No abstract

This study sought to identify novel CD8 + T cell homing markers by studying acute graft versus host disease (aGvHD), typically involving increased T cell homing to the skin and gut. FACS-sorted skin-homing (CD8β + /CLA + ), gut-homing (CD8β + /integrinβ7 + ), and reference (CD8β + /CLA -/integrinβ7 -) T cells were compared in patients affected by cutaneous and/or gastrointestinal aGVHD. Microarray analysis, qPCR, and flow cytometry revealed increased expression of peptidase inhibitor 16 (PI16) in skin-homing CD8 + T cells. Robust association of PI16 with skin homing was confirmed in all types of aGvHD and in healthy controls, too. PI16 was not observed on CLA + leukocytes other than T cells. Induction of PI16 expression on skin-homing T cells occurred independently of vitamin D3. Among skin-homing T cells, PI16 expression was most pronounced in memory-like CD45RO + /CD127 + /CD25 + /CD69 − /granzyme B − cells. PI16 was confined to the plasma membrane, was GPI-anchored, and was lost upon restimulation of memory CD8 + T cells. Loss of PI16 occurred by downregulation of PI16 transcription, and not by Phospholipase C (PLC)-or Angiotensin-converting enzyme (ACE)-mediated shedding, or by protein recycling. Inhibitor screening and pull-down experiments confirmed that PI16 inhibits cathepsin K, but may not bind to other skin proteases. These data link PI16 to skin-homing CD8 + T cells, and raise the possibility that PI16 may regulate cutaneous cathepsin K.Keywords: CD8 + T cell r GvHD r Homing r PI16 r Skin Additional supporting information may be found online in the Supporting Information section at the end of the article.

Accurate risk prediction of acute graft versus host disease (aGvHD) is currently an unmet clinical need. This study sought to analyze whether three plasma proteins expressed in a largely skin- and gut-restricted manner would be affected by the development of acute cutaneous and gastrointestinal aGvHD. The diagnostic sensitivity, specificity, and prognostic value of plasma cytokeratin-15 (KRT15) cytokeratin-20 (KRT20), and occludin (OCLN) were evaluated in a discovery and a validation cohort using ELISA in comparison with elafin (PI3) and regenerating family member 3 alpha (REG3A), two established markers of skin- and gut aGvHD. The discovery cohort (n = 39) revealed that at the time of diagnosis, plasma KRT20 showed a progressive decrease from unaffected individuals to patients with single-, and patients with multi-organ aGvHD. KRT20 was affected by cutaneous (p = 0.0263) and gastrointestinal aGvHD (p = 0.0242) independently and in an additive manner. Sensitivity and specificity of KRT20 for aGvHD involving both target organs (AUC = 0.852) were comparable to that of PI3 for skin-aGvHD (AUC = 0.708) or that of REG3A for gut-aGvHD (AUC = 0.855). Patient follow-up in the validation cohort (n = 67) corroborated these observations (p < 0.001), and linked low KRT20 to grade 2+ disease (p < 0.001), but failed to confirm low KRT20 as an independent risk factor. These data established a link between low plasma KRT20 levels and moderate to severe aGvHD involving multiple target organs.

T-cell receptor (TCR)-transgenic models of acute graft versus host disease (aGvHD) offer a straightforward and highly controlled approach to studying mechanisms and consequences of T-cell activation following allogeneic hematopoietic stem cell transplantation (aHSCT).Here we report that aHSCT involving OT-I mice as donors, carrying an ovalbumin-specific CD8+ TCR, and ActmOVA mice as recipients, expressing membrane-bound ovalbumin driven by the -actin promoter, induces lethal aGvHD in a CD8+ T cell-dependent, highly reproducible manner, within 4-7 days. Tracking of UBC-GFP/OT-I graft CD8+ T-cells disclosed heavy infiltration of the gastrointestinal tract, liver and lungs at the onset of the disease, and histology confirmed hallmark features of gastrointestinal aGVHD, hepatic aGvHD, and aGvHDassociated lymphocytic bronchitis in infiltrated organs. However, T-cell infiltration was virtually absent in the skin, a key target organ of human aGvHD, and histology confirmed absence of cutaneous aGVHD, as well. We show that the model allows studying CD8+ T-cell responses in situ, as selective recovery of graft CD45.1/OT-I CD8+ T-cells from target organs is simple and feasible by automated tissue dissociation and subsequent cell sorting. Assessment of interferon-gamma production by flow cytometry, granzyme-B release by ELISA, TREC assay and whole genome gene expression profiling confirmed that isolated graft CD8+ T-cells remained intact, underwent clonal expansion and exerted effector functions in all affected tissues.Taken together, these data demonstrate that the OT-I/Act-mOVA model is suitable to studying CD8+ T cellmediated effector mechanisms in a disease closely resembling fatal human gastrointestinal and hepatic aGVHD that may develop after aHSCT using HLA-matched unrelated donors.