title: Cellular allo reactivity against paternal HLA antigens in normal multiparous females as detected by intracellular cytokine flow cytometry remains elevated over years despite diminution of anti-HLA antibody levels.
abstract: BACKGROUND
  Sensitization to allo-antigens (Ags) resulting from previous transplants, blood transfusion or pregnancy (PG), is a significant obstacle to kidney transplantation and risk factor for antibody-mediated rejection (AMR). We have previously shown that allo-Ag-specific CD3 negative (-) cells as analyzed by cytokine flow cytometry (CFC) are elevated in many highly HLA-sensitized (HS) patients, but not most normal individuals. HS patients with high(+) CFC, especially to donor Ags, may be at high risk for AMR and may need additional pre-transplant desensitization. Here, we investigate allo-sensitization that results from paternal HLA (pHLA) Ag exposure in normal multiparous females and assess the utility of the CFC assay in detecting allo-Ag-specific immune cell activity.
  
  
  METHODS
  Whole blood from 8 normal females with previous PG (pPG), 8 normal females without pPG and 5 normal males were incubated with irradiated normal peripheral blood mononuclear cells (PBMC). IFNgamma+/CD3- cells were enumerated and results expressed as a ratio against un-stimulated cells.
  
  
  RESULTS
  4/8 females with pPG showed elevated CFC reactivity against at least 1 PBMC tested (ratio 4.7+/-1.4), while the remaining 4 (0.76+/-0.42), 8 females without pPG (0.65+/-0.24) and 5 males (0.99+/-0.59) showed minimal reactivity. The reactivity against the same PBMC was fairly consistent over months and years. Anti-HLA antibody was detected in females with elevated CFC, but the levels as analyzed by ELISA did not correlate with CFC reactivity. Individuals with minimal reactivity consistently show negative anti-HLA antibody levels. Two females with pPG with elevated CFC (#1 and #2) showed high reactivity to PBMC from their husbands, one offspring and 3rd-party normals carrying antigenic pHLA-Ags (#1: 13.2, 9.4 and 22.9; #2: 8.2, 8.0 and 8.8-12.6, respectively). Antibody specificity for antigenic pHLA-Ags was found by Luminex in anti-HLA antibody(+) samples in these 2 females.
  
  
  CONCLUSIONS
  1) The CFC is a novel way to measure allo-specific CD3- cell responses and can detect allo-sensitization resulting from PG, 2) the CFC detects allo-specific memory cell activity as reactivity is consistent over years regardless of anti-HLA antibody levels, 3) high(+) CFC in normal multiparous females may explain increased risk for AMR in female HS patients, and 4) analysis of donor- or allo-specific CFC in multiparous females awaiting kidney transplant may identify those at risk for AMR.
authors.affiliation: Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., Los Angeles, CA 90048, USA. Mieko.Toyoda@cshs.org
authors.forename: Mieko
authors.initials: M
authors.lastname: Toyoda
authors.forename: Shili
authors.initials: S
authors.lastname: Ge
authors.forename: Andy
authors.initials: A
authors.lastname: Pao
authors.forename: Ashley
authors.initials: A
authors.lastname: Vo
authors.forename: Nathan
authors.initials: N
authors.lastname: Deer
authors.forename: Angela
authors.initials: A
authors.lastname: Aguiluz
authors.forename: Artur
authors.initials: A
authors.lastname: Karasyov
authors.forename: Stanley C
authors.initials: SC
authors.lastname: Jordan
mesh_terms: D017252:CD3 Complex / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D002469:Cell Separation; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006648:Histocompatibility; D006801:Humans; D007114:Immunization; D007371:Interferon-gamma / Q000378:metabolism; D007518:Isoantibodies / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D007519:Isoantigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation; D008297:Male; D010298:Parity / Q000276:immunology*; D011247:Pregnancy / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
chemical_list: D017252:CD3 Complex; D016827:CD8 Antigens; D006680:HLA Antigens; D007518:Isoantibodies; D007519:Isoantigens; D007371:Interferon-gamma