Uncovering the Molecular Signature of Pathogenic Tissue-Infiltrating T Cells during Acute Graft-Versus-Host Disease

One of the major barriers to developing targeted therapies for aGVHD control is the difficulty in identifying T cell signatures specific for GVHD pathology while distinguishing these from the pathways essential for tissue-specific T cell immune reconstitution. To address this need we have interrogated the migration patterns, as well as the phenotypic and transcriptomic characteristics of allogeneic T cells infiltrating aGVHD target organs in non-human primates. To rigorously study T cell migration during aGVHD we tracked T cells labeled following in vivo infusion with fluorescently tagged anti-CD45 antibodies given to NHP transplant recipients with aGVHD on day 8 post-HCT, during active disease. Anti-CD45 antibodies with distinct fluorescent tags were given at 6 hours before necropsy (anti-CD45-AlexaFluor647) and 5 minutes before necropsy (anti-CD45-AlexaFluor488), in order to measure T cells that were in the circulation and those migrating to GVHD target tissues, based on their labeling with one or both fluorescently-tagged antibodies. These experiments identified increased migration of both allogeneic CD8 T cells (Figure 1A) and CD4 T cells (not shown) during aGVHD, with trafficking into secondary lymphoid organs as well as non-lymphoid GVHD target organs (intestine and kidney). While migration was increased during aGVHD, these T cells, which demonstrated some phenotypic similarities to CD8 T cells in the peripheral blood (Figure 1B), also adopted tissue-specific phenotypes as measured by flow cytometry (Figure 1C), including the expression of canonical markers of resident-memory T cells (CD69 + CD103 -/+ ). However, unlike the tissue-resident T cells in healthy controls during homeostasis, tissue-infiltrating T cells during aGVHD expressed multiple markers of activation, including Ki67 and Granzyme B (Figure 1D). These flow cytometric characteristics suggested that the phenotype of organ-infiltrating T cells during aGVHD included attributes of both tissue-residency and of pathogenic alloreactivity. To further identify aGVHD-specific signatures, we performed transcriptomic analysis of tissue-infiltrating T cells during aGVHD. Using an unsupervised weighted gene correlation network analysis (WGCNA) we characterized the gene sets associated with individual GVHD target organs (Figure 2). We found that tissue-infiltrating T cells during aGVHD could be characterized by divergent features: First, they maintained a core tissue localization signature, which included genes previously linked to tissue-resident T cells (e.g. RUNX3, IFNG, CXCR6 ). Importantly, however, they also acquired an aGVHD-specific transcriptional signature including expression of IL1RL1 (encoding ST2), ICOS , TNFRSF9 (CD137) and TNFRSF4 (OX40). This signature also included enrichment for transcripts encoding the cytotoxic mediators GRMB and GRMA , the proliferation markers MKI67 and AURKA , as well as cytokines and cytokine receptors ( IL18, IL18R, IL21, IL21R ). Proteins encoded by each of these transcripts have been linked to aGVHD-causing T cells, strengthening the inference that these constitute a robust transcriptomic signature of aGVHD pathogenesis. Thus, for the first time in a large-animal model, we have been able to directly measure both the kinetics and the protein and RNA expression signatures of T cells during their migration into aGVHD target organs, This study provides new evidence for the evolution of a phenotypic and transcriptomic dichotomy during aGVHD-mediated tissue infiltration, in which T cells take on both tissue- and aGVHD-specific characteristics. These data provide novel insights into the spatial organization of systemic alloimmunity during aGVHD, which should enable more precise targeting of pathogenic T cell populations while preserving normal tissue immune reconstitution after transplantation. Disclosures Tkachev: Regeneron Pharmaceuticals, Inc.: Research Funding. Blazar: Kadmon Corporation, LLC: Consultancy, Research Funding. Kean: Regeneron Pharmaceuticals, Inc.: Research Funding.