bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Targeted Therapy Given after Anti–PD-1 Leads to Prolonged Responses in Mouse Melanoma Models through Sustained Antitumor Immunity
Authors: Phadke Manali S. , Chen Zhihua , Li Jiannong , Mohamed Eslam , Davies Michael A. , Smalley Inna , Duckett Derek R. , Palve Vinayak , Czerniecki Brian J. , Forsyth Peter A. , Noyes David , Adeegbe Dennis O. , Eroglu Zeynep , Nguyen Kimberly T. , Tsai Kenneth Y. , Rix Uwe , Burd Christin E. , Chen Yian A. , Rodriguez Paulo C. , Smalley Keiran S.M.
Published: April 2023

Abstract
Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti–PD-1)→ TT (ceritinib–trametinib or dabrafenib–trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.
Data from Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma
Authors: Eom Jennifer , Park Saem Mul , Feisst Vaughan , Chen Chun-Jen J. , Mathy Joanna E. , McIntosh Julie D. , Angel Catherine E. , Bartlett Adam , Martin Richard , Mathy Jon A. , Cebon Jonathan S. , Black Michael A. , Brooks Anna E.S. , Dunbar P. Rod
Published: April 2023

Abstract
Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2− pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36− pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.
Data from CD28 Costimulatory Domain–Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function
Authors: Boucher Justin C. , Li Gongbo , Kotani Hiroshi , Cabral Maria L. , Morrissey Dylan , Lee Sae Bom , Spitler Kristen , Beatty Nolan J. , Cervantes Estelle V. , Shrestha Bishwas , Yu Bin , Kazi Aslamuzzaman , Wang Xuefeng , Sebti Said M. , Davila Marco L.
Published: April 2023

Abstract
An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1. Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.
Data from Tumor-Derived IL33 Promotes Tissue-Resident CD8+ T Cells and Is Required for Checkpoint Blockade Tumor Immunotherapy
Authors: Chen Lujun , Sun Runzi , Xu Junchi , Zhai Wensi , Zhang Dachuan , Yang Min , Yue Cuihua , Chen Yichao , Li Song , Turnquist Heth , Jiang Jingting , Lu Binfeng
Published: April 2023

Abstract
Immune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment by prolonging overall survival of patients with cancer. Despite advances in the clinical setting, the immune cellular network in the tumor microenvironment (TME) that mediates such therapy is not well understood. IL33 is highly expressed in normal epithelial cells but downregulated in tumor cells in advanced carcinoma. Here, we showed that IL33 was induced in tumor cells after treatment with ICB such as CTL antigen-4 (CTLA-4) and programmed death-1 (PD-1) mAbs. ST2 signaling in nontumor cells, particularly CD8+ T cells, was critical for the antitumor efficacy of ICB immunotherapy. We demonstrated that tumor-derived IL33 was crucial for the antitumor efficacy of checkpoint inhibitors. Mechanistically, IL33 increased the accumulation and effector function of tumor-resident CD103+CD8+ T cells, and CD103 expression on CD8+ T cells was required for the antitumor efficacy of IL33. In addition, IL33 also increased the numbers of CD103+ dendritic cells (DC) in the TME and CD103+ DC were required for the antitumor effect of IL33 and accumulation of tumor-infiltrating CD8+ T cells. Combination of IL33 with CTLA-4 and PD-1 ICB further prolonged survival of tumor-bearing mice. Our study established that the “danger signal” IL33 was crucial for mediating ICB cancer therapy by promoting tumor-resident adaptive immune responses.
Data from Self-Maintaining CD103+ Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses
Authors: Abd Hamid Megat , Colin-York Huw , Khalid-Alham Nasullah , Browne Molly , Cerundolo Lucia , Chen Ji-Li , Yao Xuan , Rosendo-Machado Samara , Waugh Craig , Maldonado-Perez David , Bowes Emma , Verrill Clare , Cerundolo Vincenzo , Conlon Christopher P. , Fritzsche Marco , Peng Yanchun , Dong Tao
Published: April 2023

