ARTICLE https://doi.org/10.1038/s41467-021-22433-4 OPEN Epigenetic modulation of immune synaptic- cytoskeletal networks potentiates γδ T cell- mediated cytotoxicity in lung cancer Rueyhung R. Weng 1, Hsuan-Hsuan Lu 1, Chien-Ting Lin2,3, Chia-Chi Fan4, Rong-Shan Lin2,3, Tai-Chung Huang 1, Shu-Yung Lin 1, Yi-Jhen Huang 4, Yi-Hsiu Juan1, Yi-Chieh Wu4, Zheng-Ci Hung1, Chi Liu5, Xuan-Hui Lin2,3, Wan-Chen Hsieh 6,7, Tzu-Yuan Chiu8, Jung-Chi Liao 8, Yen-Ling Chiu9,10,11, ✉ Shih-Yu Chen6, Chong-Jen Yu 1,12 & Hsing-Chen Tsai 1,4,11 1234567890():,; γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyl- transferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordi- nated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management. 1 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. 2 Tai Cheng Stem Cell Therapy Center, National Taiwan University, Taipei, Taiwan. 3 Pell Biomedical Technology Ltd, Taipei, Taiwan. 4 Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan. 5 Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan. 6 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. 7 Genome and Systems Biology Degree Program, National Taiwan University, Taipei, Taiwan. 8 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan. 9 Graduate Program in Biomedical Informatics, Department of Computer Science and Engineering, College of Informatics, Yuan Ze University, Taoyuan, Taiwan. 10 Department of Medical Research, Far Eastern Memorial Hospital, New Taipei City, Taiwan. 11 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan. 12 Department of Internal Medicine, College of Medicine, ✉ National Taiwan University, Taipei, Taiwan. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:2163 | https://doi.org/10.1038/s41467-021-22433-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22433-4 mmune-based therapy has become a promising strategy in the isolating cell surface proteins from A549 human lung cancer cells Itreatment of lung cancer. Nevertheless, only a small subset of before and after DAC treatment using the EZ-link Sulfo-NHS-SS- patients respond to current checkpoint blockade therapies. biotin-assisted biotinylation method, followed by a SILAC-based Therefore, the adoptive transfer of immune cells targeting tumor quantitative proteomics approach (Fig. 1a)14. We employed a low- cells has emerged as an attractive therapeutic option1–3. γδ dose treatment protocol established in our previous study to T cells, a distinct subset of T cells that sense molecular stress manifest the drug’s epigenetic effects over cytotoxicity15. First, signals from pathogen-infected or transformed cells, can display lung cancer cells were differentially labeled by growing in cell 13 broad tumor-targeting capabilities in a major histocompatibility culture media containing heavy arginine (L- Arginine- C6)/lysine 4 13 complex (MHC)-independent manner . These cells provide (L-Lysine- C6,) or normal arginine/lysine. The SILAC-labeled potential therapeutic opportunities for patients who are inher- cells (heavy) and the unlabeled cells (light) were treated with ently unresponsive to checkpoint blockade due to low mutational phosphate-buffered saline and 100 nM DAC, respectively, for burden, or have acquired defects along the TCR-MHC axis. three consecutive days (D3) and grown in a drug-free medium for However, the adoptive transfer of autologous or allogeneic γδ another three days (D3R3) (Fig. 1b). After biotinylation of the cell T cells in earlier clinical trials showed limited efficacy5–7. Thus, a surface proteins, total cell lysates of DAC-treated cells at D3 and better understanding of tumor lytic interactions between γδ T D3R3 were 1:1 mixed with their corresponding mock-treated and cancer cells are required to maximize the therapeutic efficacy counterparts and subjected to surface protein isolation followed by of γδ T-based immunotherapy. quantitative proteomics. We identified 666 and 831 Gene DNA methyltransferase inhibitors (DNMTis) have been Ontology (GO)-annotated surface proteins (corresponding to approved by the U.S. FDA for the treatment of hematological 8791 and 11,898 unique peptides) in A549 cells at D3 and D3R3, malignancies. Nevertheless, the therapeutic efficacies of these