A Single-Cell Atlas of Nonhematopoietic Cells in Human Lymph Node and Lymphoma Reveals Landscape of Stromal Remodeling
Authors: Abe Yoshiaki , Sakata-Yanagimoto Mamiko , Fujisawa Manabu , Miyoshi Hiroaki , Suehara Yasuhito , Hattori Keiichiro , Kusakabe Manabu , Sakamoto Tatsuhiro , Nishikii Hidekazu , Nguyen Tran B. , Yoshida Chikashi , Owada Yohei , Enomoto Tsuyoshi , Bando Hiroko , Nakayama Masahiro , Ohshima Koichi , Usuki Kensuke , Oda Tatsuya , Matsue Kosei , Chiba Shigeru 
Published: November 2021

Abstract
Background:
Activities of nonhematopoietic cells (NHCs) reportedly underlie lymphomagenesis. In follicular lymphoma (FL), mesenchymal stromal cells (SCs) including follicular dendritic cells (FDCs) have been shown to facilitate FL expansion. However, comprehensive understanding of lymphoma NHC activities have been hampered by indefinite NHC heterogeneity even in normal human lymph node (LN). Indeed, human LN blood endothelial cells (BECs) and non-endothelial stromal cells (NESCs) have not been analyzed at single-cell resolution. Here, we aimed to construct a single-cell atlas of NHCs in human LN applicable to lymphoma researches. We also sought to reveal the landscape of stromal remodeling in lymphomas, particularly in FL, to advance understanding of stromal contributions in lymphomagenesis.

Methods:
We prospectively performed single-cell RNA sequencing of NHCs (&gt;100,000 cells) extracted from 27 human samples including metastasis-free LN (MFLN; n=9), nodal FL (n=10), peripheral T-cell lymphoma (PTCL; n=5), and diffuse large B-cell lymphoma transformed from FL (tDLBCL; n=3). Data from MFLN samples were used for the construction of NHC atlas. Immunofluorescence (IF) staining was performed to investigate the existence and topological localizations of each NHC subcluster in the LN. Using the NHC atlas, we performed comprehensive comparative analysis with FL NHCs by differentially-expressed gene (DEG) and intercellular ligand-receptor analyses. We also investigated the prognostic impact of putative stroma-derived biomarkers using deposited microarray data of FL patients. Finally, we examined the applicability of the atlas to NHCs from other lymphoma subtypes by analyzing PTCL and tDLBCL NHCs. Data analysis was performed through multiple pipelines including Seurat, Monocle3, and CellphoneDB.
Results:
Graph-based clustering analysis revealed that the transcriptional features of NHC subpopulations in MFLN are detectable in FL NHCs. Unsupervised sub-clustering analysis of BECs, lymphatic endothelial cells (LECs), and NESCs revealed 10, 8, and 12 subclusters, respectively, including some lacking mouse counterpart. IF staining successfully identified each NHC subcluster and its localization in the LN. In FL NHCs, the proportion of arterial BEC subclusters markedly increased relative to MFLN, while the proportion of LECs decreased. In FL NESCs, the proportion of marginal reticular cells (MRCs) as well as FDCs greatly increased. DEG analysis revealed that the greatest changes in gene expression occurs in NESC subclusters, particularly in MRCs, T-zone reticular cells (TRCs), pericytes, and FDCs. Notably, in some NESC subclusters, we observed marked upregulation of genes relevant to solid cancers but previously not described in lymphomas (e.g. POSTN, EGFL6, and FAP). Combined interactome and DEG analysis revealed 60 FL-specific interactions between NHC subclusters and malignant B cells. For example, interactions mediated through stroma-derived CD70 were enhanced at medullary SC subclusters and SCs at LN capsule adventitia. Additionally, the CCR7-CCL19 interaction and interactions via B-cell activating factor (BAFF) were unexpectedly upregulated at non-TRC SC and medullary SC subclusters, respectively. Also, the CXCL13-CXCR5 axis was highly activated in MRCs, collectively indicating that non-FDC SCs vigorously participate in FL cell expansion and/or infiltration into extra-follicular lesions. Some intercellular interactions were functionally validated by in vitro binding assays. Based on this dataset, we identified putative stroma-derived biomarkers linked to unfavorable prognosis in FL patients including TDO2, encoding immune-modulators, and LY6H and LOX, tip cell markers. We finally confirmed that NHC subclusters identified in our atlas were also detectable in NHCs of more aggressive lymphoma subtypes including PTCL and tDLBCL. Notably, we found that extra-follicular SCs had further differentiated into follicular SCs in tDLBCL, likely representing a terminal form of stromal remodeling in FL.

Conclusion:
We constructed a comprehensive single-cell atlas of NHCs in human LN highly applicable to lymphoma NHC researches and revealed a total of 30 NHC subclusters. Our study largely updates NHC taxonomy in LNs and provides a rich resource and deeper insights into lymphoma biology, a contribution that should advance lymphoma management and therapy.
Figure 1 Figure 1.

Disclosures
Usuki: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Research Funding; Celgene K.K.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Nippon-Boehringer-Ingelheim Co., Ltd.: Research Funding; Mundipharma K.K.: Research Funding; Amgen-Astellas Biopharma K.K.: Research Funding; Nippon-Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Kyowa-Kirin Co., Ltd.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Research Funding, Speakers Bureau; Yakult Honsha Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Research Funding; Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Apellis Pharmaceuticals, Inc.: Research Funding; AbbVie GK: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Incyte Biosciences Japan G.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sanofi K.K.: Speakers Bureau; Amgen K.K.: Research Funding.