bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Immune Remodeling of the Extracellular Matrix Drives Loss of Cancer Stem Cells and Tumor Rejection
Authors: Pires Ana , Greenshields-Watson Alexander , Jones Emma , Smart Kathryn , Lauder Sarah N. , Somerville Michelle , Milutinovic Stefan , Kendrick Howard , Hindley James P. , French Rhiannon , Smalley Matthew J. , Watkins William J. , Andrews Robert , Godkin Andrew , Gallimore Awen
Published: April 2023

Abstract
The nature of the tumor microenvironment (TME) influences the ability of tumor-specific T cells to control tumor growth. In this study, we performed an unbiased comparison of the TME of regulatory T-cell (Treg)–replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T-cell infiltrates where responding tumors were T-cell rich and ECM poor, whereas the converse was observed in non-responder tumors. For this reason, we hypothesized that the ECM acted as a barrier to successful T-cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T-cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell–like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells but in widescale remodeling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity.
Data from Prevalent and Diverse Intratumoral Oncoprotein-Specific CD8+ T Cells within Polyomavirus-Driven Merkel Cell Carcinomas
Authors: Jing Lichen , Ott Mariliis , Church Candice D. , Kulikauskas Rima M. , Ibrani Dafina , Iyer Jayasri G. , Afanasiev Olga K. , Colunga Aric , Cook Maclean M. , Xie Hong , Greninger Alexander L. , Paulson Kelly G. , Chapuis Aude G. , Bhatia Shailender , Nghiem Paul , Koelle David M.
Published: April 2023

Abstract
Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag–specific T cells. Strategies to increase CD8+ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8+ T cells recognizing 1–8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8+ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV+ MCC had HLA alleles with the genetic potential that restrict CD8+ T-cell responses to MCPyV T-Ag. The LT AA 70–110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag–expressing DCs was documented. Recovery of MCPyV oncoprotein–specific CD8+ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.
Data from Paclitaxel Induces Immunogenic Cell Death in Ovarian Cancer via TLR4/IKK2/SNARE-Dependent Exocytosis
Authors: Lau Tat San , Chan Loucia Kit Ying , Man Gene Chi Wai , Wong Chi Hang , Lee Jacqueline Ho Sze , Yim So Fan , Cheung Tak Hong , McNeish Iain A. , Kwong Joseph
Published: April 2023

Abstract
Emerging evidence shows that the efficacy of chemotherapeutic drugs is reliant on their capability to induce immunogenic cell death (ICD), thus transforming dying tumor cells into antitumor vaccines. We wanted to uncover potential therapeutic strategies that target ovarian cancer by having a better understanding of the standard-of-care chemotherapy treatment. Here, we showed in ovarian cancer that paclitaxel induced ICD-associated damage-associated molecular patterns (DAMP, such as CALR exposure, ATP secretion, and HMGB1 release) in vitro and elicited significant antitumor responses in tumor vaccination assays in vivo. Paclitaxel-induced TLR4 signaling was essential to the release of DAMPs, which led to the activation of NF-κB–mediated CCL2 transcription and IkappaB kinase 2–mediated SNARE-dependent vesicle exocytosis, thus exposing CALR on the cell surface. Paclitaxel induced endoplasmic reticulum stress, which triggered protein kinase R–like ER kinase activation and eukaryotic translation initiation factor 2α phosphorylation independent of TLR4. Paclitaxel chemotherapy induced T-cell infiltration in ovarian tumors of the responsive patients; CALR expression in primary ovarian tumors also correlated with patients' survival and patient response to chemotherapy. These findings suggest that the effectiveness of paclitaxel relied upon the activation of antitumor immunity through ICD via TLR4 and highlighted the importance of CALR expression in cancer cells as an indicator of response to paclitaxel chemotherapy in ovarian cancer.
Data from A DNA-Launched Nanoparticle Vaccine Elicits CD8+ T-cell Immunity to Promote In Vivo Tumor Control
Authors: Xu Ziyang , Chokkalingam Neethu , Tello-Ruiz Edgar , Wise Megan C. , Bah Mamadou A. , Walker Susanne , Tursi Nicholas J. , Fisher Paul D. , Schultheis Katherine , Broderick Kate E. , Humeau Laurent , Kulp Daniel W. , Weiner David B.
Published: April 2023