respectively. (Fig. 1c and Supplementary Fig. 1a). The continued drugs used as a single agent in solid tumors have been less than increase in identified surface proteins after drug withdrawal is satisfactory. Compelling evidence has indicated the immunomo- consistent with our prior report of epigenetic memory effects dulatory effects of DNMTis that target MHC and T cell receptor following transient drug exposure15. Among all the identified (TCR) axis8–12. On the other hand, little is known about whether proteins, we focused on 314 proteins that showed sustained and how DNMTis may reshape surface proteins of cancer cells to upregulation upon DAC treatment by at least 1.4-fold for both D3 facilitate MHC-unrestricted recognition and tumor lysis in cell- and D3R3, for their continued induction after drug withdrawal based immunotherapies. Moreover, molecular characteristics of suggested a possible epigenetic regulation (Fig. 1d). Many of these patients that may potentially benefit from this kind of immuno- are immune-related proteins. In addition to MHC molecules and modulation have not been identified. certain immune checkpoint proteins that were previously known Here we show that DNMTis markedly alter the surface pro- to be upregulated by DAC11,12, we uncovered a plethora of teome of lung cancer cells using stable isotope labeling by amino proteins that participate in innate immunity or MHC-unrestricted acids in cell culture (SILAC)-based quantitative proteomics. immunity, such as MHC class I polypeptide-related sequences A These experiments show upregulation of multiple MHC- and B (i.e., MICA, MICB), which are ligands of NKG2D on γδ T unrestricted immune recognition molecules following transient and NK cells (Fig. 1e)16,17. DAC also upregulates UL16-binding DNMTi treatment at low doses. Gene ontology analysis of the proteins 2 and 3 (i.e., ULBP2 and ULBP3), another group of DNMTi-induced surface proteome reveals a high association with NKG2D ligands that are expressed in many cancers and in γδ T cell activation. We demonstrate that DNMTi treatment of stressed/damaged tissues (Fig. 1e)18–20. These molecules have been cancer cells enhances immune synapse formation between ex vivo identified as targets for tumor immunosurveillance by the innate expanded allogeneic γδ T cells and cancer cells and potentiates immune system and may elicit antitumor immunity without the antitumor immunity by γδ T cells. A combined genome-wide requirement for conventional MHC-restricted antigen analysis of the DNA methylome, mRNA-seq, and Omni-ATAC- presentation21. In addition, the two death receptors (i.e., TRAIL- seq reveals coordinated epigenetic regulatory patterns of immune R1 and TRAIL-R2) for TNF-related apoptosis-inducing ligand synaptic cytoskeleton networks. Additionally, primary lung can- (TRAIL) that induce cancer apoptosis as part of immune cer tissues can be stratified by immune cytoskeleton patterns surveillance22 were significantly upregulated at D3R3 (Fig. 1e). indicative of responses to γδ T-mediated cytotoxicity, which may Interestingly, analysis of SILAC-based surfaceomes in another aid in patient selection in future clinical trials for precision two human lung cancer cell lines, H1299 and CL1-0, revealed immunotherapy. These findings support the broad applicability highly similar profiles of DAC-induced surface proteins. There of DNMTis in remodeling the cancer cell cytoskeleton for MHC- were 431 proteins commonly upregulated by DAC in all three lung unrestricted γδ T cell-based therapy. cancer cell lines for D3R3 (Fig. 1f and Supplementary Fig. 1b). The involvement of DAC-induced surface molecules in the innate immune response is evident, and we mapped these proteins Results against the innate immune interactomes from InnateDB, an DNMTi upregulates surface molecules related to γδ T cell extensively curated database of innate immune pathways and activation. Two FDA-approved DNMTis, decitabine (Dacogen, interactions23. We uncovered DAC-mediated innate immune DAC), and azacytidine (Vidaza, AZA) are cytidine analogs that molecules that participate in adhesion/cell-cell interactions (e.g., incorporate into DNA/RNA and deplete DNMTs through irre- CD97 and ICAM-1), the cytoskeleton (e.g.,