Abstract
Enrichment of CD103+ tumor-infiltrating T lymphocytes (TIL) is associated with improved outcomes in patients. However, the characteristics of human CD103+ cytotoxic CD8+ T cells (CTL) and their role in tumor control remain unclear. We investigated the features and antitumor mechanisms of CD103+ CTLs by assessing T-cell receptor (TCR)–matched CD103+ and CD103− cancer-specific CTL immunity in vitro and its immunophenotype ex vivo. Interestingly, we found that differentiated CD103+ cancer-specific CTLs expressed the active form of TGFβ1 to continually self-regulate CD103 expression, without relying on external TGFβ1-producing cells. The presence of CD103 on CTLs improved TCR antigen sensitivity, which enabled faster cancer recognition and rapid antitumor cytotoxicity. These CD103+ CTLs had elevated energetic potential and faster migration capacity. However, they had increased inhibitory receptor coexpression and elevated T-cell apoptosis following prolonged cancer exposure. Our data provide fundamental insights into the properties of matured human CD103+ cancer-specific CTLs, which could have important implications for future designs of tissue-localized cancer immunotherapy strategies.
Data from Cross-talk between Colon Cells and Macrophages Increases ST6GALNAC1 and MUC1-sTn Expression in Ulcerative Colitis and Colitis-Associated Colon Cancer
Authors: Kvorjak Michael , Ahmed Yasmine , Miller Michelle L. , Sriram Raahul , Coronnello Claudia , Hashash Jana G. , Hartman Douglas J. , Telmer Cheryl A. , Miskov-Zivanov Natasa , Finn Olivera J. , Cascio Sandra
Published: April 2023

Abstract
Patients with ulcerative colitis have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including ulcerative colitis, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage–associated cytokines compared with normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A coculture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of ulcerative colitis and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL13 and CCL17. We demonstrated that IL13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of ulcerative colitis and CACC.

See related Spotlight on p. 160
Data from SHP-2 and PD-L1 Inhibition Combined with Radiotherapy Enhances Systemic Antitumor Effects in an Anti–PD-1–Resistant Model of Non–Small Cell Lung Cancer
Authors: Chen Dawei , Barsoumian Hampartsoum B. , Yang Liangpeng , Younes Ahmed I. , Verma Vivek , Hu Yun , Menon Hari , Wasley Mark , Masropour Fatemeh , Mosaffa Sara , Ozgen Tugce , Klein Katherine , Cortez Maria Angelica , Welsh James W.
Published: April 2023

Abstract
Immune checkpoint inhibitors, such as anti–PD-1/PD-L1, have emerged as promising therapies for advanced non–small cell lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD-1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with anti–PD-L1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti–PD-1–resistant mouse model. We treated 129Sv/Ev mice with anti–PD-1–resistant 344SQ NSCLC adenocarcinoma with oral SHP099 (a SHP-2 inhibitor) combined with XRT and intraperitoneal anti–PD-L1. Primary tumors were treated with XRT (three fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. XRT in combination with SHP099 and anti–PD-L1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. XRT also increased SHP-2+ M1 tumor-associated macrophages in abscopal tumors (P = 0.019). The addition of SHP099 also associated with a higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells. This triple-combination therapy had strong antitumor effects in a mouse model of anti–PD-1–resistant NSCLC and may be a novel therapeutic approach for anti–PD-1–resistant NSCLC in patients.
Data from iPSC-Derived Neoantigen-Specific CTL Therapy for Ewing Sarcoma
Authors: Ishii Midori , Ando Jun , Yamazaki Satoshi , Toyota Tokuko , Ohara Kazuo , Furukawa Yoshiki , Suehara Yoshiyuki , Nakanishi Mahito , Nakashima Kazutaka , Ohshima Koichi , Nakauchi Hiromitsu , Ando Miki
Published: April 2023

