RESEARCH ARTICLE Single-Cell Analysis of the Multicellular Ecosystem in Viral Carcinogenesis by HTLV-1 Junji Koya1, Yuki Saito1,2, Takuro Kameda3, Yasunori Kogure1, Mitsuhiro Yuasa1,4, Joji Nagasaki5, Marni B. McClure1, Sumito Shingaki1, Mariko Tabata1,6, Yuki Tahira3, Keiichi Akizuki3, Ayako Kamiunten3, Masaaki Sekine3, Kotaro Shide3, Yoko Kubuki3, Tomonori Hidaka3, Akira Kitanaka7, Nobuaki Nakano8, Atae Utsunomiya8, Yosuke Togashi5, Seishi Ogawa9, Kazuya Shimoda3, and Keisuke Kataoka1,10 Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on October 1, 2021. Copyright 2021 American Copyright 2021 by AssociationAmerican for Association Cancer Research. for Cancer Research. ABSTRACT Premalignant clonal expansion of human T-cell leukemia virus type-1 (HTLV-1)– infected cells occurs before viral carcinogenesis. Here we characterize premalig- nant cells and the multicellular ecosystem in HTLV-1 infection with and without adult T-cell leukemia/ lymphoma (ATL) by genome sequencing and single-cell simultaneous transcriptome and T/B-cell recep- tor sequencing with surface protein analysis. We distinguish malignant phenotypes caused by HTLV-1 infection and leukemogenesis and dissect clonal evolution of malignant cells with different clinical behavior. Within HTLV-1–infected cells, a regulatory T-cell phenotype associates with premalignant clonal expansion. We also delineate differences between virus- and tumor-related changes in the nonmalig- nant hematopoietic pool, including tumor-specific myeloid propagation. In a newly generated conditional knockout mouse model recapitulating T-cell–restricted CD274 (encoding PD-L1) gene lesions found in ATL, we demonstrate that PD-L1 overexpressed by T cells is transferred to surrounding cells, leading to their PD-L1 upregulation. Our findings provide insights into clonal evolution and immune landscape of multistep virus carcinogenesis. SIGNIFICANCE: Our multimodal single-cell analyses comprehensively dissect the cellular and molecular alterations of the peripheral blood in HTLV-1 infection, with and without progression to leukemia. This study not only sheds light on premalignant clonal expansion in viral carcinogenesis, but also helps to devise novel diagnostic and therapeutic strategies for HTLV-1–related disorders. INTRODUCTION Besides viral carcinogenesis, recent studies described somatic mutations in normal tissues, such as the hematopoietic and Oncogenic viral infections, such as human T-cell leukemia gastrointestinal systems, pointing to a critical role of prema- virus type-1 (HTLV-1), are responsible for 10% to 15% of human lignant clonal expansion in diverse oncogenic processes (5, cancers worldwide (1). These viruses can induce carcinogenesis 6). However, the biological properties and molecular basis by various mechanisms, such as chronic inflammation, cellu- of premalignant clonal expansion are not well characterized. lar transformation, and immune suppression (2). Accumulat- HTLV-1 is a retrovirus infecting approximately 5 to 20 million ing evidence suggests that clonal expansion of infected cells people worldwide, particularly in endemic regions (7). This precedes malignancy and contributes to tumorigenesis (3, 4). virus preferentially targets CD4+ T cells and is associated with a variety of diseases, ranging from inflammatory disorders to adult T-cell leukemia/lymphoma (ATL). ATL is an aggressive 1Division of Molecular Oncology, National Cancer Center Research Insti- T-cell neoplasm evolving after a long latency of >30 to 50 tute, Tokyo, Japan. 2Department of Gastroenterology, Keio University years. Even before ATL onset, premalignant HTLV-1–infected School of Medicine, Tokyo, Japan. 3Division of Hematology, Diabetes, and clones bearing driver mutations can be detected (4), offering Endocrinology, Department of Internal Medicine, Faculty of Medicine, Uni- a unique opportunity to functionally dissect premalignant 4 versity of Miyazaki, Miyazaki, Japan. Department of Pathology, Graduate clonal expansion in multistep viral carcinogenesis. However, School of Medicine, The University of Tokyo, Tokyo, Japan. 5Chiba Cancer Center, Research Institute, Chiba, Japan. 6Department of Urology, Graduate as only a small proportion (usually <1%–5%) of peripheral School of Medicine, The University of Tokyo, Tokyo, Japan. 7Department of blood mononuclear cells (PBMC) of asymptomatic carriers Laboratory Medicine, Kawasaki Medical School, Kurashiki, Japan. 8Depart- (AC) contain integrated proviral DNA (8), the phenotypical ment of Hematology, Imamura General Hospital, Kagoshima, Japan. and functional evaluation of HTLV-1–infected cells is difficult. 9Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 10Division of Hematology, Department of Efficient cytotoxic T-lymphocyte (CTL) response to HTLV-1 Medicine, Keio University School of Medicine, Tokyo, Japan. has been reported to limit the proviral load and the risk of Note: Supplementary data for this article are available at Blood Cancer associated diseases (9). By contrast, ATL patients are severely Discovery Online (https://bloodcancerdiscov.aacrjournals.org/). immunocompromised, frequently contracting opportunistic J. Koya and Y. Saito contributed equally to this article. infections (10). These observations emphasize the relevance K. Kataoka is the senior author of this article. of immune dynamics in HTLV-1–related diseases. This view Corresponding Author: Keisuke Kataoka, Division of Molecular Oncology, is supported by genetic studies showing frequent alterations National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo in immune molecules, such as structural variations (SV) trun- 104-0045, Japan. Phone: 81-3-3542-2511; Fax: 81-3-5565-0727; E-mail: cating the CD274 3′-untranslated regions (UTR) in ATL (11, [email protected] 12). However, although many efforts have been devoted to Blood Cancer Discov 2021;2:1–18 CTL characterization, the entire composition and functional doi: 10.1158/2643-3230.BCD-21-0044 heterogeneity of the immune microenvironment in HTLV-1 This open access article is distributed under the Creative Commons Attribution- infection and ATL remain elusive. NonCommercial-NoDerivatives License 4.0 International (CC BY-NC-ND). Recent advances in single-cell analysis provide an avenue to ©2021 The Authors; Published by the American Association for Cancer Research elucidate phenotypic features at a cellular resolution, allowing SEPTEMBER 2021 BLOOD CANCER DISCOVERY | OF2 Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on October 1, 2021. Copyright 2021 American Association for Cancer Research. RESEARCH ARTICLE Koya et al. profound understanding of tumor heterogeneity and microen- ples were grouped into the same clusters (Fig. 1B and C). vironment in cancer (13). Particularly, cellular indexing of tran- By contrast, nonmalignant cells, including those from ACs scriptomes and epitopes by sequencing (CITE-seq) is a recently and HDs, were classified into the same clusters according to developed tool for exploring transcriptomic and surface prot- their lineage, suggesting their transcriptomic similarity among eomic states in the same single cell (14). Also, single-cell T/B- patients. Almost all malignant clusters expressed HTLV-1 HBZ cell receptor (TCR/BCR) sequencing (scTCR/BCR-seq) enables (Fig. 1C). Although almost no cells were classified into malig- detection of T/B-cell clonal expansion patterns through identi- nant clusters in ACs or HDs, malignant cells accounted for cal TCR/BCR sequence detection (15), which can be utilized to 15% to 99% (median 82%) of the total cells in ATL, which were track clonal evolution in lymphoid malignancy. lower in smoldering than in other subtypes (Supplementary Here, to investigate the cellular and molecular architecture in Fig. S1D and S1E). Single-cell mutation and CNA estimation HTLV-1 infection and ATL, we performed multimodal single-cell revealed somatic alterations almost exclusively in malignant analysis, evaluating transcriptome and many surface markers. clusters, consistent with the bulk sequencing and SNP array Combined with scTCR-seq, our approach deciphers functional results (Supplementary Fig. S1F and S1G), confirming success- heterogeneity of HTLV-1–infected premalignant cells and the ful separation of malignant from nonmalignant cells. hierarchy and clonal evolution of malignant cells. Simultaneous mRNA and protein assessment delineates discordant regula- Lineage-Specific Regulation of mRNA and tion of their expression levels, particularly in myeloid lineage. In ADT Levels addition, we reveal variegated alterations in the immune micro- Protein abundance is a more direct determinant of cellular environment, including a novel mode of PD-L1 upregulation in functions but cannot necessarily be inferred from mRNA surrounding cells by CD274 SV-harboring tumors. level due to posttranscriptional processes (16). CITE-seq anal- ysis in healthy, virally infected, and leukemic states enables a RESULTS detailed comparison of mRNA and ADT level changes in vari- ous cell types. At the single-cell level, ADT was less prone to Landscape of the Multicellular Ecosystem in dropout than mRNA, conferring higher sensitivity for many HTLV-1 Infection and ATL surface markers (Supplementary Fig. S2A; Supplementary To delineate the multicellular ecosystem of HTLV-1 carrier Table S2). As expected, more than two thirds of genes showed state and ATL, we constructed transcriptomic, surface phe- strong positive