Scientists Map Two Distinct T Cell Exhaustion Programs Driving AML Relapse Before and After Transplant

Acute myeloid leukemia remains one of the most stubborn enemies in hematology. Even with targeted drugs and allogeneic stem cell transplantation, most patients eventually relapse, and the five-year survival rate hovers around 30 percent. Now a research team at Peking University People’s Hospital has used single-cell RNA sequencing to expose, in unprecedented molecular detail, how leukemia cells quietly disarm the immune system both before and after transplantation — and their findings suggest that the two forms of immune failure are not the same disease at all.

The study, published in iScience, followed four AML patients through two critical stages: before transplantation, when their disease had failed to reach remission, and after transplantation, when their leukemia had relapsed with donor-derived immune cells now patrolling the bone marrow. The researchers profiled 95,426 individual cells from these patients alongside four healthy donors, capturing the complete transcriptomic landscape of the bone marrow microenvironment at both time points. What they found was a bone marrow ecosystem fundamentally reshaped by malignancy, with T cell frequencies dramatically lower than in healthy donors and leukemia-like cells dominating the cellular terrain.

Using an integrated computational strategy combining reference-based annotation, copy number variation analysis, and differentiation-state prediction, the team classified the leukemia-like cells into eight distinct subtypes, including hematopoietic stem cell-like blasts, progenitor-like blasts, granulocyte-monocyte progenitor-like blasts, and actively cycling blasts. Pseudotime analysis revealed a developmental trajectory flowing from stem cell-like blasts toward more differentiated subtypes, confirming that the leukemia maintained a cellular hierarchy mirroring normal hematopoiesis. Critically, the stem cell-like blasts carried the highest leukemic stem cell 17-gene score, a validated measure of stemness and treatment resistance, and their proportion increased after transplantation.

These stem cell-like blasts were not merely more primitive — they were more immunosuppressive. Compared with their non-stem counterparts, they upregulated the expression of macrophage migration inhibitory factor (MIF), transforming growth factor beta 1 (TGFB1), galectin-9 (LGALS9), and HLA-E, while simultaneously downregulating HLA class II molecules. This molecular cocktail is a known inducer of T cell and natural killer cell dysfunction. The researchers also noted that antigen-presentation changes after transplantation were molecule-specific rather than uniformly suppressed, with HLA-DR upregulated while HLA-DP and HLA-DQ were downregulated, suggesting a nuanced immune escape strategy rather than blanket silencing.

Turning to the T cell compartment, the team identified three clusters characterized by high expression of inhibitory receptors such as PD-1, LAG-3, and TIGIT, which they defined as IR-high T cells. Clonal expansion analysis showed that highly expanded T cell subsets concentrated mainly in the CD8-positive effector and IR-high clusters in AML patients, while limited expansion was focused in naive and memory populations in healthy donors. A pairwise transition index revealed that CD8-positive IR-high T cells had the highest transition likelihood from effector cells, confirming that these exhausted populations emerged directly from the cytotoxic effector pool. Compared with effector cells, IR-high T cells expressed lower levels of the cytotoxic molecules granzyme B and perforin but higher levels of inhibitory receptors and the transcription factors TOX and NR4A2.

The most striking discovery came when the researchers compared the transcriptional programs of self-derived exhausted T cells before transplantation with donor-derived exhausted T cells after relapse. Self-derived exhausted T cells were characterized by enrichment of NFATC1 and NFATC3 transcriptional programs together with increased TOX and EOMES expression, whereas donor-derived exhausted T cells exhibited enrichment of NFATC2-associated signatures accompanied by elevated NR4A2 expression. Gene regulatory network analysis showed that in self-derived exhausted cells, NFATC1 primarily regulated TOX, EOMES, and multiple inhibitory receptors, while in donor-derived cells, NFATC2 primarily regulated NR4A2 and various inhibitory receptors. This fundamental divergence in regulatory architecture means that the two forms of T cell exhaustion, though phenotypically similar, run on entirely different molecular engines.

