Uncovering the Molecular Signature of Pathogenic Tissue-Infiltrating T Cells during Acute Graft-Versus-Host Disease
Authors: Tkachev Victor , Furlan Scott N , Potter E. Lake , Zheng Hengqi , Hunt Daniel J , Colonna Lucrezia , Taraseviciute Agne , Carlson Judith M , Betz Kayla , Yu Alison , Hoffman Michelle , Baldessari Audrey , Herrin Sarah , Olvera Joe , Littlewood Chad , English Christopher , Blazar Bruce R. , Roederer Mario , Kean Leslie 
Published: October 2019

Abstract
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.