title: HLA monomers as a tool to monitor indirect allorecognition.
abstract: BACKGROUND
  Recognition of donor antigens can occur through two separate pathways: the direct pathway (non-self HLA on donor cells) and the indirect pathway (self-restricted presentation of donor derived peptides on recipient cells). Indirect allorecognition is important in the development of humoral rejection; therefore, there is an increasing interest in the monitoring of indirect alloreactive T-cells. We have used an in vitro model to determine the optimal requirements for indirect presentation and assessed the risk for semidirect presentation in this system.
  
  
  METHODS
  HLA-typed monocyte-derived dendritic cells (moDCs) were incubated with cellular fragments or necrotic cells and incubated with either indirect or direct alloreactive T-cell clones. T-cell reactivity was measured through proliferation or cytokine secretion. HLA-typed moDC, monocytes, or PBMCs were incubated with HLA class I monomers, in combination with either direct/indirect T-cell clones.
  
  
  RESULTS
  Although both were efficiently taken up, alloreactivity was limited to the semi-direct pathway, as measured by allospecific CD4 (indirect) and CD8 T-cell clones (direct) when cells were used. In contrast, HLA-A2 monomers were not only efficiently taken up but also processed and presented by HLA-typed moDC, monocytes, and PBMCs. Activation was shown by a dose-dependent induction of IFN-γ production and proliferation by the CD4 T-cell clone. Antigen presentation was most efficient when the monomers were cultured for longer periods (24-48 hr) in the presence of the T-cells. Using this method, no reactivity was observed by the CD8 T-cell clone, confirming no semidirect alloreactivity.
  
  
  CONCLUSIONS
  We have developed a system that could be used to monitor indirect alloreactive T-cells.
authors.affiliation: 1 Department of Nephrology, Leiden University Medical Center (LUMC), Leiden, the Netherlands. 2 Department of Immunohematology and Blood Transfusion, LUMC, Leiden, the Netherlands. 3 Department of Hematology, LUMC, Leiden, The Netherlands. 4 Address correspondence to: Cees van Kooten, MD, PhD, Albinusdreef 2, C07-35 2333ZA Leiden, The Netherlands.
authors.forename: Eytan
authors.initials: E
authors.lastname: Breman
authors.forename: Paula P
authors.initials: PP
authors.lastname: van Miert
authors.forename: Dirk M
authors.initials: DM
authors.lastname: van der Steen
authors.forename: Mirjam H
authors.initials: MH
authors.lastname: Heemskerk
authors.forename: Ilias I
authors.initials: II
authors.lastname: Doxiadis
authors.forename: Dave
authors.initials: D
authors.lastname: Roelen
authors.forename: Frans H
authors.initials: FH
authors.lastname: Claas
authors.forename: Cees
authors.initials: C
authors.lastname: van Kooten
mesh_terms: D017951:Antigen Presentation / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D049109:Cell Proliferation; D003713:Dendritic Cells / Q000166:cytology; D000939:Epitopes / Q000737:chemistry; D006084:Graft Rejection / Q000276:immunology; D006680:HLA Antigens / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007519:Isoantigens / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D009336:Necrosis; D016377:Organ Transplantation; D010455:Peptides / Q000276:immunology; D010587:Phagocytosis
chemical_list: D000939:Epitopes; D006680:HLA Antigens; D015789:HLA-A2 Antigen; D007519:Isoantigens; D010455:Peptides