IFNγ production by NK cells from HLA-sensitized patients after in vitro exposure to allo-antigens

https://doi.org/10.1016/j.trim.2011.11.001Get rights and content

Abstract

Using a novel cytokine flow cytometry test (allo-CFC), we have previously shown that incubation of allogeneic cells with peripheral blood from highly-HLA sensitized (HS) patients results in reproducible gamma-interferon (IFNγ production in CD3 cells, and high (+) allo-CFC levels correlated with risk for antibody-mediated rejection (AMR). Here we report on identification of the cells and mechanisms responsible. The allo-CFC with/without modification was performed using blood from HS or normal individuals. IFNγ producing cells were CD3/CD19, but CD3/CD56+. In vitro and in vivo B cell-depletion did not affect IFNγ production, demonstrating NK cells as the cells responsible for IFNγ production. NK cells from allo-CFC(+) or (−) individuals released significant amounts of IFNγ against target cells treated with serum from allo-CFC(+) individuals, but not allo-CFC(−) individuals. IFNγ release was abrogated by protein A/G treatment of the pretreated target cells, suggesting mediation by antibodies via FcγRIIIa (CD16). In conclusion, NK cell IFNγ release after allo-antigen exposure is mediated primarily through antibody-dependent cellular cytotoxicity (ADCC)-like mechanisms, suggesting that NK cells may be partially responsible for graft injury during AMR including C4d(−) AMR via ADCC, and could be a potential target for modification of this process.

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.

References (34)

  • M.J. Glennie et al.

    Mechanisms of killing by anti-CD20 monoclonal antibodies

    Mol Immunol

    (2007)
  • W. van der Touw et al.

    Natural killer cells and the immune response in solid organ transplantation

    Am J Transplant

    (2010)
  • A.A. Vo et al.

    Rituximab and intravenous immune globulin for desensitization during renal transplantation

    N Engl J Med

    (2008)
  • L.C. Racusen et al.

    Antibody-mediated rejection in renal allografts: lessons from pathology

    Clin J Am Soc Nephrol

    (2006)
  • S.C. Jordan et al.

    Advances in diagnosing and managing antibody-mediated rejection

    Pediatr Nephrol

    (2010)
  • L.D. Truong et al.

    Acute antibody-mediated rejection of renal transplant: pathogenetic and diagnostic considerations

    Arch Pathol Lab Med

    (2007)
  • A.A. Vo et al.

    Use of intravenous immune globulin and rituximab for desensitization of highly HLA-sensitized patients awaiting kidney transplantation

    Transplantation

    (2010)
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