Background: For patients with hematologic malignancies, relapse is the primary treatment-related outcome of allogeneic hematopoietic stem cell transplantation (HSCT). The prognosis was quite poor for early relapses in particular. Furthermore, T cells that infiltrate tumors are crucial to the development and recurrence of illness. When comparing early relapsed patients to those who had complete remission (CR) earlyowing HSCT, there is a dearth of information on the marrow-infiltrating T cells in the former group. Thus, in the early phases of allogeneic HSCT, we examined the differentiation stage and expression of inhibitory receptors of the T cell invading the marrow.
Methods: The study collected 40 samples from patients at Chungnam National University Hospital between April 2021 and October 2024. T cells were classified into five subtypes using CD45RA, CD95, and CCR7 and regulatory T cells were analyzed using CD25 and CD45RA by flow cytometry. Subtypes of T cell included: aïve, stem cell memory (SCM), central memory (CM), effector memory (EM) and effector memory with positive CD45RA (EMRA). Early relapse was defined as relapse within 6 months of allogeneic HSCT. High-throughput single-cell donor assays were performed to determine the expression and composition of markers. Unsupervised clustering idpatient 23 distinct clusters, with each re-annotated into immune cell types and subtypes.
Results: Using this method, 40 patients with a median age of 57 donors (ranging from 30 to 71) and 1donorsients with early recurrence had their marrow-infiltrating T cells examined. At 60%, AML is the most prevalent patients; MDS (20%), ALL (15%), and PMF (2.5%) are the next most donors. The sources of stem cells were matched unrelated donor (27.5%), haplo-identical donor (37.5%), and matched sibling donor (32.5%). All patients underwent transplantation with mobilized peripheral blood stem cells. Patients undergoing allogeneic hematopoietic stem cell transplantation had reduced marrow-infiltrating CD3+T cell numbers as compared to healthy individuals (CD3+T cells: 13.89% in HSCT patients and 9.116% in ER patients vs. 34.19% in healthy people, p<0.0001). Furthermore, CR patients had more CD3+CD8+T cells than ER patients did (CD3+CD8+T cells: 50.39% in CR vs. 30.41% in ER patients, p< 0.0033). While EM and EMRA were lower in HSCT patients compared to normal subjects, naïve CD4+T cells were higher in these patients (8.629% in ER patients and 5.119% in CR patients vs 45.76% in normal subjects, p< 0.0001/EM; 54.04% in ER patients and 55.93% in CR patients vs 18.61 in normal subject, p= 0.0003 and p< 0.0001, respectively). With regard to CD3+T cells, ER patients had a greater percentage and expression level of TIM-3 than CR patients (56.33% in ER patients vs 39.20% in CR patients, p= 0.0185/MFI; 1873 in ER patients vs. 826.7 in CR patients, p= 0.0331). The proportion of TIM-3 in CD4+T cells was lower in normal participants (7.794% in normal versus 30.11% in CR patients and 26.04% in ER patients, respectively) than in allogeneic HSCT patients. Within the CD8+T cell population, naïve and EM were greater in ER patients compared to CR patients (naïve: 8.680% in ER patients vs 4.066% in CR patients, p = 0.4001 / EM: 54.44% in ER patients vs 44.33% in CR patients, p = 0.3039). The expression of TIM-3 on CD8+ EMRA T cells was greater in ER patients compared to CR patients (MFI; 1441 in HSCT patients vs. 421.7 in normal, p=0.1038). In contrast, ER patients had a greater proportion of TIM3 among CD3+CD4-CD8-than CR patients (80.33 % in ER patients compared 62.49% in CR patients, p= 0.0333). Treg levels were not significantly different between complete response (CR) and early relapse (ER) patients. Single-cell sequencing data showed a higher proportion of Hematopoietic Stem Cell (HCS)-like cells in relapsed patients, with higher expression of TIM3 ligand.
Conclusion: Among IRs, TIM3 had the highest expression in all cell groups (CD4 T cells, CD8 T cells, and Treg) compared with other IRs in the early stages of allogeneic HSCT. In particular, TIM3 was highly present in the double-negative T cell group. Therefore, in the future, it is necessary to continue research on blocking the expression of TIM3 on double negative T to destroy cancer cells, helping patients prolong life.