Development of a Multivariable Gene-Expression Signature of T-Cell-Mediated Rejection in Peripheral Blood of Kidney Transplant Recipients Validated in Cross-Sectional and Longitudinal Samples

Abstract

Background: Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage. Methods: We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first post-transplant year from rejectors, non-rejectors and stable patients. Findings: A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve for validation data-set 0·86 (0·74-0·95), sensitivity 0·70 (0·52-0·85) and positive predictive value 0·79 (0·65-0·94). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive. Interpretation: Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel T-cell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results. Funding: EU: FP7-HEALTH-2012-INNOVATION-1 (project-305147: BIO-DrIM) and FP7 grant agreement no HEALTH-F5–2010–260687. Medical Research Council: G0600698, MR/J006742/1; G0802068; MR/K002996/1 and G0801537/ID: 88245) NIHR Biomedical Research Centre at Guy’s and St Thomas’ and King’s College London. Declaration of Interest: MHF and IRB are employees of UCB Celltech, a pharmaceutical company based in the UK. Their involvement in the conduct of this research was solely in their capacity as academics whilst at King’s College London. Ethical Approval: Blood samples were collected serially from 455 consecutive KTRs, transplanted at a single regional transplant centre (Guy’s Hospital) in the Kidney Allograft Immunological Biomarkers of Rejection (KALIBRE) study (Research Ethics No.09.H0711.58).