Abstract
The prognosis of Ewing sarcoma caused by EWS/FLI1 fusion is poor, especially after metastasis. Although therapy with CTLs targeted against altered EWS/FLI1 sequences at the gene break/fusion site may be effective, CTLs generated from peripheral blood are often exhausted because of continuous exposure to tumor antigens. We addressed this by generating induced pluripotent stem cell (iPSC)–derived functionally rejuvenated CTLs (rejT) directed against the neoantigen encoded by the EWS/FLI1 fusion gene. In this study, we examined the antitumor effects of EWS/FLI1-rejTs against Ewing sarcoma. The altered amino acid sequence at the break/fusion point of EWS/FLI1, when presented as a neoantigen, evokes an immune response that targets EWS/FLI1+ sarcoma. Although the frequency of generated EWS/FLI1-specific CTLs was only 0.003%, we successfully established CTL clones from a healthy donor. We established iPSCs from a EWS/FLI1-specific CTL clone and redifferentiated them into EWS/FLI1-specific rejTs. To evaluate cytotoxicity, we cocultured EWS/FLI1-rejTs with Ewing sarcoma cell lines. EWS/FLI1-rejTs rapidly and continuously suppressed the proliferation of Ewing sarcoma for >40 hours. Using a Ewing sarcoma xenograft mouse model, we verified the antitumor effect of EWS/FLI1-rejTs via imaging, and EWS/FLI1-rejTs conferred a statistically significant survival advantage. “Off-the-shelf” therapy is less destructive and disruptive than chemotherapy, and radiation is always desirable, particularly in adolescents, whom Ewing sarcoma most often affects. Thus, EWS/FLI1-rejTs targeting a Ewing sarcoma neoantigen could be a promising new therapeutic tool.
Data from Complement C1s and C4d as Prognostic Biomarkers in Renal Cancer: Emergence of Noncanonical Functions of C1s
Authors: Daugan Marie V. , Revel Margot , Russick Jules , Dragon-Durey Marie-Agnès , Gaboriaud Christine , Robe-Rybkine Tania , Poillerat Victoria , Grunenwald Anne , Lacroix Guillaume , Bougouin Antoine , Meylan Maxime , Verkarre Virginie , Oudard Stephane M. , Mejean Arnaud , Vano Yann A. , Perkins Geraldine , Validire Pierre , Cathelineau Xavier , Sanchez-Salas Rafael , Damotte Diane , Fremeaux-Bacchi Veronique , Cremer Isabelle , Sautès-Fridman Catherine , Fridman Wolf H. , Roumenina Lubka T.
Published: April 2023

Abstract
The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade–unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade–independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression.

See related Spotlight by Magrini and Garlanda, p. 855.

See article by Daugan et al., p. 909 (40).
Data from ST8Sia6 Promotes Tumor Growth in Mice by Inhibiting Immune Responses
Authors: Friedman David J. , Crotts Sydney B. , Shapiro Michael J. , Rajcula Matthew , McCue Shaylene , Liu Xin , Khazaie Khashayarsha , Dong Haidong , Shapiro Virginia Smith
Published: April 2023

Abstract
Many tumors exhibit increased incorporation of sialic acids into cell-surface glycans, which impact the tumor microenvironment. Sialic acid immunoglobulin-like lectins (Siglec) are receptors that recognize sialic acids and modulate immune responses, including responses to tumors. However, the roles of individual sialyltransferases in tumorigenesis and tumor growth are not well understood. Here, we examined the sialyltransferase ST8Sia6, which generated α2,8-linked disialic acids that bind to murine Siglec-E and human Siglec-7 and -9. Increased ST8Sia6 expression was found on many human tumors and associated with decreased survival in several cancers, including colon cancer. Because of this, we engineered MC38 and B16-F10 tumor lines to express ST8Sia6. ST8Sia6-expressing MC38 and B16-F10 tumors exhibited faster growth and led to decreased survival, which required host Siglec-E. ST8Sia6 expression on tumors also altered macrophage polarization toward M2, including upregulation of the immune modulator arginase, which also required Siglec-E. ST8Sia6 also accelerated tumorigenesis in a genetically engineered, spontaneous murine model of colon cancer, decreasing survival from approximately 6 months to 67 days. Thus, ST8Sia6 expression on tumors inhibits antitumor immune responses to accelerate tumor growth.
Data from NKG7 Is Required for Optimal Antitumor T-cell Immunity
Authors: Li Xian-Yang , Corvino Dillon , Nowlan Bianca , Aguilera Amelia Roman , Ng Susanna S. , Braun Matthias , Cillo Anthony R. , Bald Tobias , Smyth Mark J. , Engwerda Christian R.
Published: April 2023

Abstract
Tumor antigen-specific CD8+ T cells play a critical role in antitumor immunity. Clinical trials reinvigorating the immune system via immune checkpoint blockade (ICB) have shown remarkable clinical promise. Numerous studies have identified an association between NKG7 expression and patient outcome across different malignancies. However, aside from these correlative observations, very little is known about NKG7 and its role in antitumor immunity. Herein, we utilized single-cell RNA sequencing (scRNA-seq) datasets, NKG7-deficient mice, NKG7-reporter mice, and mouse tumor models to investigate the role of NKG7 in neoantigen-mediated tumor rejection and ICB immunotherapy. scRNA-seq of tumors from patients with metastatic melanoma or head and neck squamous cell carcinoma revealed that NKG7 expression is highly associated with cytotoxicity and specifically expressed by CD8+ T cells and natural killer (NK) cells. Furthermore, we identified a key role for NKG7 in controlling intratumor T-cell accumulation and activation. NKG7 was upregulated on intratumor antigen-specific CD8+ T cells and NK cells and required for the accumulation of T cells in the tumor microenvironment. Accordingly, neoantigen-expressing mouse tumors grew faster in Nkg7-deficient mice. Strikingly, efficacy of single or combination ICB was significantly reduced in Nkg7-deficient mice.

