title: Modified ELISPOT technique--highly significant inverse correlation of post-Tx donor-reactive IFNgamma-producing cell frequencies with 6 and 12 months graft function in kidney transplant recipients.
abstract: BACKGROUND
  Upcoming trials for immunosuppression minimization and tolerance induction require the development of reliable in vitro assays for monitoring cellular alloimmunity in transplant patients. The IFN-gamma ELISPOT assay represents a promising tool for monitoring alloreactive memory/effector T cells. As T lymphopenia is a common finding during the early post-transplant (post-Tx) period, the IFN-gamma ELISPOT technique was here modified by using ELISPOT responder cells with enhanced percentage and standardized number of 200,000 T cells per well.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMNC) of kidney transplant recipients were depleted of CD14+ and CD15+ cells to increase the percentage of T cells from average 47.8% to 71.5% before transplantation (pre-Tx) and from 39.7% to 74.9% post-Tx. The assay was tested in a population of 23 de novo renal transplant patients for clinical relevance. Before and at 2-3 times during the first 6 months post-Tx, IFN-gamma-producing donor-reactive as well as recall antigen-reactive cell frequencies (Candida, tuberculin, tetanus) were determined and correlated with outcome.
  
  
  RESULTS
  Pre-Tx donor-reactive ELISPOT frequencies were enhanced in patients with acute rejection compared to non-rejectors. Moreover, mean post-Tx donor-reactive ELISPOT frequencies showed a highly significant inverse correlation with renal function at 6 and 12 months. In contrast, recall antigen-reactive ELISPOT frequencies did not correlate with outcome.
  
  
  CONCLUSIONS
  Our results suggest that the modified donor-reactive ELISPOT approach might provide a useful surrogate marker for renal transplant outcome with possible utility especially in T-lymphopenic patients.
authors.affiliation: Institute of Medical Immunology, Charité Campus Mitte, Schumannstrasse 20/21, 10098 Berlin, Germany.
authors.forename: Bele Johanna
authors.initials: BJ
authors.lastname: Näther
authors.forename: Peter
authors.initials: P
authors.lastname: Nickel
authors.forename: Gantuja
authors.initials: G
authors.lastname: Bold
authors.forename: Franziska
authors.initials: F
authors.lastname: Presber
authors.forename: Constanze
authors.initials: C
authors.lastname: Schönemann
authors.forename: Johann
authors.initials: J
authors.lastname: Pratschke
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Volk
authors.forename: Petra
authors.initials: P
authors.lastname: Reinke
mesh_terms: D015415:Biomarkers / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006084:Graft Rejection / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D016030:Kidney Transplantation / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
chemical_list: D015415:Biomarkers; D007371:Interferon-gamma