PB1928: SINGLE-CELL TRANSCRIPTOMICS REVEALS IMMUNE PROFILE ACROSS TREATMENT IN T-CELL LARGE GRANULAR LYMPHOCYTIC LEUKEMIA

Topic: 6. Chronic lymphocytic leukemia and related disorders - Clinical Background: T-cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder, characterized by monoclonal T-cell large granular lymphocytosis. Cyclophosphamide (CTX), cyclosporine (CsA) and methotrexate (MTX) are the main immunosuppressive agents used in treating T-LGLL. However, because of the rarity of T-LGLL, no holistic research has been done to show the immunological profiling across treatment in T-LGLL. In this study, single-cell RNA sequencing (scRNA-seq) and V(D)J profiling were performed to illustrate the immune profile of T-LGLL. Aims: To identify the dynamic immune profile of T-LGLL across immunosuppressive treatment. Methods: Peripheral blood samples were collected from five T-LGLL patients prior and post immunotherapy. Surface proteins were specifically labeled for epitopes sequencing, scRNA-seq was performed on the 10x Genomics Chromium Single Cell V(D)J + 5’ Gene Expression platform. Immune cells were annotated and transcriptome characteristics of each group of cells were analyzed, then T cells clonotype and clone-size were identified by V(D)J sequences. Furthermore, differential expression genes were identified and pathway enrichment was conducted. Results: ScRNA-seq and V(D)J profiling data of 67,237 immune cells, including 22137 of T cells with intact TCRα and β chains, revealed that: (1) The function of antigen presenting cells (APCs) was enhanced in disease condition. Antigen process, presentation pathway and the signaling pathways related to innate immunity were up-regulated in monocytes and dendritic cells. The binding ability to antigen and the regulatory of T cell activation and differentiation were enhanced in B lymphocyte. (2) During disease condition, the expression of cytotoxicity related genes in NK cluster was decreased, but the regulation of T cell receptor signaling pathway, T cell proliferation, activation and differentiation was enhanced. (3) The immune cell composition of patients before treatment and in non-response condition was similar, with increased proportion of CD8 T cell population. The proportion of terminal effector CD8 T cells (CD8_TE) in G2/M phase was significantly higher in patients than in healthy individuals. Moreover, in patients prior treatment, cell killing, interferon γ signaling pathway, cytokine production and exhaustion related genes were upregulated in CD8 TE. But cell proliferation and toxicity of CD8 TE in responders were decreased. (4) The dominant clones drifted with disease status. The clonal diversity of CD8 TE cells was significantly reduced and the dominant clones were amplified in disease condition. Increased cytotoxicity, up-regulated T cell activation and cytokine secretion were observed in monoclonal T cells. The proportion of dominant clones decreased after treatment, but the previous dominant clones still existed in remission state, with a decreased toxic function. (5) In addition, for patient prior treatment, the proportion of Treg was decreased significantly and apoptosis pathway was enriched. After response, the proportion of Treg was restored, metabolism-related pathway was up-regulated and apoptosis-related pathways were down-regulated. Summary/Conclusion: The immune homeostasis was unbalanced in T-LGLL, which demonstrated by stronger cytotoxicity in CD8 TE and less elevated antigen presentation effect of APCs. Immunosuppressive agents can partially remove the pathogenic LGL cells and improve immune disorders. Keywords: RNA-seq, TCR, Large granular lymphocytic leukaemia