Intracellular IFNgamma production in CD3 negative cells exposed to allo-antigens is an indicator of prior sensitization.
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.
Toyoda
Mieko
M
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
Pao
Andy
A
Vo
Ashley
A
Lukovsky
Marina
M
Radha
Raju
R
Sado
Tetsu
T
Ge
Shili
S
Reinsmoen
Nancy
N
Chai
Ning Ning
NN
Baden
Lara
L
Petrosyan
Anna
A
Skinner
Edwina
E
Mirocha
James
J
Jordan
Stanley C
SC
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
D017252:CD3 Complex; D006680:HLA Antigens; D007371:Interferon-gamma