Multiomic Analyses Uncover Immune Signatures of Steroid-Refractory Graft-Versus-Host Disease

Acute graft-versus-host disease (aGvHD) is the main cause of non-relapse mortality in the early stage after allogeneic hematopoietic cell transplantation (alloHCT). 40-60% of aGvHD cases were identified as steroid refractory (SR)-aGvHD, which has a fulminant prognosis. However, the underlying associations between immune reconstitution and SR-aGvHD remain unclear. To address this knowledge gap, samples of steroid-naïve aGvHD and non-aGvHD patients were analyzed with multi-omics approaches. Peripheral blood mononucleated cells (PBMCs) isolated from 26 non-aGvHD, 17 steroid-sensitive (SS)-aGvHD and 19 SR-aGvHD patients at d16 (one week before aGvHD fully brown) were analyzed by mass cytometry (CyTOF). The proportions of CD28^high CD8⁺ effector memory T cells (Tem), Th1-like CD4⁺ memory T cells, and CD56^high NK cells were significantly expanded in SR-aGvHD samples. CD28^high CD8⁺ Tem cells from SR-aGvHD samples showed higher expressions of ICOS, HLA-DR, IL2RB and IL27RA. However, the proportions of monocyticdendritic cells (DCs), plasmacytoid DCs, CD15^- monocytes and CCR4⁺ monocytes were decreased. Inflammatory factors related to T-cell activation (such as IL-2, IL-27, IL15RA), co-stimulation (CD40), and chemotaxis were elevated in the plasma of SR-aGvHD patients. Taken the increased proportions of T-cell subsets and elevated levels of T-cell related inflammatory factors of SR-aGvHD, we further characterized the functional status of peripheral CD3⁺ T cells by single-cell RNA sequencing. The GZMK⁺ CD8⁺ Tem cells were characterized by co-stimulation (CD27), expansion (MKI67, MCM5) and inflammation (HMGB1, HMGB2) from SR-aGvHD samples. GZMK⁺ CD8⁺ Tem cells of SR-aGvHD samples received increased amounts of interactions between HLA-I molecules and CD8A/B, which reflected an immune active status. The results were validated with external cohort. PBMCs of 144 alloHCT recipients were analyzed by flow-cytometric analysis at a median time of 16 days. The absolute number of peripheral CD28^high CD8⁺ Tem cells were significantly higher in SR-aGvHD group, compared with non-aGvHD and SS-aGvHD groups (90.8/ul vs. 19.7/ul and 7.2/ul, P ＜0.001). The area under ROC curve of 0.914 identified 11.078/ul of peripheral CD28^high CD8⁺ Tem as the optimal cutoff value predicting SR-aGvHD with 100% sensitivity and 82.7% specificity.

Functional defect of glucocorticoid receptor (GR) pathway is one of the major mechanisms for glucocorticoid resistance. We observed the decreased activity score of GR pathway in GZMK⁺ CD8⁺ Tem cells from SR-aGvHD samples. Among the genes of GR pathway, we found that NR3C1 gene, which encoding GR, was lower expressed in CD8⁺ Tem cells from SR-aGvHD samples. We investigated the expression of GR in PBMCs by flow-cytometric analysis. CD8⁺ Tem cells isolated from SR-aGvHD patients showed a significant lower expression of GR compared with CD8^- cells from SR-aGvHD patients (median fluorescence intensity [MFI]: 1214 vs. 3020, P ＜0.001), CD8⁺ Tem cells from non-aGvHD and SS-aGvHD patients (MFI: 1214 vs. 3092 and 2292, P = 0.001). To characterize the mechanism underlying GR reduction, we analyzed the overlap of SR-associated differentially expressed genes in the comparison of SR-aGvHD with SS-aGvHD and non-aGvHD. A total of 297 common upregulated and downregulated genes showed the enrichment of immune regulation and cytokine signaling pathways. Cytokine receptors, including IL27RA, IL15RA, and IL2RB and co-stimulation molecules including TNFRSF9 and CD27 were increased in CD8⁺ Tem cells of SR-aGvHD samples. The protein kinase profilers demonstrated that STAT1, STAT3, PYK2, and AKT1/2/3 were significantly phosphorylated in CD8⁺ T cells from SR-aGvHD samples. The activities of STAT1, JUNB and FOSB regulons which regulate GR expression were significantly enriched in steroid-refractory CD8⁺ Tem cells. Furthermore, the higher phosphorylation of STAT1 was confirmed in SR-aGvHD samples by flow-cytometric analysis and western-blot. Primary CD8⁺ T cells treated with IL-2, IL-15 or IL-27 showed resistant to dexamethasone-induced apoptotic effects. For the first time, our data showed that cytokine pathways of IL-2, IL-15 or IL-27 promoted overactivation of CD28^high CD8⁺ Tem cells, and the following phosphorylation of STAT1 reduced GR expression contributed to SR. Overactivated STAT1 in CD8⁺ Tem in the peri-engraftment phase might be a reliable indicator for SR-aGvHD.

No relevant conflicts of interest to declare.