Introduction
Efforts to enhance T cell-mediated acute myeloid leukemia (AML) eradication face challenges possibly due to unique mechanisms of leukemia-induced T-cell dysfunction compared to exhaustion observed in solid tumors(Penter et al., Blood 2023). We previously showed the efficacy of Wilms' Tumor (WT) Antigen 1-specific T-cell receptor (TCR C4) inserted into CD8 + T cells (T TCR-C4) in preventing AML recurrence after hematopoietic cell transplantation (HCT) (Chapuis et al., Nat Med 2019). This led us to investigate the safety and efficacy of T TCR-C4 in post-HCT relapsed patients.
Methods
In this Phase I/II study conducted at Fred Hutchinson Cancer Center, 15 patients were enrolled and prospectively monitored for disease response. We manufactured T TCR-C4 using a lentiviral transduction system (Chapuis et al, Nat Med 2019) and performed comprehensive analyses of bone marrow (BM) and peripheral blood (PB) samples using single-cell RNA sequencing (scRNAseq) and TCRseq.
Results
T TCR-C4 treatment showed no survival advantage compared to matched controls (n =17). We hypothesized that active AML exacerbates T-cell dysfunction compared to patients who received prophylactic T TCR-C4 treatment.
Identifying tumor-specific T cells poses a challenge in scRNAseq. By utilizing the WT1-transgene (WT1tg) sequence, we built a custom reference genome to dissect the transcriptional state of leukemia-specific cells. In PB scRNAseq profiles of 4 patients (24,472 CD8 + T cells) with post-infusion persistent blasts and T TCR-C4, we identified 5 main CD8 + T-cell signatures: Naïve, Stem-like, Activated-Exhausted, WT1tg positive, and NK-like (Figure 1). The NK-like signature has been associated with dysfunction in CAR-T cells (Good et al., Cell 2021). T TCR-C4 cells exhibited an NK-like skewing, although the expression of these markers was not as high as in endogenous NK-like CD8 + T cells. Trajectory inference revealed that NK-like endogenous CD8 + T cells represented the terminal state of differentiation, while T TCR-C4, despite expressing some NK-like markers, were in an intermediate state of differentiation along the trajectory (Figure 2). Combining scTCRseq data with proliferation signatures (Zheng et al., Science 2021) we found that WT1tg exhibited clonal expansion and proliferation, suggesting a functional intermediate state. We validated the 5 CD8 + T-cell differentiation states in BM samples (7,719 CD8 + T cells) from our cohort and in a publicly available large-scale BM CD8 + T-cell scRNAseq dataset (68,545 CD8+ T cells), with NK-like as the last differentiation state along the trajectory.
Differential gene expression (DGE) of CD8 + T cells between active AML (AML+) and blasts-not-detectable (AML-) timepoints revealed reduced expression of stem-like and increased expression of NK-like markers in AML+ vs. AML- suggesting that leukemia drives NK-like terminal differentiation in T TCR-C4. To investigate if the NK-like derangement is AML-specific or a general dysfunction mechanism, we performed DGE between T TCR-C4 vs. TCR-T cells mesothelin-specific (T MSLN). The analysis revealed overexpression of exhaustion markers (CTLA4, PDCD1, TOX, CXCR6, NR4A2) in T MSLN and NK-like markers (NKG7, KLRD1, KLRG1, S1PR5) in T TCR-C4.
To explore leukemia immune-escape mechanisms, we studied blasts gene expression. One patient who initially responded to T TCR-C4, exhibited monocytic blast differentiation upon relapse. We found overexpression of immune suppression genes (IL10, TNF, reactive oxygen species) in monocytic blasts at relapse vs. blasts before T TCR-C4 infusion. Published AML scRNAseq confirmed NK-like skewing in monocytic leukemia patients. Ongoing in-vitro assays will help to dissect the mechanisms of T TCR-C4 dysfunction.
Discussion
Our study reveals a novel AML-specific NK-like signature of T-cell dysfunction, exacerbated when AML blasts are present, potentially contributing to the restricted efficacy observed in TCR-T cell therapies. We observed that monocytic blast differentiation and the subsequent formation of an immune-suppressive environment imply the existence of intricate immune escape mechanisms. This study provides a critical foundation for the development of innovative interventions aimed at circumventing the dysfunction observed in cell-based therapies and fostering long-term remission in AML patients.
Disclosures
Wu:BioNTech Inc: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Greenberg:Juno Therapeutics: Patents & Royalties, Research Funding. Chapuis:Juno Therapeutics: Research Funding.
