The Expression of TIM3 on Marrow-Infiltrating T Cells Is Increased in Patients with Blood Cancers Who Relapse Early after Hematopoietic Stem Cell Transplantation

Background: Relapse is the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT) in patients with blood cancer. And bone marrow-infiltrating lymphocytes may play an important role in the anti-tumor effect of immune cells. However, few reports have analyzed changes in marrow-infiltrating T cells in patients with early relapse after allogeneic HSCT. 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.

Results: The age of patients from 18 to 72 years. The median patient age was 57 years. AML accounts for 42% of all cases, followed by with MDS & PMF (18%) and ALL (14%). The sources of stem cells were from HLA matched donor (66.2%), haplo-identical donor (23.8%). Marrow infiltrating CD3+T cell counts were lower in patients with ER compared to CR group (CD3 + T cells: 12.04% in ER patients vs 23.56% in CR patients, p =0.0284). In addition, CD3+CD8+T cells were also lower in ER than in CR patients (CD3+CD8+T cells: 32.51% in ER patients vs 55.21% in CR, p <0.0001) while in CD4+ T cells had no difference between the CR and ER groups. There was no significant difference in the differentiation stage of T cells between CR and ER patients. Among inhibitory receptors, we observed a very high proportion of TIM3 expression on CD3+ T cells, with a clear difference between ER and CR patients (29.97% in ER vs. 17.56% in CR, p=0.0130/MFI; 1746 in ER vs 622.2 in CR, p=0.0005). Similar results were seen when we analyzed by donor type. In the subset of CD3+T cells, we found that TIM3 is highly expressed in the ER group compared to the CR group on double-negative T cells (76.03% in ER vs 54.27% in CR, p= 0.015/MFI; 2865 in ER vs 1637 in CR, p=0.0111). In addition, TIM-3 in CD4+CD25+Treg was also highly expressed in patients with early relapse (46.71% in ER vs 24.41 % in CR, p= 0.0009/ MFI; 2658 in ER vs 826.2 in CR, p=0.0069. And there was an significant increase in IL-6 level in the ER group as compared to the CR patients (152.8 pg/ml in ER, p=0.0437/ 1.661 pg/ml in CR vs 152.8 pg/ml in ER, p=0.0090), although there was no significant increase in TNF- α or IFN- γ between two group. Bioinformatical scRNA-seq analysis result show that Tim-3 expression was uniquely high compare with the other IRs. Remarkable, this expression enriched on the CD3+ IKZF2+ CD4- CD8- double negative T cells. Consistent with our ELISA results, gene set enrichment analysis showed upregulated inflammation related signal such as PI3K-AKT-MTOR signaling (NES = 2.66), Inflammatory response (NES = 1.78) and down regulated by KRAS signaling (NES = 1.86) in ER double negative T cells.

Conclusion: In patients who relapsed early after allogeneic HSCT, TIM3 expression was higher on double negative T cell, CD8+T cell, and regulatory T cells compared to CR patients. Based on these findings, TIM3 blockade may have therapeutic benefit in patients with blood cancers who relapse early after transplantation.