Assessment of Immune Status of Liver Transplant Recipients by Peripheral Blood Mononuclear Cells in Response to Stimulation by Donor-specific Alloantigen.

Abstract

27 Objective. The role of assessing cytokine patterns in predicting clinical graft rejection or immunological graft stability (absence of rejection) remains unclear. The purpose of this study was to examine the effect of donor-specific alloantigenic stimulation of peripheral blood mononuclear cells (PBMC) from orthotopic liver transplant (OLT) recipients on cellular proliferative responses and cytokine profiles for a prospective correlation with clinical assessment of graft status. Methods. Donor alloantigens obtained at time of organ retrieval were stored at −70°C until utilized. PBMC from OLT recipients (n=45) were isolated and examined for proliferation and cytokine mRNA expression following stimulation by donor-specific alloantigen, third-party alloantigen or phytohemaglutinin (PHA). Recipient PBMC proliferation in response to stimulation was assessed by one-way mixed lymphocyte reaction. mRNA extracted from resting or stimulated recipient PBMC was amplified by reverse transcriptase PCR with oligo-specific primer pairs for interleukins (IL)-2, -4, -6, -10, IFN-γ, TNF-α and TGF-β. The level of cytokine gene expression was performed by semiquantitative analysis with β-actin expression set at 1000 units/μg and negative control at 0 units/μg of cDNA. Results were correlated prospectively over a 60 day interval with the patients' clinical and histological allograft status. Results. Increased IL-4 and TGF-β and decreased IL-2, IFN-γ and TNF-α mRNA expression by PBMC in response to donor-specific alloantigen stimulation predicted immunological graft stability (absence of rejection, n=30) over a minimum 60 day interval (p<0.05 compared to mRNA expression of PBMC from patients with unsuspected rejection or those who experienced a rejection episode within a 30 day period from the time of the cytokine analysis, n=15). Stimulation of recipient PBMC with third-party alloantigens or PHA yielded similar but less specific results. Recipient PBMC proliferation to the varying antigenic stimulation did not correlate with clinical graft status, nor did cytokine production by unstimulated PBMC from the OLT recipients. Conclusions. Prospective assessment of cytokine expression by PBMC from OLT recipients in response to stimulation by donor alloantigen is helpful for predicting the clinical status of the allograft and may be useful in the development of more precise immunological monitoring protocols.