Impact of tacrolimus on interferon gamma ELISpot assay results for the assessment of T-cell immunity: Proof-of-concept

Abstract

SOT patients require immunosuppressors to avoid graft rejection. Therapeutic drug monitoring is insufficient to find the optimal balance with immunosuppression. The evaluation of cell-mediated immunity by enzyme-linked immunospot (ELISpot) assay enumerating interferon-gamma (IFN-γ) is increasingly use. ELISpot assays are performed on peripheral blood mononuclear cells (PBMC) isolated from blood and brought into contact with specific peptides in an immunosuppressor-free environment. This study aims to determine the in vitro diffusion of tacrolimus in PBMC and to assess whether prior in vitro incubation of PBMC with tacrolimus modifies the IFN-γ ELISpot results when assessing the T-cell immune response. PBMC from healthy volunteers were obtained. Tacrolimus was added to the ELISpot wells at increasing concentration and quantification was obtained using liquid chromatography mass spectrometry. Results showed that the in vitro PBMC diffusion rate of tacrolimus was measured at 32%. A decrease in T-cell reactivity occurred with increasing tacrolimus concentration. The intra-PBMC concentration of tacrolimus able to inhibit 50% of T-cell reactivity was 163 pg/10⁶ PBMC, which is in the range of the in vivo intra-PBMC concentration in SOT recipients. T-cell functional assessment using ELISpot in patients treated with immunosuppressors may require the addition of immunosuppressors in vitro to better reflect the in vivo situation.

Keywords: cell-mediated immunity; immunosupressor diffusion rate; interferon-gamma ELISpot assay; peripheral blood mononuclear cells; tacrolimus.

Figure 1.

Correlation between the in vitro added tacrolimus concentration and the intra-PBMC tacrolimus concentration measured by LC-MS/MS after 1-h incubation, in pg/10⁶ PBMC (Pearson correlation coefficient of correlation R = 0.85, p < 0.001). One million PBMC were incubated with increasing amounts of tacrolimus: 0.0, 80.0, 300.0, 500.0, 800.0, and 1000.0 pg/10⁶ PBMC. Each condition was tested in triplicate (Data missing for one replicate at 500.0 and 1000.0 pg/10⁶).

Figure 2.

Dose-response curve of T-cell immune reactivity after PBMC stimulation with anti-CD3 antibody measured by IFN-γ ELISpot assay according to the tacrolimus concentration in the well. The immune response is depicted by the number of spots obtained in each well (Pearson correlation coefficient R = −0.94, p < 0.001).