title: Intracellular IFNgamma production in CD3 negative cells exposed to allo-antigens is an indicator of prior sensitization.
abstract: BACKGROUND
  Sensitization to HLA antigens (Ags) is a significant obstacle to kidney transplantation and risk factor for antibody-mediated rejection (AMR). Current screening methods to assess HLA Ag exposure include various antibody assays. However, tools to accurately measure cell-mediated immunity to allo-Ags in a clinical setting are lacking. Here we report on an intracellular cytokine flow cytometry (CFC) assay that detects intracellular gamma-interferon (IFNgamma) production in non-T cell populations (CD3-) that appears to assess sensitization from previous allo-Ag exposure.
  
  
  METHODS
  Blood from 106 highly-HLA sensitized (HS) patients (pre-, post-IVIG-treatment [Rx] and/or post-transplant) and 14 3(rd) party normal controls (3(rd)N) were incubated with donor or 3(rd)N peripheral blood mononuclear cells (PBMCs), and IFNgamma+/CD3- cells were enumerated.
  
  
  RESULTS
  The percentage of IFNgamma+ cells in CD3- cells without stimulation in pre-IVIG-Rx HS patients was similar to normals, but significantly increased with incubation with donor and/or 3(rd)N PBMCs. Reactivity in normals was minimal. Reactivity was higher in HS females than HS males. Normal females with previous pregnancy (PG) showed significantly higher response than females without PG or non-sensitized normal males. Donor-specific reactivity in the CFC assay better correlated with donor-specific B cell crossmatch than total anti-HLA antibody levels or PRA. HS patients who developed AMR post-transplant showed significantly higher reactivity than those without AMR.
  
  
  CONCLUSIONS
  The CFC assay measures IFNgamma production in CD3- cells that may indicate a memory response to allo-Ags. This response is limited to HS patients and normal females with previous PG. Patients undergoing AMR show significantly higher reactivity. This assay may represent a novel approach to measurement of allo-sensitization with clinical utility in predicting those at risk for AMR.
authors: Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., SSB111, Los Angeles, CA 90048, USA. Mieko.Toyoda@cshs.org
authors: Mieko
authors: M
authors: Toyoda
authors: Andy
authors: A
authors: Pao
authors: Ashley
authors: A
authors: Vo
authors: Marina
authors: M
authors: Lukovsky
authors: Raju
authors: R
authors: Radha
authors: Tetsu
authors: T
authors: Sado
authors: Shili
authors: S
authors: Ge
authors: Nancy
authors: N
authors: Reinsmoen
authors: Ning Ning
authors: NN
authors: Chai
authors: Lara
authors: L
authors: Baden
authors: Anna
authors: A
authors: Petrosyan
authors: Edwina
authors: E
authors: Skinner
authors: James
authors: J
authors: Mirocha
authors: Stanley C
authors: SC
authors: Jordan
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D017252:CD3 Complex / Q000096:biosynthesis; D002452:Cell Count; D002648:Child; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006084:Graft Rejection / Q000175:diagnosis / Q000453:epidemiology / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007156:Immunologic Memory; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism*; D016030:Kidney Transplantation; D008297:Male; D008875:Middle Aged; D011247:Pregnancy; D012307:Risk Factors; D012737:Sex Factors
chemical_list: D017252:CD3 Complex; D006680:HLA Antigens; D007371:Interferon-gamma