Zengkai Pan, MD, PhD1*, Yujun Deng, M.S.2*, Jingtao Huang3*, Aijie Huang, MD4*, Xiong Ni, MD, PhD5*, Lei Gao, MD, PhD6*, Chuanhe Jiang, MD, PhD7*, Luxiang Wang, MD, PhD7*, Zilu Zhang, MD, PhD7*, Quan Gu, PhD8*, Hui Cheng9*, Jianmin Wang, MD, PhD10, Hongbo Hu, PhD11* and Xiaoxia Hu7*
1Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital，Shanghai Jiao Tong University School of Medicine, Shanghai, China
2Center for Hematology and Immunology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Collaborative Innovation Center for Biotherapy, Chengdu, CHN
3Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, Nati, Shanghai, China
4The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Shanghai, China
5Department of Hematology, Institute of Hematology, Changhai Hospital, Shanghai, CHN
6Department of Hematology, Institute of Hematology, Changhai Hospital, Shanghai, China
7Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
8Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, tianjin, China
9State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences &Peking Union Medical College, Tianjin, China
10Changhai Hospital, Shanghai, China
11Center For Hematology and Immunology, State Key Laboratory of Biotherapy, West C, Chengdu, CHN
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 CD28high 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.
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