Further subclustering revealed four major exhausted T cell populations: a progenitor population, an effector population, a terminally exhausted population, and an actively dividing population. Slingshot trajectory inference showed transitions flowing from the progenitor exhausted subtype toward the other three states. Each exhausted subtype carried higher TOX and EOMES expression before transplantation but higher NR4A2 after relapse. In the progenitor exhausted cells, NFATC3 mainly regulated HAVCR2, EOMES, SELL, and TCF7; in the effector exhausted cells, NFATC2 regulated effector-related genes such as TBX21, ZEB2, and GZMB; and in the terminally exhausted cells, NFATC1 regulated TOX, EOMES, CTLA4, HAVCR2, and PDCD1. These findings suggest that the progenitor exhausted population can differentiate into other exhausted subtypes through activation of different transcription factors whose downstream effects differ dramatically.

The bone marrow microenvironment itself was profoundly immunosuppressive at both disease stages. Monocytes expressed high levels of PD-L1 and interleukin-10 but low levels of the costimulatory ligand ICOSLG compared with healthy donors. Dendritic cells similarly upregulated PD-L1, PD-L2, interleukin-10, and HLA-E while downregulating HLA class II molecules. Regulatory T cells expressed elevated levels of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, TGFB1, granzyme B, perforin, and the key transcription factors FOXP3, STAT5, and NFKB1, with most of these molecules more prominently expressed after transplantation. Hematopoietic stem cell-like blasts upregulated CD274, CD48, FGL1, interleukin-10, CD47, TGFB1, LGALS9, and HLA class I molecules while downregulating the costimulatory ligand TNFSF4. Together, these cellular components constituted an immunosuppressive bone marrow niche that persisted across both pre- and post-transplantation settings.

Cell-cell communication analysis using CellChat and scMLnet revealed that leukemia-like cells interacted most strongly with CD8-positive effector and exhausted T cells among all T cell subtypes. In the pre-transplant setting, ligand-receptor pairs including HLA-E:CD8A/B, MIF:(CD74+CD44), LGALS9:PTPRC, and TGFB1:TGFBR1/2 were enriched in these interactions, and computational modeling predicted that these signaling pathways further regulated the activation of NFAT family transcription factors in the exhausted T cells. In the post-transplant setting, similar interactions were detected except that the TGFB1:TGFBR1/2 pathway was absent. Leukemia cells also communicated indirectly with exhausted T cells through intermediary populations — regulatory T cells, monocytes, and dendritic cells — modulating their immunosuppressive transcriptional programs and downstream target genes such as LCK, CD86, TNFSF13, and GRN, which in turn participated in signaling interactions associated with NFAT-related transcriptional activity in the exhausted T cells.

Among all leukemia cell subtypes, hematopoietic stem cell-like blasts had the most comprehensive ligand-receptor pairs interacting with exhausted T cells, regulatory T cells, monocytes, and dendritic cells, and their communication probabilities were relatively high at both disease stages. Survival analysis using The Cancer Genome Atlas AML dataset revealed that a gene set consisting of only three ligands — MIF, LGALS9, and TGFB1 — had the best prognostic value for overall survival, outperforming TGFB1 alone and gene sets including HLA class I molecules. The researchers conclude that targeting TOX in self-derived exhausted T cells, NR4A2 in donor-derived exhausted T cells, and the signaling molecules MIF, galectin-9, and TGF-β1 could represent a strategy for reversing T cell exhaustion and improving outcomes in AML patients, though they acknowledge that the findings are primarily computational and will require experimental validation in larger cohorts.

Subject of Research: Single-cell dissection of T cell exhaustion niches in acute myeloid leukemia

Article Title: Single-cell dissection of self-derived versus donor-derived T cell exhaustion niches in patients with acute myeloid leukemia

Article References: Yu, C.-Z., Chen, Y.-L., Zhang, X.-H., Xu, L.-P., Wang, Y., Chen, H., Chen, Y.-H., Wang, F.-R., Sun, Y.-Q., Zhang, Y.-Y., Lv, M., Mo, X.-D., Cheng, Y.-F., Liu, K.-Y., Zhao, X.-S., Sun, X.-Q., Zhao, X.-Y., Huang, X.-J., & Chang, Y.-J. (2026). Single-cell dissection of self-derived versus donor-derived T cell exhaustion niches in patients with acute myeloid leukemia. iScience, 29(11), Article 117918. https://doi.org/10.1016/j.isci.2026.117918

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117918

Keywords: acute myeloid leukemia, T cell exhaustion, single-cell sequencing, stem cell transplantation, immunotherapy, tumor microenvironment, Single-cell, dissection, self-derived, versus, donor-derived, cell