Abstract
Cytolytic T cells (CTL) play a pivotal role in surveillance against tumors. Induction of CTL responses by vaccination may be challenging, as it requires direct transduction of target cells or special adjuvants to promote cross-presentation. Here, we observed induction of robust CTL responses through electroporation-facilitated, DNA-launched nanoparticle vaccination (DLnano-vaccines). Electroporation was observed to mediate transient tissue apoptosis and macrophage infiltration, which were deemed essential to the induction of CTLs by DLnano-vaccines through a systemic macrophage depletion study. Bolus delivery of protein nano-vaccines followed by electroporation, however, failed to induce CTLs, suggesting direct in vivo production of nano-vaccines may be required. Following these observations, new DLnano-vaccines scaffolding immunodominant melanoma Gp100 and Trp2 epitopes were designed and shown to induce more potent and consistent epitope-specific CTL responses than the corresponding DNA monomeric vaccines or CpG-adjuvanted peptide vaccines. DNA, but not recombinant protein, nano-vaccinations induced CTL responses to these epitopes and suppressed melanoma tumor growth in mouse models in a CD8+ T-cell–dependent fashion. Further studies to explore the use of DLnano-vaccines against other cancer targets and the biology with which they induce CTLs are important.
Data from Combined CD44- and CD25-Targeted Near-Infrared Photoimmunotherapy Selectively Kills Cancer and Regulatory T Cells in Syngeneic Mouse Cancer Models
Authors: Maruoka Yasuhiro , Furusawa Aki , Okada Ryuhei , Inagaki Fuyuki , Fujimura Daiki , Wakiyama Hiroaki , Kato Takuya , Nagaya Tadanobu , Choyke Peter L. , Kobayashi Hisataka
Published: April 2023

Abstract
Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a mAb conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is a surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, CD25-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Treg), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44- and CD25-targeted NIR-PIT also resulted in some complete remissions. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.
Data from Quantification of Early-Stage Myeloid-Derived Suppressor Cells in Cancer Requires Excluding Basophils
Authors: Khan ANM Nazmul H. , Emmons Tiffany R. , Wong Jerry T. , Alqassim Emad , Singel Kelly L. , Mark Jaron , Smith Brandon E. , Tario Joseph D. , Eng Kevin H. , Moysich Kirsten B. , Odunsi Kunle , Abrams Scott I. , Segal Brahm H.
Published: April 2023

Abstract
Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123high expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.
Data from Dual Relief of T-lymphocyte Proliferation and Effector Function Underlies Response to PD-1 Blockade in Epithelial Malignancies
Authors: Balança Camille-Charlotte , Scarlata Clara-Maria , Michelas Marie , Devaud Christel , Sarradin Victor , Franchet Camille , Martinez Gomez Carlos , Gomez-Roca Carlos , Tosolini Marie , Heaugwane Diana , Lauzéral-Vizcaino Françoise , Mir-Mesnier Lucile , Féliu Virginie , Valle Carine , Pont Frédéric , Ferron Gwénaël , Gladieff Laurence , Motton Stéphanie , Tanguy Le Gac Yann , Dupret-Bories Agnès , Sarini Jérôme , Vairel Benjamin , Illac Claire , Siegfried-Vergnon Aurore , Mery Eliane , Fournié Jean-Jacques , Vergez Sébastien , Delord Jean-Pierre , Rochaix Philippe , Martinez Alejandra , Ayyoub Maha
Published: April 2023

