Longitudinal analysis of whole blood transcriptomes to explore molecular signatures associated with acute renal allograft rejection

Abstract

In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature.

Keywords: blood transcriptomics; kidney transplant rejection; microarray; neutrophil to lymphocyte ratio; peripheral whole blood.

Figures

Figure 1

Comparison of 24 ARs at rejection and 24 matched NRs. A. PCA plot of 24 AR patient samples at the time of rejection and their 24 matched NR patient samples. AR and NR samples do not separate clearly. B. The same PCA plot of 24 AR and 24 NR patient samples as in A. Samples are highlighted by the time (days) of rejection (ie, time of sample collection since transplant). Sample separation based on the time of collection can be seen indicating the presence of a time-dependent gene expression signature. C. PCA plot of samples from days 2 to 10 posttransplant generated using the time-dependent gene expression signature. Overall, a strong separation of samples from days 2 to 4 and days 6 to 10 posttransplant is observed (the number shown next to each sample point is the number of days posttransplant for that sample). D. PCA plot of 8 late ARs (week 3 and beyond, posttransplant) and the matched NRs using all the filtered probe set data. These late ARs separate from NRs more clearly compared to the early ARs and NRs.

Figure 2

Genes upregulated approaching rejection are highly neutrophil enriched while genes downregulated are highly lymphocyte enriched. A. Comparison of gene enrichment scores (Benita et al36) for genes that are upregulated approaching rejection in neutrophils and monocyte CD14+ versus peripheral CD4+ and CD8+ T cells. These genes are highly enriched in neutrophils and monocytes compared to peripheral T cells. A positive score indicates enrichment and negative score indicates absence. B. Comparison of gene enrichment scores for genes that are downregulated approaching rejection in neutrophils and monocytes versus peripheral CD4+ and CD8+ T cells indicates these genes are highly enriched in T lymphocytes. C and D. Sum of enrichment scores for various blood cell and different tissue types shows that upregulated genes represent a neutrophil signature (C) and that downregulated genes represent a T lymphocyte signature (D).

Figure 3

A. PCA plot generated by the representative set of the most significant genes from both neutrophil and lymphocyte signatures shows clear separation of ARs from NRs. B. Average gene expression levels in 3 time periods: pretransplant (BL), posttransplant up to rejection (Pre-RJ and RJ), and postrejection (Post-RJ). A representative set of the neutrophil signature genes show upregulation from BL towards rejection and downregulation after rejection treatment in AR samples; these same genes show a more stable level of expression in NR samples. C. A representative set of the lymphocyte signature genes show downregulation from BL towards rejection and upregulation after rejection treatment in AR samples; these same genes show more stable expression in NR samples.

Figure 4

Validation of the rejection-associated signatures using external kidney transplant study data. Validation study microarray data were generated using the GeneST 1.1 platform on 15 AR and 22 NR sample sets (BiT2). A. SAM analysis of 15 AR samples when compared with the 22 NR samples indicates a general upregulation of neutrophil specific genes, of which approximately half are statistically significant (red data points). B. A general downregulation of most lymphocyte specific genes is observed, of which approximately a third are statistically significantly downregulated (green data points).

Figure 5

Pattern matching of gene expression changes over time reveals a molecular signature associated with AR. A. PTM analysis showing genes matching the expression signature of upregulation from pretransplant to posttransplant in AR patients only. Five AR patients and 5 matched NR patients with the precisely matched time points were used for the analysis. The 3 time points used (plotted consecutively for each patient) are pretransplant (BL), 7 days prerejection (−7), and at rejection (RJ). B. PCA plot visualizing the expression signature described in A. The AR patients (red squares, with week [W] posttransplant data indicated) are moving away from their pretransplant time point (BL) as well as from control patients (green circles) and NR patients (blue triangles) as they are approaching rejection.