See related article by Wen et al., p. 162.
Data from Viral Molecular Mimicry Influences the Antitumor Immune Response in Murine and Human Melanoma
Authors: Chiaro Jacopo , Kasanen Henna H.E. , Whalley Thomas , Capasso Cristian , Grönholm Mikaela , Feola Sara , Peltonen Karita , Hamdan Firas , Hernberg Micaela , Mäkelä Siru , Karhapää Hanna , Brown Paul E. , Martins Beatriz , Fusciello Manlio , Ylösmäki Erkko O. , Greco Dario , Kreutzman Anna S. , Mustjoki Satu , Szomolay Barbara , Cerullo Vincenzo
Published: April 2023

Abstract
Molecular mimicry is one of the leading mechanisms by which infectious agents can induce autoimmunity. Whether a similar mechanism triggers an antitumor immune response is unexplored, and the role of antiviral T cells infiltrating the tumor has remained anecdotal. To address these questions, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we demonstrated that, in mice, preexisting immunity toward specific viral epitopes enhanced the efficacy of cancer immunotherapy via molecular mimicry in different settings. To understand whether this mechanism could partly explain immunotherapy responsiveness in humans, we analyzed a cohort of patients with melanoma undergoing anti-PD1 treatment who had a high IgG titer for cytomegalovirus (CMV). In this cohort of patients, we showed that high levels of CMV-specific antibodies were associated with prolonged progression-free survival and found that, in some cases, peripheral blood mononuclear cells (PBMC) could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell receptor sequencing revealed expansion of the same CD8+ T-cell clones when PBMCs were expanded with tumor or homologous viral peptides. In conclusion, we have demonstrated that preexisting immunity and molecular mimicry could influence the response to immunotherapies. In addition, we have developed a free online tool that can identify tumor antigens and neoantigens highly similar to pathogen antigens to exploit molecular mimicry and cross-reactive T cells in cancer vaccine development.
Data from CDK2 Inhibition Enhances Antitumor Immunity by Increasing IFN Response to Endogenous Retroviruses
Authors: Chen Yu , Cai Qiaomei , Pan Chaohu , Liu Wancheng , Li Lili , Liu Junxiao , Gao Meiling , Li Xiaorong , Wang Liguo , Rao Yu , Yang Heng , Cheng Genhong
Published: April 2023

Abstract
Inhibitors of cyclin-dependent kinase-2 (CDK2) are commonly used against several solid tumors, and their primary mechanisms of action were thought to include cell proliferation arrest, induction of cancer cell apoptosis and induction of differentiation. Here, we found that CDK2 inhibition by either small molecular inhibitors or genetic Cdk2 deficiency promoted antitumor immunity in murine models of fibrosarcoma and lung carcinoma. Mechanistically, CDK2 inhibition reduced phosphorylation of RB protein and transcription of E2F-mediated DNA methyltransferase 1 (DNMT1), which resulted in increased expression of endogenous retroviral RNA and type I IFN (IFN-I) response. The increased IFN-I response subsequently promoted antitumor immunity by enhancing tumor antigen presentation and CD8+ T-cell infiltration. Our studies provide evidence that inhibition of CDK2 in cancer cells suppresses tumor growth by enhancing antitumor immune responses in the tumor microenvironment, suggesting a new mechanism to enhance antitumor immunity by CDK2 inhibitors.
Data from The Fibronectin–ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells
Authors: Paavola Kevin J. , Roda Julie M. , Lin Vicky Y. , Chen Peirong , O'Hollaren Kyle P. , Ventura Richard , Crawley Suzanne C. , Li Betty , Chen Hung-I H. , Malmersjö Seth , Sharkov Nikolai A. , Horner Geoffrey , Guo Wei , Kutach Alan K. , Mondal Kalyani , Zhang Zhen , Lichtman Joshua S. , Song Christina , Rivera Lee B. , Liu Wenhui , Luo Jian , Wang Yan , Solloway Mark J. , Allan Bernard B. , Kekatpure Avantika , Starck Shelley R. , Haldankar Raj , Fan Bin , Chu Chun , Tang Jie , Molgora Martina , Colonna Marco , Kaplan Daniel D. , Hsu Jer-Yuan
Published: April 2023