Abstract
Although understanding of T-cell exhaustion is widely based on mouse models, its analysis in patients with cancer could provide clues indicating tumor sensitivity to immune checkpoint blockade (ICB). Data suggest a role for costimulatory pathways, particularly CD28, in exhausted T-cell responsiveness to PD-1/PD-L1 blockade. Here, we used single-cell transcriptomic, phenotypic, and functional approaches to dissect the relation between CD8+ T-cell exhaustion, CD28 costimulation, and tumor specificity in head and neck, cervical, and ovarian cancers. We found that memory tumor–specific CD8+ T cells, but not bystander cells, sequentially express immune checkpoints once they infiltrate tumors, leading, in situ, to a functionally exhausted population. Exhausted T cells were nonetheless endowed with effector and tumor residency potential but exhibited loss of the costimulatory receptor CD28 in comparison with their circulating memory counterparts. Accordingly, PD-1 inhibition improved proliferation of circulating tumor–specific CD8+ T cells and reversed functional exhaustion of specific T cells at tumor sites. In agreement with their tumor specificity, high infiltration of tumors by exhausted cells was predictive of response to therapy and survival in ICB-treated patients with head and neck cancer. Our results showed that PD-1 blockade–mediated proliferation/reinvigoration of circulating memory T cells and local reversion of exhaustion occur concurrently to control tumors.
Data from Inflammation-Induced Abnormal Expression of Self-molecules on Epithelial Cells: Targets for Tumor Immunoprevention
Authors: Jacqueline Camille , Lee Amanda , Frey Nolan , Minden Jonathan S. , Finn Olivera J.
Published: April 2023

Abstract
Tumor-associated antigens (TAA) are self-molecules abnormally expressed on tumor cells, which elicit humoral and cellular immunity and are targets of immunosurveillance. Immunity to TAAs is found in some healthy individuals with no history of cancer and correlates positively with a history of acute inflammatory and infectious events and cancer risk reduction. This suggests a potential role in cancer immunosurveillance for the immune memory elicited against disease-associated antigens (DAA) expressed on infected and inflamed tissues that are later recognized on tumors as TAAs. To understand probable sources for DAA generation, we investigated in vitro the role of inflammation that accompanies both infection and carcinogenesis. After exposure of normal primary breast epithelial cells to proinflammatory cytokines IL1β, IL6, and TNFα, or macrophages producing these cytokines, we saw transient overexpression of well-known TAAs, carcinoembryonic antigen and Her-2/neu, and overexpression and hypoglycosylation of MUC1. We documented inflammation-induced changes in the global cellular proteome by 2D difference gel electrophoresis combined with mass spectrometry and identified seven new DAAs. Through gene profiling, we showed that the cytokine treatment activated NF-κB and transcription of the identified DAAs. We tested three in vitro–identified DAAs, Serpin B1, S100A9, and SOD2, and found them overexpressed in premalignant and malignant breast tissues as well as in inflammatory conditions of the colon, stomach, and liver. This new category of TAAs, which are also DAAs, represent a potentially large number of predictable, shared, immunogenic, and safe antigens to use in preventative cancer vaccines and as targets for cancer therapies.
Data from The Pellino1–PKCθ Signaling Axis Is an Essential Target for Improving Antitumor CD8+ T-lymphocyte Function
Authors: Park Jihyun , Lee Si-Yeon , Jeon Yoon , Kim Kyung-Mo , Lee Jin-Kwan , Ko Jiwon , Park Eun-Ji , Yoon Joon-Sup , Kang Baeki E. , Ryu Dongryeol , Lee Ho , Shin Su-Jin , Go Heounjeong , Lee Chang-Woo
Published: April 2023

Abstract
CD8+ T cells play an important role in the elimination of tumors. However, the underlying mechanisms involved in eliciting and maintaining effector responses in CD8+ T cells remain to be elucidated. Pellino1 (Peli1) is a receptor signal-responsive ubiquitin E3 ligase, which acts as a critical mediator for innate immunity. Here, we found that the risk of developing tumors was dependent on Peli1 expression. Peli1 was upregulated in CD8+ T cells among tumor-infiltrating lymphocytes (TIL). In contrast, a deficit of Peli1 enhanced the maintenance and effector function of CD8+ TILs. The development of Peli1-deficient CD8+ TILs prevented T-cell exhaustion and retained the hyperactivated states of T cells to eliminate tumors. We also found that Peli1 directly interacted with protein kinase C-theta (PKCθ), a central kinase in T-cell receptor downstream signal transduction, but whose role in tumor immunology remains unknown. Peli1 inhibited the PKCθ pathway by lysine 48–mediated ubiquitination degradation in CD8+ TILs. In summary, the Peli1–PKCθ signaling axis is a common inhibitory mechanism that prevents antitumor CD8+ T-cell function, and thus targeting Peli1 may be a useful therapeutic strategy for improving cytotoxic T-cell activity.
Data from Neoadjuvant STING Activation, Extended Half-life IL2, and Checkpoint Blockade Promote Metastasis Clearance via Sustained NK-cell Activation
Authors: Milling Lauren E. , Garafola Daniel , Agarwal Yash , Wu Shengwei , Thomas Ayush , Donahue Nathan , Adams Josetta , Thai Nikki , Suh Heikyung , Irvine Darrell J.
Published: April 2023

