title: Alloreactive T cell clonotype recruitment in a mixed lymphocyte reaction: implications for graft engineering.
abstract: OBJECTIVE
  The selective elimination of alloreactive T cells from donor stem cell grafts prior to hematopoietic stem cell transplantation (HSCT) is an important goal in the prevention of graft-vs-host disease (GVHD). However, in HLA-identical donor-recipient pairs, it has proven difficult to identify alloreactive T cells using in vitro systems pretransplant due, in part, to their low frequency and a lack of methodological standardization. To better understand the alloresponse between HLA-identical related pairs, we characterized the alloreactive T cells generated in a mixed lymphocyte reaction (MLR) assay system.
  
  
  METHODS
  HSCT donor peripheral blood mononuclear cells (responder) were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) dye and cocultured with irradiated HSCT recipient cells (stimulator) in a one-way MLR. Alloreactive T cells were sorted by upregulation of activation markers (CD25 in most cases) and the responding clonotypes were defined by sequencing the complementarity region 3 (CDR3) of the T cell receptor beta-chain.
  
  
  RESULTS
  We show that the recruitment of alloreactive CD4(+) T cells is highly variable. Oligoclonal CD4(+) T-cell expansions in repeated MLRs performed in the same donor-recipient pair showed inconsistent recruitment of clonotypes. The recruitment of alloreactive CD8(+) T cells was more consistent in repeated assays, with the same clonotypes identified in the same donor-recipient pair performed under different conditions.
  
  
  CONCLUSIONS
  Taken together, our data show that even in culture conditions constrained to eliminate background proliferation, stochastic events and low precursor frequencies preclude reproducible elicitation of immunodominant T cell clonotypes with the potential to cause GVHD.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892-1202, USA. scheinbp@nhlbi.nih.gov
authors.forename: Phillip
authors.initials: P
authors.lastname: Scheinberg
authors.forename: David A
authors.initials: DA
authors.lastname: Price
authors.forename: David R
authors.initials: DR
authors.lastname: Ambrozak
authors.forename: A John
authors.initials: AJ
authors.lastname: Barrett
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Douek
mesh_terms: D000954:Antigens, Surface / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D049109:Cell Proliferation; D018920:Coculture Techniques; D019674:Genes, T-Cell Receptor beta / Q000276:immunology; D006082:Graft Enhancement, Immunologic; D006086:Graft vs Host Disease / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D006412:Hematopoietic Stem Cells / Q000276:immunology* / Q000473:pathology; D006801:Humans; D047589:Leukocyte Reduction Procedures; D018448:Models, Immunological; D014019:Tissue Donors; D014180:Transplantation; D014184:Transplantation, Homologous
chemical_list: D000954:Antigens, Surface