Single-cell immune analysis of chronic GVHD after hematopoietic stem cell transplantation

Abstract

Patients with life-threatening hematologic malignancies often receive hematopoietic stem cell transplants as a treatment of last resort. Unfortunately, allogeneic transplant patients regularly develop graft-versus-host disease (GVHD) when donor immune cells react to the recipient’s tissues. Chronic GVHD (cGVHD) in particular typically occurs after day 100 post-transplantation as patients are gradually weaned off immunosuppressive medication. Despite a recent clinical trial that showed costimulation blockade reduced the incidence of acute GVHD, cGVHD remains a serious complication and a significant cause of morbidity and non-relapse mortality after allogeneic hematopoietic stem cell transplants. The precise biological mechanisms underlying cGVHD remain unclear. To better understand the biology of cGVHD, we utilized a recent phase II trial that enrolled patients receiving unrelated donor hematopoietic stem cell transplants, including 8/8 HLA-matched transplant patients. Importantly, we analyzed placebo group patients who received standard post-operative care and were not administered steroids, which minimizes potential effects from confounding variables. We divided our patients into two populations: “operationally tolerant” patients who did not develop grade 2-4 acute GVHD or moderate-severe cGVHD, and “de novo cGVHD” patients who developed moderate-severe cGVHD without antecedent grade 2-4 acute GVHD. Based on longitudinal flow cytometry data, we identified a higher frequency and absolute number of naïve CD4+ T cells in patients who later develop cGVHD. This increase in naïve CD4+ T cells occurred before clinical manifestation of cGVHD and persisted over several time points. To better understand the mechanisms of cGVHD and immune tolerance, using next generation sequencing, we additionally analyzed surface protein and gene expression of patient CD4+ T cells at 100 days post-transplantation. We discovered that the expression of interferon response genes such as IFI6, ISG15, MX1, STAT1, and MT2A correlated with a future disease state while DUSP2 expression was associated with the establishment of immunological tolerance. This work hopes to elucidate the role of immune cells in initiating cGVHD pathogenesis. Additionally, these findings posit targetable mechanisms for developing prophylactic interventions and may provide new insights into T cell-mediated autoimmunity.