IFNγ production by NK cells from HLA-sensitized patients after in vitro exposure to allo-antigens
Highlights
► IFNγ producing CD3− cells in the allo-CFC assay were identified as NK cells. ► NK cell was activated by an allo-antibody-mediated ADCC-like mechanism via FcγRIIIa. ► NK cells may also be responsible for graft injuries including C4d(−) AMR via ADCC.
Introduction
High rates of AMR remain an obstacle to successful outcomes in HS patients transplanted after desensitization [[1], [2], [3]]. Current screening methods for AMR consist primarily of antibody assays to detect donor-specific antibodies (DSA) and biopsies [[4], [5], [6], [7]]. Although antibody-mediated complement-dependent cytotoxicity (CDC) is important, cellular effector pathways including antibody-dependent cellular cytotoxicity (ADCC) may also play an important role in the pathogenesis of AMR [5]. We previously reported on the allo-CFC assay which measures non-T cell (CD3−) IFNγ release in response to allo-antigens expressed on peripheral blood mononuclear cells (PBMCs) [8], [9]. Using this assay, we found that allo-antigen-specific CD3− cells were elevated in most HS patients, but not in normal individuals without history of allo-antigen exposure. Normal women with a history of previous pregnancies were allo-CFC(+) with a demonstrated specificity for paternal HLA antigens. HS patients who were allo-CFC high(+) pre-transplant were at increased risk for AMR after transplant. Based on these findings, we concluded that the allo-CFC assay measures allo-antigen-specific cell responses and can assess allo-sensitization resulting from exposure to allo-antigens through previous pregnancies, blood transfusions and transplants. This assay may also have clinical utility in predicting risk for AMR in HS patients awaiting kidney transplant. Here we report on the identification of the cells responsible for IFNγ release after allo-antigen exposure using the allo-CFC test.
Section snippets
Patient population
This study was approved by the Institutional Review Board at Cedars-Sinai Medical Center (CSMC IRB) (Protocol numbers: 10969, 12562 and 17197). The study was conducted in accordance with the ethical guideline based on federal regulations and the common rule. In addition, CSMC has a Federalwide Assurance.
To monitor the immune cell number, heparinized blood was obtained pre-, post-desensitization and post-transplant in 25 patients who underwent kidney transplantation between October 2007 and
Results
To identify cells responsible for allo-antigen-stimulated IFNγ production in the CD3− cell population, allo-CFC was performed. Here, IFNγ+/CD3− cells were 8.4% and were CD8− to CD8dim + (Fig. 1-A-c). By plotting CD3− cells against IFNγ and CD19 or CD56, we found that the IFNγ+/CD3− cells were CD19−, but CD56dim + cells (Fig. 1-A-d,e), suggesting that IFNγ+ cells are not likely B cells, but NK cells. Since NK cells can be divided into several subsets by CD56 and CD16 expression [13], another
Discussion
B cells, antibody and complement are thought to be sufficient for mediation of AMR as current therapies are directed at antibody reduction, B cell depletion and modification of antibody-dependent, complement-mediated injury [6]. To date, there is, to our knowledge, no confirmation of ADCC mediated by NK cells as an alternate pathway for mediation of AMR. We have previously shown that IFNγ production by non-T cells in response to in vitro allo-antigen stimulation predicts a high risk for AMR,
Acknowledgment
This study was supported, in part, by a grant from Talecris Biotherapeutics, Inc., a grant from the Amado Foundation and the Joyce Jillson Fund for Transplant Research.
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