Abstract
Combination immunotherapy treatments that recruit both innate and adaptive immunity have the potential to increase cancer response rates by engaging a more complete repertoire of effector mechanisms. Here, we combined intratumoral STimulator of INterferon Genes (STING) agonist therapy with systemically injected extended half-life IL2 and anti–PD-1 checkpoint blockade (hereafter CIP therapy) to drive innate and adaptive antitumor immunity in models of triple-negative breast cancer. Unlike treatment with the individual components, this trivalent immunotherapy halted primary tumor progression and led to long-term remission for a majority of animals in two spontaneously metastasizing orthotopic breast tumor models, though only as a neoadjuvant therapy but not adjuvant therapy. CIP therapy induced antitumor T-cell responses, but protection from metastatic relapse depended on natural killer (NK) cells. The combination of STING agonists with IL2/anti–PD-1 synergized to stimulate sustained granzyme and cytokine expression by lung-infiltrating NK cells. Type I IFNs generated as a result of STING agonism, combined with IL2, acted in a positive-feedback loop by enhancing the expression of IFNAR-1 and CD25 on lung NK cells. These results suggest that NK cells can be therapeutically targeted to effectively eliminate tumor metastases.

See related Spotlight by Demaria, p. 3.
Data from Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells
Authors: Pfirschke Christina , Zilionis Rapolas , Engblom Camilla , Messemaker Marius , Zou Angela E. , Rickelt Steffen , Gort-Freitas Nicolas A. , Lin Yunkang , Bill Ruben , Siwicki Marie , Gungabeesoon Jeremy , Sprachman Melissa M. , Marquard Angela N. , Rodell Christopher B. , Cuccarese Michael F. , Quintana Jeremy , Ahmed Maaz S. , Kohler Rainer H. , Savova Virginia , Weissleder Ralph , Klein Allon M. , Pittet Mikael J.
Published: April 2023

Abstract
Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R− cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor–mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell–targeted immunotherapies and promote antitumor immunity.

See related Spotlight by Burrello and de Visser, p. 4.
Data from An In Vivo Screen to Identify Short Peptide Mimotopes with Enhanced Antitumor Immunogenicity
Authors: He Xuedan , Zhou Shiqi , Quinn Breandan , Jahagirdar Dushyant , Ortega Joaquin , Long Mark D. , Abrams Scott I. , Lovell Jonathan F.
Published: April 2023

Abstract
Tumor-associated self-antigens are potential cancer vaccine targets but suffer from limited immunogenicity. There are examples of mutated, short self-peptides inducing epitope-specific CD8+ T cells more efficiently than the wild-type epitope, but current approaches cannot yet reliably identify such epitopes, which are referred to as enhanced mimotopes (“e-mimotopes”). Here, we present a generalized strategy to develop e-mimotopes, using the tyrosinase-related protein 2 (Trp2) peptide Trp2180–188, which is a murine MHC class I (MHC-I) epitope, as a test case. Using a vaccine adjuvant that induces peptide particle formation and strong cellular responses with nanogram antigen doses, a two-step method systematically identified e-mimotope candidates with murine immunization. First, position-scanning peptide microlibraries were generated in which each position of the wild-type epitope sequence was randomized. Randomization of only one specific residue of the Trp2 epitope increased antitumor immunogenicity. Second, all 20 amino acids were individually substituted and tested at that position, enabling the identification of two e-mimotopes with single amino acid mutations. Despite similar MHC-I affinity compared with the wild-type epitope, e-mimotope immunization elicited improved Trp2-specific cytotoxic T-cell phenotypes and improved T-cell receptor affinity for both the e-mimotopes and the native epitope, resulting in better outcomes in multiple prophylactic and therapeutic tumor models. The screening method was also applied to other targets with other murine MHC-I restriction elements, including epitopes within glycoprotein 70 and Wilms' Tumor Gene 1, to identify additional e-mimotopes with enhanced potency.
Data from γδ T Cells Support Antigen-Specific αβ T cell–Mediated Antitumor Responses during BCG Treatment for Bladder Cancer
Authors: Ji Niannian , Mukherjee Neelam , Shu Zhen-Ju , Reyes Ryan M. , Meeks Joshua J. , McConkey David J. , Gelfond Jonathan A. , Curiel Tyler J. , Svatek Robert S.
Published: April 2023

