High-throughput identification of potential minor histocompatibility antigens by MHC tetramer-based screening: Feasibility and limitations

Pleun Hombrink, Sine Reker Hadrup, Arne Bakker, Michel G. D. Kester, J. H. Frederik Falkenburg, Peter A. von dem Borne, Ton N. M. Schumacher, Mirjam H. M. Heemskerk

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T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1 IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance. �� 2011 Hombrink et al.
Original languageEnglish
JournalP L o S One
Issue number8
Pages (from-to)e22523
Publication statusPublished - 2011
Externally publishedYes


  • Agricultural and Biological Sciences (all)
  • Biochemistry, Genetics and Molecular Biology (all)
  • Medicine (all)
  • HLA A2 antigen
  • minor histocompatibility antigen
  • mitogen activated protein kinase
  • mitogen activated protein kinase kinase kinase kinase 1
  • unclassified drug
  • epitope
  • allogeneic stem cell transplantation
  • antigen binding
  • antigen detection
  • antigen specificity
  • article
  • binding affinity
  • cell isolation
  • controlled study
  • flow cytometry
  • graft versus leukemia effect
  • hematopoietic stem cell
  • high throughput screening
  • human
  • human cell
  • immune response
  • lymphocyte subpopulation
  • molecular recognition
  • prediction
  • protein expression
  • sensitivity analysis
  • single nucleotide polymorphism
  • T lymphocyte
  • cell line
  • DNA microarray
  • gene expression profiling
  • genetics
  • immunology
  • major histocompatibility complex
  • metabolism
  • physiology
  • Cell Line
  • Epitopes
  • Flow Cytometry
  • Gene Expression Profiling
  • HLA-A2 Antigen
  • Humans
  • Major Histocompatibility Complex
  • Minor Histocompatibility Antigens
  • Oligonucleotide Array Sequence Analysis
  • Polymorphism, Single Nucleotide
  • T-Lymphocytes

Cite this

Hombrink, P., Hadrup, S. R., Bakker, A., Kester, M. G. D., Falkenburg, J. H. F., von dem Borne, P. A., Schumacher, T. N. M., & Heemskerk, M. H. M. (2011). High-throughput identification of potential minor histocompatibility antigens by MHC tetramer-based screening: Feasibility and limitations. P L o S One, 6(8), e22523. https://doi.org/10.1371/journal.pone.0022523