel human minor histocompatibility antigens. Blood 113:5041–5048PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Spaapen RM, Lokhorst HM, van den Oudenalder K et al (2008) Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis. J Exp Med 205:2863–2872PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    van Bergen CA, Rutten CE, van der Meijden ED et al (2010) High-throughput characterization of 10 new minor histocompatibility antigens by whole genome association scanning. Cancer Res 70:9073–9083PubMedCrossRefGoogle Scholar
  43. 43.
    Akatsuka Y, Nishida T, Kondo E et al (2003) Identification of a polymorphic gene, BCL2A1, encoding two novel hematopoietic lineage-specific minor histocompatibility antigens. J Exp Med 197:1489–1500PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Kawase T, Akatsuka Y, Torikai H et al (2007) Alternative splicing due to an intronic SNP in HMSD generates a novel minor histocompatibility antigen. Blood 110:1055–1063PubMedCrossRefGoogle Scholar
  45. 45.
    Tykodi SS, Fujii N, Vigneron N et al (2008) C19orf48 encodes a minor histocompatibility antigen recognized by CD8+ cytotoxic T cells from renal cell carcinoma patients. Clin Cancer Res 14:5260–5269PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Torikai H, Akatsuka Y, Miyazaki M et al (2006) The human cathepsin H gene encodes two novel minor histocompatibility antigen epitopes restricted by HLA-A*3101 and -A*3303. Br J Haematol 134:406–410PubMedCrossRefGoogle Scholar
  47. 47.
    den Haan JM, Meadows LM, Wang W et al (1998) The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism. Science 279:1054–1057CrossRefGoogle Scholar
  48. 48.
    Mommaas B, Kamp J, Drijfhout JW et al (2002) Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus. J Immunol 169: 3131–3136PubMedCrossRefGoogle Scholar
  49. 49.
    Bleakley M, Otterud BE, Richardt JL et al (2010) Leukemia-associated minor histocompatibility antigen discovery using T-cell clones isolated by in vitro stimulation of naive CD8+ T cells. Blood 115:4923–4933PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    van Bergen CA, Kester MG, Jedema I et al (2007) Multiple myeloma-reactive T cells recognize an activation-induced minor histocompatibility antigen encoded by the ATP- dependent interferon-responsive (ADIR) gene. Blood 109: 4089–4096Google Scholar
  51. 51.
    Stumpf AN, van der Meijden ED, van Bergen CA, Willemze R, Falkenburg JH, Griffioen M (2009) Identification of 4 new HLA-DR-restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity. Blood 114:3684–3692PubMedCrossRefGoogle Scholar
  52. 52.
    Griffioen M, van der Meijden ED, Slager EH et al (2008) Identification of phosphatidylinositol 4-kinase type II beta as HLA class II-restricted target in graft versus leukemia reactivity. Proc Natl Acad Sci U S A 105: 3837–3842PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    de Rijke B, van Horssens-Zoetbrood A, Beekman JM et al (2005) A frame-shift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response associated with remission of chronic myeloid leukemia. J Clin Invest 115:3506–3516PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Brickner AG, Evans AM, Mito JK et al (2006) The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL. Blood 107:3779–3786PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Warren EH, Gavin MA, Xuereb SM et al (2002) A single nucleotide polymorphism in the SP110 nuclear phosphoprotein gene creates a minor histocompatibility antigen whose expression is regulated by interferon-gamma. Blood 100:73AGoogle Scholar
  56. 56.
    Oostvogels R, Minnema MC, van Elk M et al (2013) Towards effective and safe immunotherapy after allogeneic stem cell transplantation: identification of hematopoietic-specific minor histocompatibility antigen UTA2-1. Leukemia 27:642–649PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Vogt MH, de Paus RA, Voogt PJ, Willemze R, Falkenburg JH (2000) DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection. Blood 95:1100–1105PubMedGoogle Scholar
  58. 58.
    Pierce RA, Field ED, den Haan JM et al (1999) Cutting edge: the HLA-A*0101-restricted HY minor histocompatibility antigen originates from DFFRY and contains a cysteinylated cysteine residue as identified by a novel mass spectrometric technique. J Immunol 163:6360–6364PubMedGoogle Scholar
  59. 59.
    Meadows L, Wang W, den Haan JM et al (1997) The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity 6:273–281PubMedCrossRefGoogle Scholar
  60. 60.
    Torikai H, Akatsuka Y, Miyazaki M et al (2004) A novel HLA-A*3303-restricted minor histocompatibility antigen encoded by an unconventional open reading frame of human TMSB4Y gene. J Immunol 173:7046–7054PubMedCrossRefGoogle Scholar
  61. 61.
    Rosinski KV, Fujii N, Mito JK et al (2008) DDX3Y encodes a class I MHC-restricted H-Y antigen that is expressed in leukemic stem cells. Blood 111:4817–4826PubMedCentralPubMedCrossRefGoogle Scholar
  62. 62.
    Ivanov R, Aarts T, Hol S et al (2005) Identification of a 40S ribosomal protein S4-derived H-Y epitope able to elicit a lymphoblast-specific cytotoxic T lymphocyte response. Clin Cancer Res 11:1694–1703PubMedCrossRefGoogle Scholar
  63. 63.
    Vogt MH, Goulmy E, Kloosterboer FM et al (2000) UTY gene codes for an HLA-B60-restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T- cell recognition. Blood 96:3126–3132PubMedGoogle Scholar
  64. 64.
    Wang W, Meadows LR, den Haan JM et al (1995) Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein. Science 269:1588–1590PubMedCrossRefGoogle Scholar
  65. 65.
    Warren EH, Gavin MA, Simpson E et al (2000) The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen. J Immunol 164: 2807–2814PubMedCrossRefGoogle Scholar
  66. 66.
    Vogt MH, van den Muijsenberg J, Goulmy E et al (2002) The DBY gene codes for an HLA-DQ5 restricted human male specific minor histocompatibility antigen involved in GvHD. Blood 99:3027–3032PubMedCrossRefGoogle Scholar
  67. 67.
    Zorn E, Miklos DB, Floyd BH et al (2004) Minor histocompatibility antigen DBY elicits a coordinated B and T cell response after allogeneic stem cell transplantation. J Exp Med 199:1133–1142PubMedCentralPubMedCrossRefGoogle Scholar
  68. 68.
    Spierings E, Vermeulen C, Vogt MH et al (2003) Identification of HLA class II-restricted H-Y-specific T-helper epitope evoking CD4+ T-helper cells in H-Y-mismatched transplantation. Lancet 362:610–615PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Eric Spierings
    • 1
  1. 1.Laboratory for Translational ImmunologyUniversity Medical Center UtrechtUtrechtThe Netherlands

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