title: Detection of Virus-Specific CD8+ T Cells With Cross-Reactivity Against Alloantigens: Potency and Flaws of Present Experimental Methods.
abstract: Virus-specific T cells have the intrinsic capacity to cross-react against allogeneic HLA antigens, a phenomenon known as heterologous immunity. In transplantation, these cells may contribute to the alloimmune response and negatively impact graft outcome. This study describes the various techniques that can be used to detect heterologous immune responses of virus-specific CD8(+) T cells against allogeneic HLA antigens. The strengths and weaknesses of the different approaches are discussed and illustrated by experimental data.
  
  
  METHODS
  Mixed-lymphocyte reactions (MLRs) were performed to detect allo-HLA cross-reactivity of virus-specific CD8(+) T cells in total peripheral blood mononuclear cells. T-cell lines and clones were generated to confirm allo-HLA cross-reactivity by IFNγ production and cytotoxicity. In addition, the conventional MLR protocol was adjusted by introducing a 3-day resting phase and subsequent short restimulation with alloantigen or viral peptide, whereupon the expression of IFNγ, IL-2, CD107a, and CD137 was determined.
  
  
  RESULTS
  The accuracy of conventional MLR is challenged by potential bystander activation. T-cell lines and clones can circumvent this issue, yet their generation is laborious and time-consuming. Using the adjusted MLR and restimulation protocol, we found that only truly cross-reactive T cells responded to re-encounter of alloantigen and viral peptide, whereas bystander-activated cells did not.
  
  
  CONCLUSIONS
  The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8(+) T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard.
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Heleen
authors: H
authors: van den Heuvel
authors: Department of Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands.; Renal Transplant Unit, Division of Internal Medicine, Department of Nephrology, Academic Medical Centre, Amsterdam, The Netherlands.
authors: Kirstin M
authors: KM
authors: Heutinck
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Ellen P M W
authors: EP
authors: van der Meer-Prins
authors: Department of Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands.; Renal Transplant Unit, Division of Internal Medicine, Department of Nephrology, Academic Medical Centre, Amsterdam, The Netherlands.
authors: Si La
authors: SL
authors: Yong
authors: Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors: Frans H J
authors: FH
authors: Claas
authors: Renal Transplant Unit, Division of Internal Medicine, Department of Nephrology, Academic Medical Centre, Amsterdam, The Netherlands.
authors: Ineke J M
authors: IJ
authors: Ten Berge