Abstract
Bacillus Calmette-Guérin (BCG) is the most effective intravesical agent at reducing recurrence for patients with high-grade, non–muscle-invasive bladder cancer. Nevertheless, response to BCG is variable and strategies to boost BCG efficacy have not materialized. Prior work demonstrated a requirement for either conventional αβ or nonconventional γδ T cells in mediating BCG treatment efficacy, yet the importance of T-cell antigen specificity for BCG's treatment effect is unclear. Here, we provide direct evidence to show that BCG increases the number of tumor antigen–specific αβ T cells in patients with bladder cancer and protects mice from subsequent same-tumor challenge, supporting BCG induction of tumor-specific memory and protection. Adoptive T-cell transfers of antigen-specific αβ T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen–specific αβ T cells contributed to BCG efficacy. BCG-specific antitumor immunity, however, also required nonconventional γδ T cells. Prior work shows that the mTOR inhibitor rapamycin induces the proliferation and effector function of γδ T cells. Here, rapamycin increased BCG efficacy against both mouse and human bladder cancer in vivo in a γδ T cell–dependent manner. Thus, γδ T cells augment antitumor adaptive immune effects of BCG and support rapamycin as a promising approach to boost BCG efficacy in the treatment of non–muscle-invasive bladder cancer.
Data from Site-Dependent Immune Escape Due to Impaired Dendritic Cell Cross-Priming
Authors: Diamond Mark S. , Lin Jeffrey H. , Vonderheide Robert H.
Published: April 2023

Abstract
T-cell recognition of tumor neoantigens is critical for cancer immune surveillance and the efficacy of immunotherapy. Tumors can evade host immunity by altering their antigenicity or orchestrating an immunosuppressive microenvironment, leading to outgrowth of poorly immunogenic tumors through the well-established process of cancer immunoediting. Whether cancer immune surveillance and immunoediting depend on the tissue site of origin, however, is poorly understood. Herein, we studied T-cell–mediated surveillance of antigenic, clonal murine pancreatic adenocarcinoma cells expressing neoantigen. Whereas such tumors are robustly eliminated after subcutaneous or intravenous challenge, we observed selective immune escape within the pancreas and peritoneum. Tumor outgrowth occurred in the absence of immunoediting, and antitumor immunity could not be rescued by PD-1 or CTLA-4 checkpoint blockade. Instead, tumor escape was associated with diminished CD8+ T-cell priming by type I conventional dendritic cells (cDC1). Enhancing cDC1 cross-presentation by CD40 agonist treatment restored immunologic control by promoting T-cell priming and broadening T-cell responses through epitope spread. These findings demonstrate that immune escape of highly antigenic tumors can occur without immunoediting in a tissue-restricted manner and highlight barriers to cDC1-mediated T-cell priming imposed by certain microenvironments that must be addressed for successful combination immunotherapies.
Data from Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells
Authors: Somasundaram Ashwin , Cillo Anthony R. , Lampenfeld Caleb , Workman Creg J. , Kunning Sheryl , Oliveri Lauren , Velez Maria , Joyce Sonali , Calderon Michael , Dadey Rebekah , Rajasundaram Dhivyaa , Normolle Daniel P. , Watkins Simon C. , Herman James G. , Kirkwood John M. , Lipson Evan J. , Ferris Robert L. , Bruno Tullia C. , Vignali Dario A.A.
Published: April 2023

Abstract
Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.
Data from Immunologic Features in De Novo and Recurrent Glioblastoma Are Associated with Survival Outcomes
Authors: Alanio Cécile , Binder Zev A. , Chang Renee B. , Nasrallah MacLean P. , Delman Devora , Li Joey H. , Tang Oliver Y. , Zhang Logan Y. , Zhang Jiasi Vicky , Wherry E. John , O'Rourke Donald M. , Beatty Gregory L.
Published: April 2023

