Dissecting the Cognate Event in Human Kidney Transplants With T Cell-Mediated Rejection.

Abstract

We hypothesized that the molecular changes most associated with TCMR would reveal the cognate event - T cell-APC engagement in the graft - and distinguish this from the inflammatory response (IFNG effects, innate immunity) and the injury response. We analyzed microarray data from indication kidney transplant biopsies: 403 as a discovery set and 300 as an independent validation set. In the discovery set we generated the top 300 TCMR-associated transcripts (defined by p-value) by comparing TCMR to all other biopsies, including antibody-mediated rejection, acute kidney injury, polyomavirus nephropathy, glomerulonephritis and non-specific atrophy-fibrosis; then analyzed these transcripts for overrepresentation in the curated IPA Canonical Pathways (Ingenuity Systems). The top pathways were associated with proximal signals of T cell activation - signal 1 pathways T Cell Receptor Signaling (p=2.0x10-12) and PKC-theta Signaling (p=3.0x10-10) - and signal 2 pathways iCOS-iCOSL (p=7.9x10-14), CD28 (p=2.0x10-12), and CTLA4 Signaling (p=3.0x10-10). Shown in Figure 1, the signal 2 pathways shared many transcripts (eg CD3D, LCK, ZAP70, LAT, ITK) with signal 1. Reasoning that the TCMR-associated transcripts must reflect T cell receptor triggering, we removed the transcripts used in the T Cell Receptor Signaling Pathway and reran pathway analysis - Step 2 of Figure 1. Top pathways no longer included proximal signals of T cell activation, but rather T Helper Cell Differentiation (p=4.0x10-8) and Antigen Presentation (p=9.1x10-6), which included HLA-DMA/B, -DOA, IFNG, IL12RB1, IL21R, and STAT1. Subsequent removal of these transcripts and re-analysis of the TCMR-associated transcripts left weak pathway associations representing innate immunity - Step 3 of Figure 1. All findings were confirmed in the validation set.Figure: No Caption available.The minimal model that accounts for the pathway ontology is that the cognate event in TCMR activates both the effector T cell and various APCs (dendritic cells, macrophages, and B cells), triggering antigen presentation (HLA-DMA/B, DOA/DOB) and specific cytokine crosstalk (IL21 and IL12), including IFNG. This method of stratifying pathway analysis into distinct layers could be applied to other well phenotyped diseases. DISCLOSURES:Halloran, P.: Speaker's Bureau, Astellas, Novartis, OneLamba.