Abstract
Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3–fibronectin interaction represents a “stromal checkpoint” through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.
Data from Insights into Immune Escape During Tumor Evolution and Response to Immunotherapy Using a Rat Model of Breast Cancer
Authors: Gil Del Alcazar Carlos R. , Trinh Anne , Alečković Maša , Rojas Jimenez Ernesto , Harper Nicholas W. , Oliphant Michael U.J. , Xie Shanshan , Krop Ethan D. , Lulseged Bethlehem , Murphy Katherine C. , Keenan Tanya E. , Van Allen Eliezer M. , Tolaney Sara M. , Freeman Gordon J. , Dillon Deborah A. , Muthuswamy Senthil K. , Polyak Kornelia
Published: April 2023

Abstract
Animal models are critical for the preclinical validation of cancer immunotherapies. Unfortunately, mouse breast cancer models do not faithfully reproduce the molecular subtypes and immune environment of the human disease. In particular, there are no good murine models of estrogen receptor–positive (ER+) breast cancer, the predominant subtype in patients. Here, we show that Nitroso-N-methylurea–induced mammary tumors in outbred Sprague-Dawley rats recapitulate the heterogeneity for mutational profiles, ER expression, and immune evasive mechanisms observed in human breast cancer. We demonstrate the utility of this model for preclinical studies by dissecting mechanisms of response to immunotherapy using combination TGFBR inhibition and PD-L1 blockade. Short-term treatment of early-stage tumors induced durable responses. Gene expression profiling and spatial mapping classified tumors as inflammatory and noninflammatory, and identified IFNγ, T-cell receptor (TCR), and B-cell receptor (BCR) signaling, CD74/MHC II, and epithelium-interacting CD8+ T cells as markers of response, whereas the complement system, M2 macrophage phenotype, and translation in mitochondria were associated with resistance. We found that the expression of CD74 correlated with leukocyte fraction and TCR diversity in human breast cancer. We identified a subset of rat ER+ tumors marked by expression of antigen-processing genes that had an active immune environment and responded to treatment. A gene signature characteristic of these tumors predicted disease-free survival in patients with ER+ Luminal A breast cancer and overall survival in patients with metastatic breast cancer receiving anti–PD-L1 therapy. We demonstrate the usefulness of this preclinical model for immunotherapy and suggest examination to expand immunotherapy to a subset of patients with ER+ disease.

See related Spotlight by Roussos Torres, p. 672
Data from Targeting Macrophages with CAR T Cells Delays Solid Tumor Progression and Enhances Antitumor Immunity
Authors: Sánchez-Paulete Alfonso R. , Mateus-Tique Jaime , Mollaoglu Gurkan , Nielsen Sebastian R. , Marks Adam , Lakshmi Ashwitha , Khan Jalal A. , Wilk C. Matthias , Pia Luisanna , Baccarini Alessia , Merad Miriam , Brown Brian D.
Published: April 2023

Abstract
Tumor-associated macrophages (TAM) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably with PD-1 blockade, and significantly extended mouse survival. Antitumor effects were mediated by F4.CAR-T–produced IFNγ, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigen and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof of principle to support CAR T-cell targeting of TAMs as a means to enhance antitumor immunity.
Data from Hypophosphatemia Due to Increased Effector Cell Metabolic Activity Is Associated with Neurotoxicity Symptoms in CD19-Targeted CAR T-cell Therapy
Authors: Tang Jack Pengfei , Peters Cole W. , Quiros Crystal , Wang Xiaoyan , Klomhaus Alexandra M. , Yamada Reiko E. , Timmerman John M. , Moore Theodore B. , Nowicki Theodore S.
Published: April 2023

