Host microRNAs are decreased in pediatric solid-organ transplant recipients during EBV+ Post-transplant Lymphoproliferative Disorder

Abstract

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. Predisposing factors include primary Epstein-Barr virus (EBV) infection, reactivation of EBV in recipient B cells, and decreased T cell immunity due to immunosuppression. In our previous studies EBV infection was demonstrated to markedly alter the expression of host B cell microRNA (miR). Specifically, miR-194 expression was uniquely suppressed in EBV+ B cell lines from PTLD patients and the 3'untranslated region of IL-10 was determined to be targeted by miR-194. Although EBV has been shown to regulate host miR expression in B cell lymphoma cell lines, the expression of miRs in the circulation of patients with EBV-associated PTLD has not been studied. The objective of this study was to determine if changes in miR expression are associated with EBV+ PTLD. In this study, we have shown that miR-194 is significantly decreased in EBV+PTLD tumors and that additional miRs, including miRs-17, 19 and 106a are also reduced in EBV+PTLD as compared to EBV-PTLD. We quantitated the levels of miRs-17, 19, 106a, 155, and 194 in the plasma and extracellular vesicles (EV; 50-70 nm as determined by nanoparticle tracking analysis) from pediatric recipients of solid organ transplants with EBV+ PTLD+ that were matched 1:2 with EBV+ PTLD- pediatric transplant recipients as part of the NIH-sponsored Clinical Trials in Organ Transplantation in Children, (CTOTC-06) study. Levels of miRs-17, 19, 106a, and 194 were reduced in the plasma and extracellular vesicles (EV) of EBV+ PTLD+ group compared to matched controls, with miRs-17 (p = 0.034; plasma), miRs-19 (p = 0.029; EV) and miR-106a (p = 0.007; plasma and EV) being significantly reduced. Similar levels of miR-155 were detected in the plasma and EV of all pediatric SOT recipients. Importantly, ~90% of the cell-free miR were contained within the EV supporting that EBV+ PTLD tumor miR are detected in the circulation and suggesting that EVs, containing miRs, may have the potential to target and regulate cells of the immune system. Further development of diagnostic, mechanistic and potential therapeutic uses of the miRs in PTLD is warranted.

Keywords: Epstein-Barr Virus; Post-Transplant Lymphoproliferative Disorder; extracellular vesicles; microRNA; solid-organ transplant.

Figure 1

Decreased levels of miRs are detected in the plasma of EBV+ PTLD+ pediatric transplant recipients. (A) Schematic diagram of the workflow for quantitation of plasma derived miR from EBV+ PTLD+ pediatric SOT recipients (n = 18) and EBV+ PTLD- matched controls (n = 37) (B) Concentrations of miR-17, miR-19, miR-106a, miR-155 and miR-194 were measured by qPCR in plasma of PTLD+ patients with early lesions or monomorphic/polymorphic lesions and matched controls. PTLD+ vs controls, miR-17, p = 0.034; miR-106a, p = 0.007 by Mann-Whitney test. Monomorphic/polymorphic PTLD vs. control, miR-106a, p = 0.047 by Kruskal-Wallis test and Dunn’s multiple comparison test.

Figure 2

Longitudinal analysis demonstrates a decrease in miR levels prior to a diagnosis of PTLD. (A) Schematic diagram of the pairing of PTLD+ (n = 13) and control (n = 13) subjects by matching organ and time of sample collection post-transplant. In both PTLD and control groups, the timeline in black represents the number of days post-transplant. In the PTLD group, the timeline in blue represents the number of days prior to PTLD diagnosis, indicated by Day 0. In the PTLD+ group, the last samples were collected between 0-60 days prior to PTLD diagnosis. (B) Concentrations of miR-17, miR-19, miR-106a, miR-155 and miR-194 were measured by qPCR in plasma samples collected at three timepoints post-transplant with the last sample collected within 0-60 days prior to PTLD diagnosis (n = 5). (C) Concentrations of miR-17, miR-19, miR-106a, miR-155 and miR-194 were measured by qPCR in plasma samples collected post-transplant from control patients (n = 5) at timepoints matched with paired PTLD+ subjects.

Figure 3

miRs are localized to EVs in plasma. (A) Schematic diagram of the workflow for the isolation of EVs from plasma and qPCR analysis of EV-derived miRNA from PTLD (n = 18) and control (n = 36) pediatric SOT recipients (B) Characterization of vesicles size and concentration of EVs in EV pellets isolated from plasma fraction by nanoparticle tracking analysis. (C) Concentrations of miR-17, miR-19, miR-106a, miR-155 and miR-194 were measured by qPCR in EVs and EV-depleted plasma samples. Black bars represent the fraction of EV-derived miRNAs in whole plasma. Gray bars represent the fraction of miRNAs derived from EV-depleted plasma.

Figure 4

Decreased levels of miRs are detected in the extracellular vesicles of PTLD+ pediatric transplant recipients as compared to controls. Concentrations of miR-17, miR-19, miR-106a, miR-155 and miR-194 were measured by qPCR in isolated EVs from PTLD+ (n = 18) and control (n = 36) patients. PTLD+ vs controls, miR-19, p = 0.029; miR-106a, p = 0.007 by Mann-Whitney test. Monomorphic/polymorphic PTLD vs. control, miR-17, p = 0.0387; miR-19, p = 0.044; miR-106a, p = 0.0091 by Kruskal-Wallis test and Dunn’s multiple comparison test.