P1242: a truely leukemic subpopulation identified by single cell sequencing in t large granular lymphocytic leukemia

Topic: 20. Lymphoma Biology & Translational Research Background: T-cell large granular lymphocytic leukemia (T-LGLL) is a chronic lymphoproliferative disease of mature T cells, characterized by monoclonal T-cell large granular lymphocytosis, often clinically manifest as bone marrow failure and accompany with autoimmune symptoms. Immunosuppressive therapies are effective, but the dominant clone may persist even in responders. The pathogenesis of T-LGLL has not been fully elucidated. In this study, single-cell RNA sequencing and V(D)J profiling were performed to discern clonotypes and gene expression patterns of T lymphocytes from T-LGLL patients. Aims: To identify the biomarkers of leukemic large granular lymphocytes (LGLs) and explore novel agent for treating T-LGLL. Methods: Peripheral blood samples were collected from five T-LGLL patients, three T-cell clones of uncertain significance (T-CUS) patients and three healthy donors. single-cell RNA library was performed on the 10x Genomics Chromium Single Cell V(D)J and 5’ Gene Expression platform, and sequencing obtained on the Illumina Platform. Barcode assignment, alignment, unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 6.1.0. Immune cells were clustered and annotated, then transcriptome characteristics of each group of cells were analyzed, T cell receptor (TCR) clonetype and clonesize were identified. Results: By analyzing the characteristics of transcriptome and immune library of CD8 T cells from all samples, terminal effector T cells were divided into three clusters (which named CD8 TE1, CD8 TE2 and CD8 TE3). Almost all of TE3 cluster, which characterized by decreased clonetype diversity, were from T-LGLL patients. To identify whether TE3 cluster was pathogenic, we mapped the transcriptome on previous sequencing of T-LGLLs (Nat commun(2022)13:1981 and Nat commun (2022)13:1982) and verified TE3 cluster was the leukemic subpopulation. Compared with non-leukemic effector T cells, this leukemic subpopulation demonstrated higher toxicity and increased proliferative function. Interestingly, upregulated exhausted genes were also observed in this cluster. Furthermore, cell killing, TCR activation, interferon γ signaling pathway and cytokine production were upregulated in leukemic cluster. Remarkablely, the leukemic effector T cells demonstrated a lower level of killer cell lectinlike receptor G1 (KLRG1). We also verified the significantly decreased expression of surface KLRG1 on CD3+CD8+CD57+ cells in other five T-LGLL patients. Moreover, up-regulated PI3K-AKT pathway was observed in this KLRG1- leukemic subpopulation. Proliferation and cytotoxicity of leukemic lymphocytes from patients were significantly decreased by PI3K inhibitor. Summary/Conclusion: Large granular lymphocytes is a typical KLRG1- clonal T effector cluster, characterized by increased cytotoxicity and exhaustion. PI3K inhibitor, which could restrain proliferation and effector function of leukemic lymphocytes, is a promising agent for T-LGLL. Keywords: LGL