Abstract
A major complication of chimeric antigen receptor (CAR) T-cell therapy is immune effector cell–associated neurotoxicity syndrome (ICANS), which presents as aphasia, confusion, weakness, somnolence, seizures, and coma. This is similar to the neurologic manifestations of hypophosphatemia, which can result from sudden increases in metabolic demand for phosphorylated intermediates (e.g., refeeding syndrome and sepsis). Given these similarities, we investigated whether CAR T-cell effector metabolic activity is associated with increased extracellular phosphate consumption and a possible association between hypophosphatemia and ICANS. In vitro 4–1BB and CD28 CD19-targeted CAR T-cell effector activity was found to be associated with increased consumption of media phosphorus, which was temporally associated with increased single-cell effector secretomic activity and increased phosphorus-dependent metabolic demand of the CAR T cells. A clinical cohort of 77 patients treated with CD19-targeted CAR T-cell therapy demonstrated a significant anticorrelation between serum phosphorus and ICANS incidence and severity, with earlier onset of hypophosphatemia after CAR T-cell infusion more likely to result in neurotoxicity. These results imply phosphorous level monitoring could alert to the development of ICANS in clinical scenarios.

See related Spotlight by Tobin et al., p. 1422
Data from Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors
Authors: Bajgain Pradip , Torres Chavez Alejandro G. , Balasubramanian Kishore , Fleckenstein Lindsey , Lulla Premal , Heslop Helen E. , Vera Juan , Leen Ann M.
Published: April 2023

Abstract
T-cell immunotherapy has demonstrated remarkable clinical outcomes in certain hematologic malignancies. However, efficacy in solid tumors has been suboptimal, partially due to the hostile tumor microenvironment composed of immune-inhibitory molecules. One such suppressive agent abundantly expressed in solid tumors is Fas ligand (FasL), which can trigger apoptosis of Fas-expressing effector cells such as T cells and natural killer (NK) cells. To alleviate this FasL-induced suppression of tumor-specific immune cells in solid tumors, we describe here the development of a Fas decoy that is secreted by engineered cells upon activation and sequesters the ligand, preventing it from engaging with Fas on the surface of effector cells. We further improved the immune-stimulatory effects of this approach by creating a Fas decoy and IL15 cytokine fusion protein, which enhanced the persistence and antitumor activity of decoy-engineered as well as bystander chimeric-antigen receptor (CAR) T cells in xenograft models of pancreatic cancer. Our data indicate that secreted Fas decoys can augment the efficacy of both adoptively transferred and endogenous tumor-specific effector cells in FasL-expressing solid tumors.
Data from The Tumor Microenvironment of Clear-Cell Ovarian Cancer
Authors: Devlin Michael-John , Miller Rowan , Laforets Florian , Kotantaki Panoraia , Garsed Dale W. , Kristeleit Rebecca , Bowtell David D. , McDermott Jacqueline , Maniati Eleni , Balkwill Frances R.
Published: April 2023

Abstract
Some patients with advanced clear-cell ovarian cancer (CCOC) respond to immunotherapy; however, little is known about the tumor microenvironment (TME) of this relatively rare disease. Here, we describe a comprehensive quantitative and topographical analysis of biopsies from 45 patients, 9 with Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I/II (early CCOC) and 36 with FIGO stage III/IV (advanced CCOC). We investigated 14 immune cell phenotype markers, PD-1 and ligands, and collagen structure and texture. We interrogated a microarray data set from a second cohort of 29 patients and compared the TMEs of ARID1A-wildtype (ARID1Awt) versus ARID1A-mutant (ARID1Amut) disease. We found significant variations in immune cell frequency and phenotype, checkpoint expression, and collagen matrix between the malignant cell area (MCA), leading edge (LE), and stroma. The MCA had the largest population of CD138+ plasma cells, the LE had more CD20+ B cells and T cells, whereas the stroma had more mast cells and αSMA+ fibroblasts. PD-L2 was expressed predominantly on malignant cells and was the dominant PD-1 ligand. Compared with early CCOC, advanced-stage disease had significantly more fibroblasts and a more complex collagen matrix, with microarray analysis indicating “TGFβ remodeling of the extracellular matrix” as the most significantly enriched pathway. Data showed significant differences in immune cell populations, collagen matrix, and cytokine expression between ARID1Awt and ARID1Amut CCOC, which may reflect different paths of tumorigenesis and the relationship to endometriosis. Increased infiltration of CD8+ T cells within the MCA and CD4+ T cells at the LE and stroma significantly associated with decreased overall survival.
Data from Hippo Signaling Pathway Regulates Cancer Cell–Intrinsic MHC-II Expression
Authors: Zeng Zexian , Gu Shengqing Stan , Ouardaoui Nofal , Tymm Carly , Yang Lin , Wong Cheryl J. , Li Dian , Zhang Wubing , Wang Xiaoqing , Weirather Jason L. , Rodig Scott J. , Hodi F. Stephen , Brown Myles , Liu X. Shirley
Published: April 2023

