Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma
Authors: Fujisawa Manabu , Nguyen Tran B. , Abe Yoshiaki , Suehara Yasuhito , Fukumoto Kota , Suma Sakurako , Usuki Kensuke , Narita Kentaro , Matsue Kosei , Nakamura Naoya , Ishikawa Shumpei , Miura Fumihito , Ito Takashi , Suzuki Ayako , Suzuki Yutaka , Takahashi Satoru , Chiba Shigeru , Sakata-Yanagimoto Mamiko 
Published: November 2021

Abstract
Background
Angioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2mutations, whereas the G17V RHOA mutation in TET2-mutated immature cells facilitates development of T follicular helper (T FH)-like tumor cells. Notably, we and others have reported that immune cells derived from ACH with TET2 mutations infiltrate AITL tissues. However, how ACH-derived immune cells function as a microenvironmental niche in AITL remains largely unknown.
Objective
To elucidate the role of TET2-mutated immune cells in AITL tumorigenesis.
Methods
The G17V RHOA transgenic mice were crossed with mice lacking Tet2 in all blood cells (Mx-Crex Tet2f/f, A) and in T cells (Cd4-Crex Tet2f/f, B), respectively. Single-cell RNA sequencing (Sc-seq) was performed on &gt;60,000 cells from AITL in mice (AITLm, n=2) and human (AITLh, n=5), and their controls to reveal the immune profiles. We used Seurat and Monocle3 pipelines for analysis of Sc-seq. Whole genome bisulfite sequencing (WGBS) was used to analyze the methylome of germinal center B (GCB) cells in AITLm and control.
Results
AITLm occurred only in A, but not in B. Then, we intraperitoneally transplanted Cd4 + tumor-containing cells together with various lineages of immune cells sorted from AITLm into nude mice. AITLm developed only when B-lineage cells were cotransplanted with Cd4 + tumor-containing cells. Unsupervised clustering of the Sc-seq data identified 6 T-, 6 B- and 3 myeloid clusters in AITLm. B-cell clusters were annotated into naïve B-, memory B-, GCB-, and plasma clusters along the B-cell differentiation through Geneset variable analysis (GSVA) and trajectory analysis. We found that the aberrant GCB clusters, simultaneously exhibiting DZ-like proliferation markers (Aicda and Mki67) and LZ-like activation markers (Cd40, Cd83) were markedly expanded in AITLm. Geneset Enrichment Analysis (GSEA) revealed that MYC targets and other signaling pathways involved in cell proliferation were highly enriched in the GCB clusters in AITLm. WGBS showed that the number of hypermethylated regions (HyperDMRs) was markedly higher than that of hypomethylated regions (HypoDMRs) at all the regions; promoters, exons, introns, untranslated and intergenic regions. Among HyperDMRs, Atp13a2, Pdzd2, Rapgef4, Irf4 and Egr3 expressions were downregulated in the GCB clusters of Sc-seq in AITLm. Remarkably, the number of BCR clones in GCB of AITLm were significantly less than those in controls. In addition, in AITLm mice, the number of somatic mutations in GCB cells was significantly higher than that in T FH-like tumor cells. Remarkably, we detected unique core histone mutations in the GCB cells of AITLm, including the recurrent p.Ser87Asn Histone3 mutations. Next, In silico network analysis using Sc-seq data between GCB and T FH-like clusters identified that 11 interactions, including Cd40-Cd40lg were significantly enhanced in AITLm compared to controls. Flowcytomeric analysis revealed that cell-surface expression of Cd40 were significantly higher in the GCB cells of AITLm than those of control. Pathologically, the follicular structure was disrupted in AITLm. Consequently, Cd40lg +Cd4 +tumor cells and Cd40 +Cd19 + cells were both diffusely distributed and sometimes localized adjacent to each other. Finally, administration of an anti-Cd40lg antibody prolonged the survival of nude mice transplanted with AITLm.
In AITLh with TET2 mutations, unsupervised clustering of Sc-seq identified T-, B-, and myeloid-cell clusters and a cluster characterized by proliferative markers. In B-lineage cells, 9 clusters were re-clustered and annotated to naïve or memory B-, GCB- and plasmablast clusters under the same manner of mouse data. Gene ontology analysis from differential expression genes in each cluster showed that the GCB- and CD40-related genesets were enriched not only in the GCB cluster but also in the naive to memory B clusters. Furthermore, the AITL-B-specific geneset, which referred from genes (CD40, CD83, AICDA, MKI67) highly expressed in the GCB cluster in AITLm was enriched not only in the GCB cluster, but also in the naive to memory B clusters in AITLh.
Conclusion
This study suggests a new concept that ACH-derived GCB cells with TET2 mutations can undergo independent clonal evolution and function as microenvironmental cells to support tumorigenesis in AITL via the CD40-CD40LG axis.

Disclosures
Usuki: Astellas Pharma Inc.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Research Funding; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; 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; Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Apellis Pharmaceuticals, Inc.: Research Funding; 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.