Differentially expressed gene transcripts using RNA sequencing from the blood of immunosuppressed kidney allograft recipients.

Casey Dorr,Jonathan S Maltzman,Weihua Guan,David P Schladt,Amutha Muthusamy,William S Oetting,Steven E Scherer,Kinjal Sanghavi,Pamala A Jacobson,Ajay K Israni,Baolin Wu,Arthur J Matas,Marcia J Brott,Valquiria Bueno

doi:10.1371/journal.pone.0125045

Abstract

We performed RNA sequencing (RNAseq) on peripheral blood mononuclear cells (PBMCs) to identify differentially expressed gene transcripts (DEGs) after kidney transplantation and after the start of immunosuppressive drugs. RNAseq is superior to microarray to determine DEGs because it’s not limited to available probes, has increased sensitivity, and detects alternative and previously unknown transcripts. DEGs were determined in 32 adult kidney recipients, without clinical acute rejection (AR), treated with antibody induction, calcineurin inhibitor, mycophenolate, with and without steroids. Blood was obtained pre-transplant (baseline), week 1, months 3 and 6 post-transplant. PBMCs were isolated, RNA extracted and gene expression measured using RNAseq. Principal components (PCs) were computed using a surrogate variable approach. DEGs post-transplant were identified by controlling false discovery rate (FDR) at < 0.01 with at least a 2 fold change in expression from pre-transplant. The top 5 DEGs with higher levels of transcripts in blood at week 1 were TOMM40L, TMEM205, OLFM4, MMP8, and OSBPL9 compared to baseline. The top 5 DEGs with lower levels at week 1 post-transplant were IL7R, KLRC3, CD3E, CD3D, and KLRC2 (Striking Image) compared to baseline. The top pathways from genes with lower levels at 1 week post-transplant compared to baseline, were T cell receptor signaling and iCOS-iCOSL signaling while the top pathways from genes with higher levels than baseline were axonal guidance signaling and LXR/RXR activation. Gene expression signatures at month 3 were similar to week 1. DEGs at 6 months post-transplant create a different gene signature than week 1 or month 3 post-transplant. RNAseq analysis identified more DEGs with lower than higher levels in blood compared to baseline at week 1 and month 3. The number of DEGs decreased with time post-transplant. Further investigations to determine the specific lymphocyte(s) responsible for differential gene expression may be important in selecting and personalizing immune suppressant drugs and may lead to targeted therapies.

Highlights

Kidney allograft transplantation is the most cost-effective treatment for end stage renal disease [1,2,3]
Our hypothesis is that peripheral blood mononuclear cells (PBMCs) transcripts vary after the initiation of immunosuppression and at different times following kidney allograft transplantation
The T-cell signaling components CD3D, CD3E, and CD3G had decreased levels at week 1 post-transplant (Table 4), but levels increase towards pre-transplant levels at months 3 and 6 in the blood (Fig 1)

Summary

Introduction

Kidney allograft transplantation is the most cost-effective treatment for end stage renal disease [1,2,3]. The long-term success of transplantation is often threatened by acute rejection (AR) and chronic allograft dysfunction (CGD), which are common adverse outcomes in kidney allograft recipients despite modern immunosuppression [4]. Despite the use of better immunosuppressive regimens today than 15 years ago, lymphocytes, the primary targets of immunosuppressive drugs, still find ways to evade the immune suppression. This may be due to altered genetic mechanisms and cellular pathways that lead to insufficient T and/or B-cell suppression. To address if genetic mechanisms may be related to drug related immunosuppression we investigated if gene expression changes occur before and after the start of immune suppressant therapy and over time as therapy changes. We believe that eventually gene signatures can be used to personally tailor immune suppression therapies and predict clinical outcomes

Methods

Results

Conclusion