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LYMPHOMA

Tumor-promoting function and regulatory landscape of PD-L2 in B-cell lymphoma

Leukemia volume 37, pages 492–496 (2023)Cite this article

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Then, we compared the effects of PD-L1 and PD-L2 on the tumor microenvironment (TME) when constitutively expressed at a similar level. Hence, we generated a A20-ovalbumin (OVA) murine B-cell lymphoma line lacking Pd-l1 and introduced murine Pd-l1 or Pd-l2 expression (Supplementary Fig. 2A–E). Syngeneic transplantation revealed that Pd-l1/Pd-l2 overexpression enhanced tumor growth and attenuated CD8+ T-cell infiltration into tumors, which was restored by anti–PD-1 antibody (Supplementary Fig. 2F–J). Furthermore, we constructed single-cell transcriptomic, surface phenotypic (antibody-derived tag [ADT]), and T/B-cell receptor (TCR/BCR) repertoire maps of 22,116 nonmalignant CD45+ cells from mock–/Pd-l1–/Pd-l2–expressing A20-OVA tumors (Fig. 1C, D; Supplementary Fig. 3A,B; Supplementary Tables 6, 7) [7]. Importantly, Pd-l1–/Pd-l2–expressing tumors exhibited similar cellular dynamics and transcriptomic and surface phenotypic changes in the TME (Supplementary Tables 8–10). CD8+ T cells, particularly exhausted CD8+ T cells (CD8Tex) showing high clonality, and regulatory T cells were decreased in Pd-l1/Pd-l2–expressing tumors (Fig. 1E; Supplementary Fig. 3B). Interestingly, myeloid cells, including monocytes/macrophages (Mono/Macs) and plasmacytoid dendritic cells (pDCs), were or tended to be increased in Pd-l1/Pd-l2–expressing tumors (Fig. 1E). M1 and M2 macrophage signatures were downregulated and upregulated in Pd-l1/Pd-l2–expressing tumors, respectively, whereas other immune-related signatures were comparable (Supplementary Fig. 3D–F). Responses to bacterial molecules, including lipopolysaccharide, were transcriptionally downregulated in Mono/Macs from Pd-l1/Pd-l2–expressing tumors, suggestive of M2-like anti-inflammatory, pro-tumorigenic phenotype (Fig. 1F) [8]. Furthermore, antigen presentation pathways were suppressed in conventional DCs and pDCs. Consistently, pro-inflammatory cytokine‒inducible markers, such as Ly6A/E and I-A/I-E, were downregulated in various clusters from Pd-l1/Pd-l2–expressing tumors (Fig. 1G; Supplementary Fig. 4A, B). Notably, pDC depletion with anti-BST2 antibody inhibited growth of Pd-l1–/Pd-l2–expressing tumors (Fig. 1H; Supplementary Fig. 4C), suggesting the pro-tumorigenic role of pDCs. Together, our single-cell analysis delineates pleiotropic extrinsic effects shared by PD-L1 and PD-L2, mainly enhancing anti-inflammatory, pro-tumorigenic responses in microenvironmental cells, particularly various myeloid subsets, although experimental validation and further mechanistic investigation is warranted.

As DHS26 showed strong enrichment as the canonical TSS, this region was intensively explored. Surprisingly, GM12878 RNA-seq revealed transcriptional reads originating from DHS26 and spliced at chr9:5,517,122 into exon 2 (Fig. 2B), although this region was annotated as a putative enhancer in GM12878 [9]. The identical novel exon-containing transcript was also observed in two B-cell lymphoma lines (SLVL and HKBML) (Supplementary Fig. 7A, B). Cap analysis of gene expression with sequencing (CAGE-seq) [13] showed a strong peak at position chr9:5,517,068 (Supplementary Fig. 7C). Full-length cDNA sequencing revealed that transcripts initiating in the novel exon contained the canonical exons 2–7 and terminated at the 3′-UTR end (Supplementary Fig. 7D). Among seven cell lines intensively investigated in Encyclopedia of DNA Elements (ENCODE) [9], level of histone H3 lysine 4 trimethylation (H3K4me3), marking active promoters, was increased around the novel TSS exclusively in GM12878, while H3K4 monomethylation (H3K4me1), marking active enhancers, in this region was detected in most cell lines. Compared with other PD-L2 exons, the novel TSS-containing exon showed low evolutionary conservation (Supplementary Fig. 7E). These results suggest B-cell–specific transcriptional initiation from an intragenic enhancer within PD-L2, demonstrating dual promoter and enhancer potential [14]. Differential regulation of the canonical and novel TSSs were validated by CRISPR-mediated disruption: targeting wtCas9 and/or dCas9-KRAB to DHS26 in GM12878 and SLVL suppressed only the novel exon–containing transcripts, while the canonical transcripts were selectively downregulated by DHS20 targeting (Supplementary Figs. 5I, 7F, G). In pan-cancer transcriptome analysis, the novel exon was almost uniquely expressed in DLBCL, accounting for 13% of the samples, while nearly no expression was detected in normal tissues (Fig. 2C; Supplementary Fig. 7H; Supplementary Table 14) [2, 15]. Frequent expression of the novel exon was also observed in PMBL (Supplementary Fig. 7I).

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Fig. 1: Anti-inflammatory, pro-tumorigenic function of PD-L2 revealed by multi-omics single-cell analysis in mice.
Fig. 2: PD-L2 regulatory landscape revealed by CRISPR tiling and loss-of-function screening in human B-cells.

Data availability

Single-cell and CAGE-seq data have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession PRJDB12182. The novel PD-L2 transcript sequence has been deposited in DDBJ under accession LC649798.

Code availability

Custom code used in this paper is available upon request.

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Acknowledgements

This work is supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers JP21H05051, JP19K22573, and JP19K23979), Japan Agency for Medical Research and Development (AMED) (grant numbers JP21ck0106542 and JP19cm0106338), National Cancer Center Research and Development Funds (31-A-4), The Cell Science Research Foundation, Princess Takamatsu Cancer Research Fund, and Foundation for Promotion of Cancer Research. We thank F. Ueki, Y. Hokama, and Y. Ito for technical assistance. The sequencing resources were partly provided by Fundamental Innovative Oncology Core at the National Cancer Center. The supercomputing resources were provided by the Human Genome Center, the Institute of Medical Science, The University of Tokyo. The images of the mice in Fig. 1C and Supplementary Fig. 2F are from TogoTV (©2016 DBCLS TogoTV). We acknowledge the ENCODE Consortium, the TCGA Research Network, the GTEx Consortium, the NCICCR, and the CTSP for providing data analyzed in this paper.

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Author notes

  1. These authors contributed equally: Sumito Shingaki, Junji Koya, Mitsuhiro Yuasa.

Authors and Affiliations

  1. Division of Molecular Oncology, National Cancer Center Research Institute, Tokyo, Japan

    Sumito Shingaki, Junji Koya, Mitsuhiro Yuasa, Yuki Saito, Mariko Tabata, Marni B. McClure, Yasunori Kogure & Keisuke Kataoka

  2. Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Mitsuhiro Yuasa

  3. Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan

    Yuki Saito

  4. Department of Urology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Mariko Tabata

  5. Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Seishi Ogawa

  6. Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan

    Seishi Ogawa

  7. Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institute, Stockholm, Sweden

    Seishi Ogawa

  8. Laboratory of Sequence Analysis, Human Genome Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Kotoe Katayama

  9. Department of Tumor Microenvironment, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

    Yosuke Togashi

  10. Division of Health Medical Intelligence, Human Genome Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Seiya Imoto

  11. Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan

    Keisuke Kataoka

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  1. Sumito Shingaki
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Contributions

SS, JK, and K Kataoka designed the study. SS, JK, and YK performed experiments. MY, YS, SO, K Katayama, SI, and YK performed sequencing data analyses. MT, MBM, and YT assisted experiments and data analyses. SS, JK, MY, YS, and K Kataoka generated figures and tables and wrote the manuscript. K Kataoka led the entire project. All authors participated in discussions and interpretation of the data and results.

Corresponding author

Correspondence to Keisuke Kataoka.

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Competing interests

SO holds stock in Asahi Genomics, has patent applications for PD-L1 abnormalities as a predictive biomarker for immune checkpoint blockade therapy, and has received research funding from Dainippon-Sumitomo Pharmaceutical and Cordia Therapeutics. K Kataoka holds stock in Asahi Genomics, has patent applications for PD-L1 abnormalities as a predictive biomarker for immune checkpoint blockade therapy, and has received research funding from Otsuka Pharmaceutical, Takeda Pharmaceutical, Chugai Pharmaceutical, Chordia Therapeutics, and Bristol-Myers Squibb. The other authors declare no competing financial interests.

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Shingaki, S., Koya, J., Yuasa, M. et al. Tumor-promoting function and regulatory landscape of PD-L2 in B-cell lymphoma. Leukemia 37, 492–496 (2023). https://doi.org/10.1038/s41375-022-01772-1

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