Abstract
Glioblastoma (GBM) is an immunologically “cold” tumor characterized by poor responsiveness to immunotherapy. Standard of care for GBM is surgical resection followed by chemoradiotherapy and maintenance chemotherapy. However, tumor recurrence is the norm, and recurring tumors are found frequently to have acquired molecular changes (e.g., mutations) that may influence their immunobiology. Here, we compared the immune contexture of de novo GBM and recurrent GBM (rGBM) using high-dimensional cytometry and multiplex IHC. Although myeloid and T cells were similarly abundant in de novo and rGBM, their spatial organization within tumors differed and was linked to outcomes. In rGBM, T cells were enriched and activated in perivascular regions and clustered with activated macrophages and fewer regulatory T cells. Moreover, a higher expression of phosphorylated STAT1 by T cells in these regions at recurrence was associated with a favorable prognosis. Together, our data identify differences in the immunobiology of de novo GBM and rGBM and identify perivascular T cells as potential therapeutic targets.

See related Spotlight by Bayik et al., p. 787
Data from Imaging α-GalCer–Activated iNKT Cells in a Hepatic Metastatic Environment
Authors: Babes Liane , Shim Raymond , Kubes Paul
Published: April 2023

Abstract
Patients with colorectal cancer frequently develop liver metastases after, and perhaps as a consequence of, lifesaving surgical resection of the primary tumor. This creates a potential opportunity for prophylactic metastatic treatment with novel immunostimulatory molecules. Here, we used state-of-the-art intravital imaging of an experimental liver metastasis model to visualize the early behavior and function of invariant natural killer T (iNKT) cells stimulated with α-galactosylceramide (α-GalCer). Intravenous α-GalCer prior to tumor cell seeding in the liver significantly inhibited tumor growth. However, some seeding tumor cells survived. A multiple dosing regimen reduced tumor burden and prolonged the life of mice, whereas tumors returned within 5 days after a single dose of α-GalCer. With multiple doses of α-GalCer, iNKT cells increased in number and granularity (as did NK cells). As a result, the total number of contacts and time in contact with tumors increased substantially. In the absence of iNKT cells, the beneficial effect of α-GalCer was lost. Robust cytokine production dissipated over time. Repeated therapy, even after cytokine dissipation, led to reduced tumor burden and prolonged survival. Serial transplantation of tumors exposed to α-GalCer–activated iNKT cells did not induce greater resistance, suggesting no obvious epigenetic or genetic immunoediting in tumors exposed to activated iNKT cells. Very few tumor cells expressed CD1d in this model, and as such, adding monomers of CD1d–α-GalCer further reduced tumor growth. The data suggest early and repeated stimulation of iNKT cells with α-GalCer could have direct therapeutic benefit for patients with colorectal cancer who develop metastatic liver disease.
Data from Arid5a Promotes Immune Evasion by Augmenting Tryptophan Metabolism and Chemokine Expression
Authors: Parajuli Gyanu , Tekguc Murat , Wing James B. , Hashimoto Ari , Okuzaki Daisuke , Hirata Takeshi , Sasaki Atsushi , Itokazu Takahide , Handa Haruka , Sugino Hirokazu , Nishikawa Yoshihiro , Metwally Hozaifa , Kodama Yuzo , Tanaka Shinya , Sabe Hisataka , Yamashita Toshihide , Sakaguchi Shimon , Kishimoto Tadamitsu , Hashimoto Shigeru
Published: April 2023

Abstract
The acquisition of mesenchymal traits leads to immune evasion in various cancers, but the underlying molecular mechanisms remain unclear. In this study, we found that the expression levels of AT-rich interaction domain-containing protein 5a (Arid5a), an RNA-binding protein, were substantially increased in mesenchymal tumor subtypes. The deletion of Arid5a in tumor cell lines enhanced antitumor immunity in immunocompetent mice, but not in immunodeficient mice, suggesting a role for Arid5a in immune evasion. Furthermore, an Arid5a-deficient tumor microenvironment was shown to have robust antitumor immunity, as manifested by suppressed infiltration of granulocytic myeloid-derived suppressor cells and regulatory T cells. In addition, infiltrated T cells were more cytotoxic and less exhausted. Mechanistically, Arid5a stabilized Ido1 and Ccl2 mRNAs and augmented their expression, resulting in enhanced tryptophan catabolism and an immunosuppressive tumor microenvironment. Thus, our findings demonstrate the role of Arid5a beyond inflammatory diseases and suggest Arid5a as a promising target for the treatment of immunotolerant malignant tumors.

