Biopsy samples were cryopreserved in optimal cutting temperature (OCT) compound and stored at -80☌. Specimens were placed in phosphate-buffered saline (PBS) on ice. In this study, we sought to identify a new possible target for ATLL treatment, while also considering clonal malignancy and their TME interactions. We also examined non T-cell components of the ATLL TME including myeloid cells and stromal cells, especially cancer associated fibroblast (CAF) subtypes and their potential communications with T-cells. With this approach, we were able to identify distinct T-cell subpopulations that likely represent the malignant clones in ATLL via an integrated analysis of the transcriptome and T-cell clonal repertoire. In this study, we used scRNA-seq and TCR clonal analysis to dissect the malignant tumor cells and TME of ATLL. ScRNA-seq has provided a useful cell atlas to better understand the intra-tumoral diversity of the TME and disease-specific cellular crosstalk with malignant cells ( 11). Accordingly, the interaction with the tumor microenvironment (TME) surrounding the neoplasm is important for its regulation and growth. HTLV-1 not only infects T-cells, but also various cell types including B-cells, myeloid cells, and fibroblasts ( 9, 10). However, investigations using single-cell RNA sequencing (scRNA-seq) technology with T-cell receptor (TCR) clonal analysis have been lacking, making it difficult to understand the dynamics of the immune response during ATLL progression. Copy number abnormalities (CNAs) of ATLL are comparable to the PTCL-GATA3 subgroup ( 5), and gene expression profiling has been used to define distinct diagnostic and prognostic subtypes of PTCL ( 8). Recent advances in next-generation sequencing (NGS) technology have provided more detailed information on the unique pathogenesis of ATLL compared to other subtypes of peripheral T-cell lymphoma (PTCL) ( 5– 7). Since HTLV-1 infection is endemic in southwestern Japan, ATLL has been mainly reported in the same region ( 3, 4). These findings suggest a potential targeted therapeutic pathway to better treat this neoplasm.Īdult T-cell Leukemia/Lymphoma (ATLL) is an aggressive mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1) infection ( 1, 2). Cancer associated fibroblasts (CAFs) of ATLL play an important role for CD4 T-cell proliferation via FGF7-FGF1 and PDGFA-PDGFRA/B signaling, and CAFs, particularly EGR-enriched, are also associated with CD8 and NKT expansion by EGFR. By dissecting diverse cell types comprising the TME, we identified a novel subset of cancer-associated fibroblast, which showed enriched epidermal growth factor receptor (EGFR)-related transcripts including early growth response 1 and 2 (EGR1 and EGR2). Highly clonal tumor cells showed multiple activating pathways, suggesting dynamic evolution of the malignancy. Herein, we examined ATLL using archived fresh frozen tissue after biopsy using single-cell RNA sequencing (scRNA-seq) with T-cell receptor (TCR) clonal analysis. Difficulties in harvesting fresh tissue in a clinical setting have hampered our deeper understanding of this malignancy. Moreover, the intercellular communications between the tumor microenvironment (TME) and tumor cells in this malignancy are currently unknown. However, little is known about the underlying activated molecular pathways at the single cell level. Adult T-cell Leukemia/Lymphoma (ATLL) is a rare aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection.