Abstract
Background
Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance.
Methods
We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients’ responses to ICB in 60 ESCC patients.
Results
Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A+ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A+ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC.
Conclusions
HLA-A+ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 24 print issues and online access
$259.00 per year
only $10.79 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The RNA-seq data can be accessed on the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics repository (https://ngdc.cncb.ac.cn/gsa-human) under the accession number HRA006820. The scRNA-seq data was downloaded from the GEO repository under the accession number GSE160269 [36].
References
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.
Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66:115–32.
Liu J, Xie X, Zhou C, Peng S, Rao D, Fu J. Which factors are associated with actual 5-year survival of oesophageal squamous cell carcinoma? Eur J Cardiothorac Surg. 2012;41:e7–11.
Peng Q, Qiu X, Zhang Z, Zhang S, Zhang Y, Liang Y, et al. PD-L1 on dendritic cells attenuates T cell activation and regulates response to immune checkpoint blockade. Nat Commun. 2020;11:4835.
Kojima T, Shah MA, Muro K, Francois E, Adenis A, Hsu CH, et al. Randomized Phase III KEYNOTE-181 Study of Pembrolizumab Versus Chemotherapy in Advanced Esophageal Cancer. J Clin Oncol. 2020;38:4138–48.
Kato K, Cho BC, Takahashi M, Okada M, Lin CY, Chin K, et al. Nivolumab versus chemotherapy in patients with advanced oesophageal squamous cell carcinoma refractory or intolerant to previous chemotherapy (ATTRACTION-3): a multicentre, randomised, open-label, phase 3 trial. Lancet Oncol. 2019;20:1506–17.
Baba Y, Nomoto D, Okadome K, Ishimoto T, Iwatsuki M, Miyamoto Y, et al. Tumor immune microenvironment and immune checkpoint inhibitors in esophageal squamous cell carcinoma. Cancer Sci. 2020;111:3132–41.
Sautes-Fridman C, Petitprez F, Calderaro J, Fridman WH. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer. 2019;19:307–25.
Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. Science. 2022;375:eabf9419.
Chen Z, Wang X, Jin Z, Li B, Jiang D, Wang Y, et al. Deep learning on tertiary lymphoid structures in hematoxylin-eosin predicts cancer prognosis and immunotherapy response. NPJ Precis Oncol. 2024;8:73.
Helmink BA, Reddy SM, Gao J, Zhang S, Basar R, Thakur R, et al. B cells and tertiary lymphoid structures promote immunotherapy response. Nature. 2020;577:549–55.
Cabrita R, Lauss M, Sanna A, Donia M, Skaarup Larsen M, Mitra S, et al. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature. 2020;577:561–5.
Petitprez F, de Reynies A, Keung EZ, Chen TW, Sun CM, Calderaro J, et al. B cells are associated with survival and immunotherapy response in sarcoma. Nature. 2020;577:556–60.
Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45:228–47.
Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34:525–7.
Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation. 2021;2:100141.
Bolotin DA, Poslavsky S, Mitrophanov I, Shugay M, Mamedov IZ, Putintseva EV, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods. 2015;12:380–1.
de Masson A, O’Malley JT, Elco CP, Garcia SS, Divito SJ, Lowry EL, et al. High-throughput sequencing of the T cell receptor beta gene identifies aggressive early-stage mycosis fungoides. Sci Transl Med. 2018;10:eaar5894.
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.
Hao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S, Butler A, et al. Integrated analysis of multimodal single-cell data. Cell. 2021;184:3573–3587 e3529.
McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Syst. 2019;8:329–37.e324.
Korsunsky I, Millard N, Fan J, Slowikowski K, Zhang F, Wei K, et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods. 2019;16:1289–96.
Aran D, Looney AP, Liu L, Wu E, Fong V, Hsu A, et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol. 2019;20:163–72.
Trapnell C, Cacchiarelli D, Grimsby J, Pokharel P, Li S, Morse M, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol. 2014;32:381–6.
Newman AM, Liu CL, Green MR, Gentles AJ, Feng W, Xu Y, et al. Robust enumeration of cell subsets from tissue expression profiles. Nat Methods. 2015;12:453–7.
Chen B, Khodadoust MS, Liu CL, Newman AM, Alizadeh AA. Profiling Tumor Infiltrating Immune Cells with CIBERSORT. Methods Mol Biol. 2018;1711:243–59.
Sharonov GV, Serebrovskaya EO, Yuzhakova DV, Britanova OV, Chudakov DM. B cells, plasma cells and antibody repertoires in the tumour microenvironment. Nat Rev Immunol. 2020;20:294–307.
Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, et al. IFN-gamma-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest. 2017;127:2930–40.
Senbabaoglu Y, Gejman RS, Winer AG, Liu M, Van Allen EM, de Velasco G, et al. Tumor immune microenvironment characterization in clear cell renal cell carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures. Genome Biol. 2016;17:231.
