Elevated TIM3 expression on bone marrow T cells drives immune dysfunction in early relapsed blood cancer after allogeneic hematopoietic stem cell transplantation

Abstract

Abstract Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a curative option for hematologic malignancies, primarily through the graft-versus-leukemia effect. However, approximately one-third of patients relapse following transplantation, with particularly poor outcomes observed in early relapse (ER) cases. Currently, there are no established therapies that effectively reverse T cell exhaustion in these patients. Here, we investigated the differentiation stage and inhibitory receptor expression of marrow-infiltrating T cells in early relapsed patients after allogeneic HSCT. Method: We conducted a retrospective analysis on 74 patients who received allogeneic hematopoietic stem cell transplantation at Chungnam National University Hospital (Daejeon, South Korea) between April 2021 and December 2023. Early Relapse (ER) was defined as relapse within six months following allogeneic hematopoietic stem cell transplantation. Using CD45RA, CD95, and CCR7, we employed flow cytometry to divide T cells into five subtypes: naïve, effector memory (EM), central memory (CM), stem cell memory (SCM), and effector memory with positive CD45RA (EMRA). Alternatively, we defined regulatory T cells (Treg) as CD25highCD127low and used CD25 and CD45RA to categorize them into three groups: naïve, active, and non-suppressive Treg. And 5 inhibitory receptors (PD1. CTLA4, TIM3, LAG3, TIGIT) were also analyzed by flow cytometry. Additionally, we measured the inflammatory cytokines (IL-6, TNF-α, IFN-γ) level by ELISA. And we performed single cell RNA sequencing analysis on bone marrow samples from 2 CR and 2 ER patients. And we performed cytotoxicity assay with BM T cell from the patients using sabatolimab (TIM3 blockade). Results: Flow cytometry revealed a marked reduction in CD3+ and CD8+T cells in ER patients compared to those in complete remission (CR), while CD4+T cell levels remained similarly low in both groups. Notably, the proportion of DNT cells was significantly increased in ER patients. Among the inhibitory receptors (IRs) analyzed—TIM3, TIGIT, CTLA4, PD1, and LAG3—TIM3 showed the highest expression in CD3+T cells, with elevated levels observed specifically in the DNT subset of ER patients. To further characterize the heterogeneity of DNT cells, including TCRαβ, TCRγδ, and natural killer T (NKT) cell features, we performed additional immunophenotyping. TIM3 expression was significantly higher in both DNT-TCRαβ and DNT-TCRγδ subsets in the ER patients. Given that DNT-medicated cytotoxicity relies on DNAM1 in leukemic cells, we found that DNAM1 was reduced in DNT-TCRγδ subset in the ER patients. While overall Treg frequencies did not differ between the two groups (Figure 1K), TIM3 expression was elevated in total and active Tregs in ER patients (Figure 1L–M). Transcriptomic analysis revealed downregulation of cytotoxic granules (PRF1, GZMA, GZMB) and effector genes (CD226, TRDC) in DNT cells from ER patients. Among these, CD226 (also known as DNAM1) plays a key role in mediating cytotoxicity in DNT cells, while TRDC contributes to the cytotoxic function of γδ+DNT cells via the Fas/FasL signaling pathway. Furthermore, IKZF2 expression was lower in the ER group than in the CR group, and IKZF2 is also associated with the cytotoxic function of DNT cells. A cytotoxicity assay using sabatolimab (anti-TIM3 antibody) and BM-derived T cells from ER patients showed significantly enhanced apoptosis of THP-1 leukemia cells, indicating restored T cell cytotoxic function. Conclusion: our data reveal that early post-transplant relapse is associated with TIM3-mediated immune dysfunction in both DNT and Treg populations. Elevated TIM3 expression contributes to impaired T cell cytotoxicity and increased immunosuppressive tumor microenvironment. Importantly, the TIM3 blockade restored cytotoxic function in vitro, suggesting a promising therapeutic approach for patients experiencing early relapse after allo-HSCT.