Abstract
MHC-II is known to be mainly expressed on the surface of antigen-presenting cells. Evidence suggests MHC-II is also expressed by cancer cells and may be associated with better immunotherapy responses. However, the role and regulation of MHC-II in cancer cells remain unclear. In this study, we leveraged data mining and experimental validation to elucidate the regulation of MHC-II in cancer cells and its role in modulating the response to immunotherapy. We collated an extensive collection of omics data to examine cancer cell–intrinsic MHC-II expression and its association with immunotherapy outcomes. We then tested the functional relevance of cancer cell–intrinsic MHC-II expression using a syngeneic transplantation model. Finally, we performed data mining to identify pathways potentially involved in the regulation of MHC-II expression, and experimentally validated candidate regulators. Analyses of preimmunotherapy clinical samples in the CheckMate 064 trial revealed that cancer cell–intrinsic MHC-II protein was positively correlated with more favorable immunotherapy outcomes. Comprehensive meta-analyses of multiomics data from an exhaustive collection of data revealed that MHC-II is heterogeneously expressed in various solid tumors, and its expression is particularly high in melanoma. Using a syngeneic transplantation model, we further established that melanoma cells with high MHC-II responded better to anti–PD-1 treatment. Data mining followed by experimental validation revealed the Hippo signaling pathway as a potential regulator of melanoma MHC-II expression. In summary, we identified the Hippo signaling pathway as a novel regulator of cancer cell–intrinsic MHC-II expression. These findings suggest modulation of MHC-II in melanoma could potentially improve immunotherapy response.
Data from Adenosine-Deaminase-Acting-on-RNA-1 Facilitates T-cell Migration toward Human Melanoma Cells
Authors: Margolis Naama , Moalem Hanna , Meirson Tomer , Galore-Haskel Gilli , Markovits Ettai , Baruch Erez N. , Vizel Bella , Yeffet Avner , Kanterman-Rifman Julia , Debby Assaf , Besser Michal J. , Schachter Jacob , Markel Gal
Published: April 2023

Abstract
The effect of tumor/T-cell interactions on subsequent immune infiltration is undefined. Here, we report that preexposure of melanoma cells to cognate T cells enhanced the chemotaxis of new T cells in vitro. The effect was HLA class I–restricted and IFNγ-dependent, as it was abolished by β2M-knockdown, MHC-blocking antibodies, JAK1 inhibitors, JAK1-silencing and IFNgR1-blocking antibodies. RNA sequencing (RNA-seq) of 73 melanoma metastases showed a significant correlation between the interferon-inducible p150 isoform of adenosine-deaminase-acting-on-RNA-1 (ADAR1) enzyme and immune infiltration. Consistent with this, cocultures of cognate melanoma/T-cell pairs led to IFNγ-dependent induction of ADAR1-p150 in the melanoma cells, as visualized in situ using dynamic cell blocks, in ovo using fertilized chick eggs, and in vitro with Western blots. ADAR1 staining and RNA-seq in patient-derived biopsies following immunotherapy showed a rise in ADAR1-p150 expression concurrently with CD8+ cell infiltration and clinical response. Silencing ADAR1-p150 abolished the IFNγ-driven enhanced T-cell migration, confirming its mechanistic role. Silencing and overexpression of the constitutive isoform of ADAR1, ADAR1-p110, decreased and increased T-cell migration, respectively. Chemokine arrays showed that ADAR1 controls the secretion of multiple chemokines from melanoma cells, probably through microRNA-mediated regulation. Chemokine receptor blockade eliminated the IFNγ-driven T-cell chemotaxis. We propose that the constitutive ADAR1 downregulation observed in melanoma contributes to immune exclusion, whereas antigen-specific T cells induce ADAR1-p150 by releasing IFNγ, which can drive T-cell infiltration.
Data from In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti–PD-1/PD-L1 Therapies
Authors: Denis Morgane , Grasselly Chloé , Choffour Pierre-Antoine , Wierinckx Anne , Mathé Doriane , Chettab Kamel , Tourette Anne , Talhi Nolan , Bourguignon Aurore , Birzele Fabian , Kress Elsa , Jordheim Lars Petter , Klein Christian , Matera Eva-Laure , Dumontet Charles
Published: April 2023

