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

Abstract

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the leading cause of treatment failure in patients with hematologic malignancies. To better understand the mechanisms underlying early relapse (ER), we comprehensively explored the expression of inhibitory receptors (IRs) in bone marrow (BM) T cells at differentiation stage and examined transcriptional differences. Among the evaluated IRs, TIM3 was significantly upregulated in CD3⁺T cells of patients with ER compared to patients with complete remission (CR). Notably, double-negative T (DNT) cells, a unique subset with MHC-independent cytotoxic potential, constituted a high proportion of BM T cells and expressed increased TIM3 expression in ER patients. Moreover, regulatory T cells (Tregs) showed elevated TIM3 levels, contributing to an immunosuppressive microenvironment after allo-HSCT. Transcriptomic analysis revealed downregulation of cytotoxic granules and effector genes in DNT cells from ER patients. Functional assays demonstrated that TIM3 blockade with sabatolimab restored T cell cytotoxicity, leading to enhanced leukemia cell apoptosis in ER patients. These findings highlight TIM3 as a critical regulator of T cell exhaustion and immune suppression in patients with ER and provide a rationale for the therapeutic use of TIM3 blockade in preventing and treating relapses after allo-HSCT.

Keywords: Allogeneic hematologic stem cell transplantation (HSCT); Double negative t cells (DNTs); Early relapse (ER); Inhibitor receptors; Leukemia immune microenvironment; Regulatory t cells (Treg); Sabatolimab; T cell immunoglobulin and mucin domain 3 (TIM3).

Fig. 1

T-cell subsets and the expression level of the inhibitory receptors on the BM T cells. A Proportion of CD3⁺T cells among total lymphocytes in the control (N = 9), CR (N = 55) and ER (N = 19) groups. B Percentage of CD4⁺T cells among the total CD3⁺T cells. C Percentage of CD8⁺T cells among the total CD3⁺T cells. D Percentage of DNT cells among the total CD3⁺T cells. E Percentages of IR-positive cells in total CD3⁺T cells. F Percentages of IR-positive cells in DNT cells. G Uniform manifold approximation and projection (UMAP) visualization showing expression levels of each IR on CD3⁺T cells in each group. H Proportion of IR-positive cells within the DNT-TCRαβ subset in CR (N = 15) and ER (N = 6) groups. I Proportion of IR-positive cells within the DNT-TCRɣδ subset. J Ratio of DNAM1 expresssion across CD3⁺T cell subtypes (CD4⁺T cells, CD8⁺T cells, DNT cell subtypes including DNT-TCRαβ, DNT-TCRɣδ and DNT-NKT). K Percentage of conventional (CD4⁺CD25^hiCD127^lo) Tregs among CD4⁺T cells in the control (N = 9), CR (N = 55) and ER (N = 19) groups. L Proportion of IR-positive cells among the conventional (CD4⁺CD25^hiCD127^lo) Tregs. M TIM3 expression level in Tregs subsets between CR and ER groups

Fig. 2

Transcriptional landscape of T-cell subsets and cytotoxicity assay with TIM3 blockade. A Violin plot showing expression levels of representative genes (CD4, CD8A, IKZF2, CD160, THEMIS, HAVCR2, CD226, PRF1, GZMB, GZMA) in the DNT, CD4⁺ and CD8⁺T cell subsets. B Single-sample gene set enrichment analysis (ssGSEA) comparing DNT, CD4⁺ and CD8⁺T cells. C Volcano plot illustrates differentially expressed genes between complete remission (CR) and early relapse (ER) groups, with 321 genes upregulated in CR and 315 genes upregulated in ER. D T-cell cytotoxicity assay against THP-1 cells following TIM-3 blockade using sabatolimab