Early versus late JAK-STAT–IFN signaling distinguishes no rejection from subclinical to clinical TCMR after liver transplantation

Subclinical rejection after liver transplantation precludes immunosuppression withdrawal despite normal clinical function. To better understand the immunobiology of early subclinical rejection, we performed longitudinal, multimodal immune profiling and single-cell RNA sequencing (scRNA-seq) in adult living-donor recipients ( n  = 13) participating in an interventional immunosuppression withdrawal trial. Samples from patients with subclinical rejection (termed “nonpermissive”) at 12 months posttransplant exhibited distinct immune trajectories from those with quiescent (“permissive”) allografts despite comparable baseline profiles. Central to these distinct dynamics was biphasic Janus kinase (JAK)–signal transducer and activator of transcription (STAT)–interferon signaling. Greater interferon-stimulated gene (ISG) expression and JAK-STAT activation occurred postreperfusion in permissive allograft recipients, which reversed at 12 months, when elevated ISG expression and JAK-STAT signaling were evident in nonpermissive recipients exhibiting subclinical rejection. We then leveraged our findings to ascertain whether a comparable signature existed in internal and external bulk RNA-seq and scRNA-seq liver transplant cohorts. Across cohorts, including a rodent model, there was a similar elevation in JAK-STAT signaling in instances of allograft rejection. We also detected distinct, portal-based phosphorylated STAT1 staining in a preliminary analysis of biopsies exhibiting histologic rejection compared with nonrejecting controls. Moreover, ruxolitinib-mediated JAK inhibition suppressed alloreactive CD8 ⁺ T cell proliferation and inflammatory-mediator production in vitro. Together, these exploratory findings suggest a temporal, biphasic role for JAK-STAT signaling in the regulation and occurrence of T cell–mediated rejection after liver transplant and highlight JAK inhibition as a potential therapeutic strategy.