Jiang Y, Li Y, Zhu B. T-cell exhaustion in the tumor microenvironment. Cell Death Dis. 2015;6:e1792.
Gu-Trantien C, Loi S, Garaud S, Equeter C, Libin M, de Wind A, et al. CD4(+) follicular helper T cell infiltration predicts breast cancer survival. J Clin Invest. 2013;123:2873–92.
Zhang Y, Chen H, Mo H, Hu X, Gao R, Zhao Y, et al. Single-cell analyses reveal key immune cell subsets associated with response to PD-L1 blockade in triple-negative breast cancer. Cancer Cell. 2021;39:1578–93.e1578.
Thommen DS, Koelzer VH, Herzig P, Roller A, Trefny M, Dimeloe S, et al. A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade. Nat Med. 2018;24:994–1004.
Liu B, Hu X, Feng K, Gao R, Xue Z, Zhang S, et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nat Cancer. 2022;3:108–21.
Groeneveld CS, Fontugne J, Cabel L, Bernard-Pierrot I, Radvanyi F, Allory Y, et al. Tertiary lymphoid structures marker CXCL13 is associated with better survival for patients with advanced-stage bladder cancer treated with immunotherapy. Eur J Cancer. 2021;148:181–9.
Zhang X, Peng L, Luo Y, Zhang S, Pu Y, Chen Y, et al. Dissecting esophageal squamous-cell carcinoma ecosystem by single-cell transcriptomic analysis. Nat Commun. 2021;12:5291.
Jansen CS, Prokhnevska N, Master VA, Sanda MG, Carlisle JW, Bilen MA, et al. An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature. 2019;576:465–70.
Eberhardt CS, Kissick HT, Patel MR, Cardenas MA, Prokhnevska N, Obeng RC, et al. Functional HPV-specific PD-1(+) stem-like CD8 T cells in head and neck cancer. Nature. 2021;597:279–84.
Horton BL, Morgan DM, Momin N, Zagorulya M, Torres-Mejia E, Bhandarkar V, et al. Lack of CD8(+) T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer. Sci Immunol. 2021;6:eabi8800.
Philip M, Schietinger A. CD8(+) T cell differentiation and dysfunction in cancer. Nat Rev Immunol. 2022;22:209–23.
Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, et al. Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell. 2018;175:998–1013.e1020.
Miller BC, Sen DR, Al Abosy R, Bi K, Virkud YV, LaFleur MW, et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol. 2019;20:326–36.
Brummelman J, Mazza EMC, Alvisi G, Colombo FS, Grilli A, Mikulak J, et al. High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors. J Exp Med. 2018;215:2520–35.
Kurtulus S, Madi A, Escobar G, Klapholz M, Nyman J, Christian E, et al. Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1(-)CD8(+) Tumor-Infiltrating T Cells. Immunity. 2019;50:181–94.e186.
Prokhnevska N, Cardenas MA, Valanparambil RM, Sobierajska E, Barwick BG, Jansen C, et al. CD8(+) T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor. Immunity. 2023;56:107–24.e105.
Kwon BS, Weissman SM. cDNA sequences of two inducible T-cell genes. Proc Natl Acad Sci USA. 1989;86:1963–7.
Melero I, Bach N, Hellstrom KE, Aruffo A, Mittler RS, Chen L. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur J Immunol. 1998;28:1116–21.
Salgado FJ, Lojo J, Fernandez-Alonso CM, Vinuela J, Cordero OJ, Nogueira M. Interleukin-dependent modulation of HLA-DR expression on CD4and CD8 activated T cells. Immunol Cell Biol. 2002;80:138–47.
Alspach E, Lussier DM, Schreiber RD. Interferon gamma and Its Important Roles in Promoting and Inhibiting Spontaneous and Therapeutic Cancer Immunity. Cold Spring Harb Perspect Biol. 2019;11:a028480.
Buchan SL, Dou L, Remer M, Booth SG, Dunn SN, Lai C, et al. Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function. Immunity. 2018;49:958–70.e957.
Shaikh RB, Santee S, Granger SW, Butrovich K, Cheung T, Kronenberg M, et al. Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction. J Immunol. 2001;167:6330–7.
Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013;13:227–42.
van Dijk N, Gil-Jimenez A, Silina K, Hendricksen K, Smit LA, de Feijter JM, et al. Preoperative ipilimumab plus nivolumab in locoregionally advanced urothelial cancer: the NABUCCO trial. Nat Med. 2020;26:1839–44.
Gao J, Navai N, Alhalabi O, Siefker-Radtke A, Campbell MT, Tidwell RS, et al. Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma. Nat Med. 2020;26:1845–51.
