Gene Expression Profiles Suggest The Activation of an Interferon (IFN) Alpha Signaling in CD4+ T Cells of Chronic Antibody-Mediated Kidney Graft Rejection (CAMR).

Abstract

CAMR is the main cause of chronic graft injury and loss, but its pathogenesis is still largely unclear. The aim of the present study was to investigate the molecular mechanisms underlying the development of CAMR by the analysis of gene expression profiles of both total peripheral lymphomonocytes (PLM) and isolated CD4+ and CD8+ T lymphocytes. We enrolled 18 patients with biopsy-proven CAMR and 18 transplant recipients with normal graft histology/function (control group). Gene expression profile of PLM and CD4+ and CD8+ T lymphocytes isolated from both groups (n=8/group) was assessed by Agilent microarrays, evaluated by standard statistical and functional pathway analysis (Ingenuity Pathway Analysis, IPA) and validated by quantitative RT-PCR in an independent set of patients (n=6/group). We identified 67 probesets, corresponding to 53 genes, differentially expressed in the 2 groups (FDR<5%, fold-change ≥2). The main canonical pathway identified by these genes was the IFN-alpha pathway (p=6.0*10-5), while network analysis showed that 22/53 of the differentially-expressed genes regulate or are regulated by IFN-alpha (IPA score=52, p≤.01). Quantitative RT-PCR on the main differentially expressed genes (GBP1, CXCL9, Serping1, C1QA, SLC1A7) confirmed the results obtained by microarrays. We, then, analyzed the gene expression profile of CD4+ and CD8+T lymphocytes isolated from an independent cohort of 4 CAMR and 4 control subjects. Only one gene (TNFSF10) was differentially expressed in CAMR patients in both cell population. CD4+T cells showed a specific IFN-alpha signature (p=5.06*10-11), while CD8+T were characterized by several differentially regulated pathways (Tryacylglicerol biosynthesis p=1.4*10-3; TREM1 signaling p=5.8*10-2; Toll like receptor signaling p=5.2*10-2), but IFN-alpha signaling. Immunofluorescence analysis of graft biopsies demonstrated a statistically significant increased expression of MXA, an IFN-alpha-dependent protein, in CAMR patients (CAMR 1471.6±28.9 pixels vs control 689±44 pixels, p=.02). Our data suggest a key role for IFN-alpha in modulating CD4+T cell response during CAMR. This observation may open new perspectives for early non-invasive diagnosis of CAMR.