ZFP36L2 as a promising therapeutic target in acute graft-versus-host disease: Evidence from murine models and clinical validation

Abstract Introduction Acute graft-versus-host disease (aGVHD) remains a major cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dysfunctional regulatory T cells (Tregs) are implicated in aGVHD pathogenesis, but molecular mechanisms remain unclear. Emerging evidence suggests post-transcriptional regulation may play a pivotal role in Treg mediated immunosuppression. Here, we employed an approach combining single-cell transcriptomics (scRNA-seq) of patient CD3+ T cells with a validated aGVHD murine model to identify zinc finger protein 36-like 2 (ZFP36L2), an RNA-binding protein regulating mRNA stability, as a novel molecular checkpoint in aGVHD progression. Methods Peripheral blood CD3+ T cells were isolated by magnetic bead sorting from 6 patients received allo-HSCT (3 aGVHD vs. 3 controls). The CD3+ T cells underwent scRNA-seq (Singleron GEXSCOPE®). Differentially expressed genes (DEGs) in Tregs were cross-validated with Gene Expression Omnibus (GEO) datasets (GSE17922: genes downregulated in aGVHD, GSE36832: genes rescued post-therapy) to identify hub genes. For validation, murine aGVHD model was established using female BALB/c (H-2d) as recipients, which received 6.5 Gy total body irradiation (TBI) and infused with bone marrow (1×10⁷) and splenocytes (5×10⁶) of male C57BL/6 (H-2b) as donors. Murine aGVHD model were divided into healthy control group, TBI-only group, non-aGVHD group and aGVHD group, 5 mice in each group and 3 independent replicates. ZFP36L2 expression in target organs (liver, spleen, colon, small intestine) were assessed by qPCR, immunohistochemistry (IHC), and Western blot. Results Peripheral blood CD3+ T cells were clustered into 9 subsets (including CD8+ Effector T Cells, CD8+ Effector Memory T Cells, CD8+ Exhausted T Cells, Gamma Delta T Cells, Natural Killer Cells, Naive T Cells, Proliferating T Cells and Tregs). ScRNA-seq identified higher expression of proliferation-related genes (MKI67, GAPDH, PCLAF) in Treg cells and resulted in Tregs significantly expanded in aGVHD patients (P=0.025). Cross-analysis with GEO datasets (GSE17922/GSE36832) identified ZFP36L2 as a downregulated gene in aGVHD patients' Tregs. ZFP36L2 was significantly downregulated in aGVHD patients' PBMCs (17 aGVHD vs. 17 non-aGVHD, P=0.03) from clinically validation. In murine models, ZFP36L2 mRNA and protein were downregulated in aGVHD target organs (liver, colon, small intestine; P<0.05) compared with other 3 groups. The reduction of p-AKT/AKT ratio is consistent with the downregulation of ZFP36L2. To evaluate the impact of phosphorylation on ZFP36L2, the aGVHD group received therapeutic intervention: rapamycin (1 mg/kg/day) or vehicle with the same dose of PBS by intraperitoneal injection from d0~d15. IHC and qPCR revealed that rapamycin upregulated ZFP36L2 expression（P<0.05）and mitigated target organ damages. While heterogeneity of ZFP36L2 level increasement exists among different organs, which was more prominent in cutaneous, splenic, and small intestinal tissues compared to hepatic and colonic tissues. Concurrently, rapamycin increased splenic Treg proportions (2.59±0.23% vs. 1.22±0.07%, P=0.03) and Foxp3+ expression in tissue-derived single-cell suspensions from liver, spleen, and small intestinal from flow cytometry analysis. Conclusions We identify ZFP36L2 as a critical downregulated target in aGVHD Tregs via scRNA-seq, aGVHD murine models and clinical validation. Restoring ZFP36L2 experssion by rapamycin could recalibrate Treg-mediated immunosuppression in aGVHD. However, the precise molecular mechanisms underlying ZFP36L2-mediated immunoregulation require further investigation.