Monocyte-T cells crosstalk orchestrates early graft-versus-leukemia immunity in Acute Myeloid Leukemia patients after allogeneic transplantation Free

Background Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Hematopoietic stem cell transplantation (HSCT), with haploidentical HSCT being a key approach, stands as a leading curative approach for various malignant hematologic disorders. Post-transplant early immune reconstitution critically impacts long-term survival, yet relapse remains a major prognostic challenge. Building on our previous research (Huo, et al., Sci Immunol. 2023), this study delves into the nuances of early immune reconstitution following haplo-HSCT in AML patients and graft-versus-leukemia (GVL) effects associated with early relapse (ER).

Methods Firstly, we performed single-cell RNA sequencing on serially collected fresh peripheral blood (PB) and bone marrow (BM) samples from three AML patients and four aplastic anemia (AA) patients at 14, 30, and 60 days after haploidentical HSCT. ​​We next generated single-cell transcriptomic libraries and TCR repertoires using thawed BM samples from six additional AML patients - three maintaining continuous complete remission (CCR) and three experiencing ER within 6 months post-HSCT, at 30 days, 90 days (representing the pre-relapse phase), and at the time of relapse (for the ER group only) post-HSCT. Flow cytometry was used to assess galectin-9 (Gal-9) expression on monocytes and T cell exhaustion markers in thawed BM samples at 30 and 90 days post-HSCT from CCR and ER AML patients. Also, we conducted co-culture experiments using monocytes isolated from 30-day post-HSCT BM samples (CCR vs. ER) and T cells from healthy donor PB. Finally, we evaluated the role of Gal-9 in T cell exhaustion and tested the ability of MG-T-19 to restore T cell function.

Results AML patients exhibited fundamentally distinct post-HSCT expansion kinetics compared to AA patients, characterized by sequential monocyte (day 30) and T cell (day 60) activation phases. Monocytes from AML patients at 30 days post-transplant showed elevated expression of genes related to tumor immunity and T-cell proliferation, such as TNFSF10 and JAK2, along with their corresponding transcription factors. Concurrently, early post-transplant T cells underwent rapid activation, with significant upregulation of genes associated with antigen presentation and TCR signaling. Furthermore, we found that monocyte-T cell interactions in AML patients at 30 days post-transplant were substantially stronger than in AA patients, with heightened activity in tumor progression-related pathways such as Galectin, ADGRE, and LAIR1.

We further investigated GVL mechanisms through comparative analysis of monocyte and T cell dynamics in AML patients achieving CCR versus those experiencing ER following haplo-HSCT. ER patients displayed more pronounced T-cell proliferation post-transplant than CCR patients. CCR patients developed a distinct CD8⁺ T cell subset characterized by high FCER1Gexpression at 30 days post-HSCT, which may possess high cytotoxic potential and recognize unmutated tumor antigens via TCR-mediated mechanisms (Chou C, et al. Nature. 2022), contributing to potent GVL effects. In contrast, CD8⁺ T cells in ER patients exhibited more exhausted phenotypes, with impaired signal-incoming capacity, while their monocytes remained hyperactivated and overexpressed Gal-9. Flow cytometry confirmed that monocytes from ER patients at 30 days post-transplant highly expressed Gal-9 protein, while CD8⁺ T cells exhibited greater exhaustion at both 30 and 90 days. Co-culture experiments demonstrated that ER-derived monocytes exacerbated T-cell exhaustion. Exogenous Gal-9 suppressed the reversal of CD8⁺ T cells exhaustion in a dose-dependent manner, an effect that was significantly rescued by MG-T-19.

Finally, TCR repertoire analysis revealed distinct TRBV gene usage preference between CCR and ER patients. Affinity prediction indicated that dominant TCR clones in both groups retained some affinity for AML antigenic peptides.

Conclusion Hematopoietic and early immune reconstitution differ between AML and AA post-HSCT. TCR clones from both CCR and ER AML patients retain leukemia-associated antigen reactivity, but T cells in ER group exhibit a significantly enhanced exhaustion signature. This phenomenon is likely driven by monocyte-derived Gal-9 upregulation at 30 days post-HSCT, impairing CD8⁺ T cell-mediated GVL effects and promoting relapse. Pharmacologic inhibition (MG-T-19) significantly reduces T cells exhaustion, highlighting its therapeutic potential.