Abstract
Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti–PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti–PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti–PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.
Data from CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer
Authors: Nishii Kazuya , Ohashi Kadoaki , Tomida Shuta , Nakasuka Takamasa , Hirabae Atsuko , Okawa Sachi , Nishimura Jun , Higo Hisao , Watanabe Hiromi , Kano Hirohisa , Ando Chihiro , Makimoto Go , Ninomiya Kiichiro , Kato Yuka , Kubo Toshio , Ichihara Eiki , Hotta Katsuyuki , Tabata Masahiro , Toyooka Shinichi , Udono Heiichiro , Maeda Yoshinobu , Kiura Katsuyuki
Published: April 2023

Abstract
Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non–small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti–PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.
Data from BCG-Induced Tumor Immunity Requires Tumor-Intrinsic CIITA Independent of MHC-II
Authors: Redelman-Sidi Gil , Binyamin Anna , Antonelli Anthony C. , Catalano Will , Bean James , Al-Ahmadie Hikmat , Jungbluth Achim A. , Glickman Michael S.
Published: April 2023

Abstract
For decades, BCG immunotherapy has been the standard of care for non–muscle-invasive bladder cancer. Despite this clinical experience, the mechanism by which BCG stimulates tumor-eliminating immunity is unclear, and there is still a need for more accurate prediction of clinical outcomes in advance of treatment initiation. We have shown that BCG stimulates tumor-specific T-cell immunity that requires tumor cell expression of the IFNγ receptor (IFNGR); however, the downstream components of IFNGR signaling responsible for responsiveness to BCG are unknown. Here, we demonstrate that the IFNγ-driven, tumor cell intrinsic expression of the class II transactivator CIITA is required for activation of a tumor-specific CD4 T-cell response and BCG-induced tumor immunity. Despite the established role for CIITA in controlling MHC-II antigen presentation machinery, the requirement for CIITA is independent of MHC-II and associated genes. Rather, we find that CIITA is required for a broader tumor-intrinsic transcriptional program linked to critical pathways of tumor immunity via mechanisms that remain to be determined. Tumor cell intrinsic expression of CIITA is not required for a response to immunotherapy targeting programmed cell death protein 1 (PD-1), suggesting that different modalities of immunotherapy for bladder cancer could be employed based on tumor-intrinsic characteristics.
Data from Radiofrequency Ablation Remodels the Tumor Microenvironment and Promotes Neutrophil-Mediated Abscopal Immunomodulation in Pancreatic Cancer
Authors: Faraoni Erika Y. , O'Brien Baylee J. , Strickland Lincoln N. , Osborn Baron K. , Mota Victoria , Chaney Jarod , Atkins Constance Lynn , Cen Putao , Rowe Julie , Cardenas Jessica , Poulsen Kyle L. , Wray Curtis J. , Thosani Nirav C. , Bailey-Lundberg Jennifer M.
Published: April 2023

Abstract
Pancreatic ductal adenocarcinoma (PDAC) presents a 5-year overall survival rate of 11%, despite efforts to improve clinical outcomes in the past two decades. Therapeutic resistance is a hallmark of this disease, due to its dense and suppressive tumor microenvironment (TME). Endoscopic ultrasound-guided radiofrequency ablation (EUS-RFA) is a promising local ablative and potential immunomodulatory therapy for PDAC. In this study, we performed RFA in a preclinical tumor-bearing KrasG12D; Trp53R172H/+; Pdx1:Cre (KPC) syngeneic model, analyzed local and abscopal affects after RFA and compared our findings with resected PDAC specimens. We found that RFA reduced PDAC tumor progression in vivo and promoted strong TME remodeling. In addition, we discovered tumor-infiltrating neutrophils determined abscopal effects. Using imaging mass cytometry, we showed that RFA elevated dendritic cell numbers in RFA-treated tumors and promoted a significant CD4+ and CD8+ T-cell abscopal response. In addition, RFA elevated levels of programmed death-ligand 1 (PD-L1) and checkpoint blockade inhibition targeting PD-L1 sustained tumor growth reduction in the context of RFA. This study indicates RFA treatment, which has been shown to increase tumor antigen shedding, promotes antitumor immunity. This is critical in PDAC where recent clinical immunotherapy trials have not resulted in substantial changes in overall survival.