Liu Z, Meng X, Tang X, Zou W, He Y. Intratumoral tertiary lymphoid structures promote patient survival and immunotherapy response in head neck squamous cell carcinoma. Cancer Immunol Immunother. 2023;72:1505–21.
Barros LRC, Souza-Santos PT, Pretti MAM, Vieira GF, Bragatte MAS, Mendes MFA, et al. High infiltration of B cells in tertiary lymphoid structures, TCR oligoclonality, and neoantigens are part of esophageal squamous cell carcinoma microenvironment. J Leukoc Biol. 2020;108:1307–18.
Meylan M, Petitprez F, Becht E, Bougouin A, Pupier G, Calvez A, et al. Tertiary lymphoid structures generate and propagate anti-tumor antibody-producing plasma cells in renal cell cancer. Immunity. 2022;55:527–41.e525.
Hudson WH, Gensheimer J, Hashimoto M, Wieland A, Valanparambil RM, Li P, et al. Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1(+) Stem-like CD8(+) T Cells during Chronic Infection. Immunity. 2019;51:1043–58.e1044.
Beltra JC, Manne S, Abdel-Hakeem MS, Kurachi M, Giles JR, Chen Z, et al. Developmental Relationships of Four Exhausted CD8(+) T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms. Immunity. 2020;52:825–41.e828.
Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol. 2003;3:292–303.
Bergomas F, Grizzi F, Doni A, Pesce S, Laghi L, Allavena P, et al. Tertiary intratumor lymphoid tissue in colo-rectal cancer. Cancers. 2011;4:1–10.
Di Caro G, Bergomas F, Grizzi F, Doni A, Bianchi P, Malesci A, et al. Occurrence of tertiary lymphoid tissue is associated with T-cell infiltration and predicts better prognosis in early-stage colorectal cancers. Clin Cancer Res. 2014;20:2147–58.
Zhou F. Molecular mechanisms of IFN-gamma to up-regulate MHC class I antigen processing and presentation. Int Rev Immunol. 2009;28:239–60.
Zhang S, Kohli K, Black RG, Yao L, Spadinger SM, He Q, et al. Systemic Interferon-gamma Increases MHC Class I Expression and T-cell Infiltration in Cold Tumors: Results of a Phase 0 Clinical Trial. Cancer Immunol Res. 2019;7:1237–43.
Sznarkowska A, Mikac S, Pilch M. MHC Class I Regulation: The Origin Perspective. Cancers. 2020;12:1155.
Li S, Li K, Tian F, Li H, Xia Q, Li T, et al. A high interferon gamma signature of CD8(+) T cells predicts response to neoadjuvant immunotherapy plus chemotherapy in gastric cancer. Front Immunol. 2022;13:1056144.
Grasso CS, Tsoi J, Onyshchenko M, Abril-Rodriguez G, Ross-Macdonald P, Wind-Rotolo M, et al. Conserved Interferon-gamma Signaling Drives Clinical Response to Immune Checkpoint Blockade Therapy in Melanoma. Cancer Cell. 2020;38:500–15.e503.
Karachaliou N, Crespo G, Aldeguer E, Drozdowskyj A, Gimenez Capitan A, Teixido C, et al. Interferon-gamma (INFG), an important marker of response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) and melanoma patients. J Clin Oncol. 2017;35:11504.
Acknowledgements
We thank Shaoyang Sun at Fudan University for the assistance with the experiment.
Funding
This work was supported by funding from the National Key R&D Program of China (2021YFC2501004), the National Natural Science Foundation of China (82171837, 82188102, 82030089, 82225033), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-067), the Fundamental Research Funds for the Central Universities (3332021091), Non-profit central research institute fund of Chinese Academy of Medical Sciences (2022-RC310-08), the Science and Technology Commission of Shanghai Municipality (23JS1400400), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01 and 2018SHZDZX01) and ZJLab.
Author information
Authors and Affiliations
Contributions
ZL, YL and YJ conceived and supervised the study. DZ performed experiments. DZ, JM and XX analyzed and interpreted the data, with assistance from ZC, MJ, WY, JW, WM and WQ. DJ, LJ and XW recruited patients under the supervision of YH and JH. DJ and LJ gathered clinical data. DZ, JM and YL wrote the manuscript with assistance from all authors. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
Written informed consent was obtained from each participant. The study was conducted following the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committees of Cancer Hospital, Chinese Academy of Medical Science, Beijing, China (Approval No. 16-008/1087), and Zhongshan Hospital, Fudan University, Shanghai, China (Approval No. B2022-632).
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zhang, D., Jiang, D., Jiang, L. et al. HLA-A+ tertiary lymphoid structures with reactivated tumor infiltrating lymphocytes are associated with a positive immunotherapy response in esophageal squamous cell carcinoma. Br J Cancer (2024). https://doi.org/10.1038/s41416-024-02712-9
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41416-024-02712-9