Immunosuppressive T-cell landscape in myelodysplastic Neoplasms with TP53 mutations

Abstract Background TP53 mutations were common genomic alterations in myelodysplastic neoplasms (MDS) and associated with adverse prognosis. While recent studies suggested that TP53 mutations may promote an immunosuppressive microenvironment in MDS and acute myeloid leukemia (AML), the role of T-cell dysregulation in the bone marrow (BM) niche of TP53-mutated MDS remain poorly characterized. Methods Fresh bone marrow were obtained from MDS patients and healthy donors. Flow cytometry was performed to examine the expression of inhibitory checkpoint receptors or stimulatory checkpoint receptors on T-cell subsets. Single-cell RNA sequencing (scRNA-seq) was conducted on magnetically sorted CD3+ T cells to assess transcriptional profiles of T-cell differentiation and exhaustion. Results Flow cytometry revealed high expressions of checkpoint receptors inT-cell subsets of MDS with TP53mutations We performed comprehensive flow cytometry analyses of bone marrow samples from 108 MDS patients (including 92 with NGS data) and 15 healthy donors. Our analysis demonstrated that neither bone marrow blast percentages nor IPSS-R classification correlated with immune checkpoint expression on CD8+ T cells. However, when stratifying patients by TP53 mutational status, we observed significantly higher percentages of CTLA4+, PD-1+, TIM-3+, LAG3+, and TIGIT+ cells within CD8+ T-cell populations in TP53-mutated MDS patients compared to TP53 wild-type cases or healthy donors. Notably, this checkpoint upregulation pattern was exclusive to TP53 mutations, as no significant associations were observed with other commonly mutated genes in MDS, including TET2, ASXL1 and DNMT3A. Furthermore, parallel alterations were observed in the CD4+ T-cell compartment of TP53-mutated cases. MDS patients with TP53 mutations exhibited markedly elevated frequencies of checkpoint-positive CD4+ T cells compared to wild-type counterparts or healthy donors, including CTLA4+, TIM-3+, and LAG3+ populations. Additionally, the expressions of co-stimulatory molecules including ICOS and 4-1BB on Treg cells were substantially increased in TP53-mutated MDS. ScRNA-seq revealed T-cell differentiation abnormalities and exhaustion signatures inMDS with TP53mutations To gain deeper insight into T-cell landscape within the bone marrow microenvironment, we performed scRNA-seq on isolated CD3+ T cells from 3 healthy donors, 6 TP53 wild-type MDS, and 6 TP53-mutated MDS patients (all included were MDS-BiTP53). Following rigorous quality control, transcriptional profiles of 41,730 CD8+ and 37,256 CD4+ T cells were analyzed. At the transcriptional level, both CD8+ and CD4+ T-cell populations in TP53-mutated MDS demonstrated significantly elevated expression of multiple inhibitory receptors, including CTLA4, PD-1, TIM3, LAG3, and TIGIT. Also, Tregs in TP53-mutated MDS possessed enhanced expression of ICOS and 4-1BB. These findings corroborated our flow cytometry results at the protein level. Further subcluster analysis of T cells revealed a striking reduction in naive CD8+ and CD4+ T-cell frequencies in TP53-mutated cases compared to wild-type controls, suggesting impaired T-cell differentiation potential. Notably, we identified a distinct population of PD-1hi CD8+ memory T cells exhibiting exhausted phenotypes that was significantly expanded in TP53-mutated cases. Similarly, the percentages of PD-1hi CD4+ effector T cells with suppressive markers were obviously elevated in TP53-mutated patients. Moreover, an increased frequency of Treg cells in TP53-mutated MDS was observed, suggesting heightened regulatory activity. Conclusion Our study revealed that TP53-mutated MDS exhibited profound T-cell dysfunction within the bone marrow microenvironment, including impaired naive T-cell population, expansion of exhausted T-cell subset with elevated checkpoint molecule expression, and enrichment of functionally suppressive regulatory T cells. Our findings collectively suggested that immunomodulatory therapeutic strategies especially targeting checkpoint receptors may be beneficial for MDS with TP53 mutations.