ADGRG1/GPR56 As a Marker of CD8+ Tumor-Reactive T Cells in Acute Myeloid Leukemia

Key words: AML; tumor-reactive T cell; ADGRG1

Objective: Given the promising clinical efficacy of T cell-based immunotherapy in solid tumors, there is a growing interest in harnessing T cells for treating acute myeloid leukemia (AML). However, the characteristics of tumor-reactive T cells and their unique molecular markers remain unclear. In this study, we aim to explore the specific marker of AML tumor-reactive T cells.

Methods: Bone marrow (BM) T cells from newly diagnosed AML patients were collected for scRNA-seq and scTCR-seq. These data were integrated with publicly available healthy donors' single-cell data. Using the STARTRAC-like algorithm, tumor-reactive T cells were identified based on the tumor enrichment index, clonal expansion index, proliferation index, and activation of TCR signaling pathways. Further validation was conducted on experimental approaches like CD33 CAR-T and target cell co-culture models and IFN-γ release assays. RNA-seq data from 42 AML patients were used for further validation by deconvolution.

Results:

By integrating single-cell data from healthy donors and AML patients, T cells were clustered with specific markers and we focused on CD8⁺ T cells for further analysis. Using the STARTRAC-like algorithm, these subgroups were categorized into tumor-reactive T cells and bystander T cells. Unlike solid tumors, tumor-reactive T cells in AML were predominantly terminally differentiated effector T cells and did not show significant upregulation of exhaustion markers like PD-1. Instead, they exhibited a senescent-like cytotoxic profile (CD27^-CD28^-KLRG1⁺) with upregulation of NK cell-associated molecules. Notably, ADGRG1 was significantly elevated and almost specifically expressed in tumor-reactive T cells compared to bystander T cells. In a conditional knock-in leukemia mouse model, high expression of ADGRG1 was also observed in CD8⁺ tumor-reactive T cells at the single-cell level. This suggested that ADGRG1 may serve as a unique AML CD8⁺ tumor-reactive T cell marker.

To further validate this hypothesis, anti-CD33 CAR-T and Molm13 were applied as an artificial effector-target cell model. When stimulated by CD3/CD28 Dynabeads or cocultured with K562 (CD33-), neither CAR-T nor vector T cells showed significant ADGRG1 expression. However, cocultured with Molm13 (CD33⁺), the ADGRG1 was significantly upregulated on anti-CD33 CAR-T cells (p = 0.016), suggesting the TCR-antigen interaction induces the evident ADGRG1 upregulation. Then ADGRG1^+/-CD8⁺ T cells were isolated from the BM samples of newly diagnosed AML patients from various subtypes and co-cultured with the matched patient's BM CD34⁺ leukemia blast cell. It was observed that ADGRG1⁺CD8⁺ T cells secreted significantly higher levels of IFN-γ upon co-culture 48h with blasts, demonstrating the tumor reactivity of ADGRG1⁺CD8⁺ T cells. The characteristic of ADGRG1 as a marker for tumor-reactive T cells might be a common feature of tumor-reactive T cells in AML.

We also conducted a preliminary exploration into the clinical significance of the ADGRG1⁺CD8⁺ T cell subset. By deconvolution based on the single-cell landscape of AML patients, relapsed/refractory patients had lower levels of ADGRG1⁺CD8⁺ T cells in their BM (p = 0.004), suggesting that a lower proportion of these cells at the initial diagnosis might indicate a poor prognosis.

Conclusions: By drawing on experiences from solid tumor research, we identified tumor-reactive T cells with unique phenotypes in AML and detected ADGRG1 as their marker. Our findings provide a simpler, more feasible approach for future tumor-reactive TCR screening.