See related Spotlight by Van den Eynde, p. 854.
Data from Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4+ Th1 Responses and Potential Use as a Cancer Vaccine
Authors: Basu Amrita , Albert Gabriella K. , Awshah Sabrina , Datta Jashodeep , Kodumudi Krithika N. , Gallen Corey , Beyer Amber , Smalley Keiran S.M. , Rodriguez Paulo C. , Duckett Derek R. , Forsyth Peter A. , Soyano Aixa , Koski Gary K. , Lima Barros Costa Ricardo , Han Heather , Soliman Hatem , Lee Marie Catherine , Kalinski Pawel , Czerniecki Brian J.
Published: April 2023

Abstract
The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II–binding HER3 peptides, which can generate HER3-specific CD4+ Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II–binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide–pulsed dendritic cell vaccination resulted in anti-HER3 CD4+ Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4+ T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4+ Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors.
Data from BET Inhibition Enhances TNF-Mediated Antitumor Immunity
Authors: Wellinger Lisa C. , Hogg Simon J. , Newman Dane M. , Friess Thomas , Geiss Daniela , Michie Jessica , Ramsbottom Kelly M. , Bacac Marina , Fauti Tanja , Marbach Daniel , Jarassier Laura , Thienger Phillip , Paehler Axel , Cluse Leonie A. , Kearney Conor J. , Vervoort Stephin J. , Trapani Joseph A. , Oliaro Jane , Shortt Jake , Ruefli-Brasse Astrid , Rohle Daniel , Johnstone Ricky W.
Published: April 2023

Abstract
Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.
Data from Tumor MHC Class I Expression Associates with Intralesional IL2 Response in Melanoma
Authors: Pourmaleki Maryam , Jones Caitlin J. , Ariyan Charlotte E. , Zeng Zheng , Pirun Mono , Navarrete Daniel A. , Li Yanyun , Zhang Mianlei , Nandakumar Subhiksha , Campos Carl , Nadeem Saad , Klimstra David S. , Temple-Oberle Claire F. , Brenn Thomas , Lipson Evan J. , Schenk Kara M. , Stein Julie E. , Taube Janis M. , White Michael G. , Traweek Raymond , Wargo Jennifer A. , Kirkwood John M. , Gasmi Billel , Goff Stephanie L. , Corwin Alex D. , McDonough Elizabeth , Ginty Fiona , Callahan Margaret K. , Schietinger Andrea , Socci Nicholas D. , Mellinghoff Ingo K. , Hollmann Travis J.
Published: April 2023

Abstract
Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1−LAG-3−TIM-3−). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as “extreme responders”) were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.
Data from Sensory Nerves Impede the Formation of Tertiary Lymphoid Structures and Development of Protective Antimelanoma Immune Responses
Authors: Vats Kavita , Kruglov Oleg , Sahoo Bikram , Soman Vishal , Zhang Jiying , Shurin Galina V. , Chandran Uma R. , Skums Pavel , Shurin Michael R. , Zelikovsky Alex , Storkus Walter J. , Bunimovich Yuri L.
Published: April 2023

Abstract
Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.
Data from Design and Validation of Inducible TurboCARs with Tunable Induction and Combinatorial Cytokine Signaling
Authors: Lin Regina J. , Nager Andrew R. , Park Spencer , Sutton Janette , Lay Cecilia , Melton Zea , Zhang Yi , Boldajipour Bijan , Blarcom Thomas J. Van , Panowski Siler H. , Sasu Barbra J. , Chaparro-Riggers Javier
Published: April 2023

Abstract
Although cytokine support can enhance CAR T-cell function, coadministering cytokines or engineering CAR T cells to secrete cytokines can result in toxicities. To mitigate these safety risks, we engineered iTurboCAR T cells that coexpress a novel inducible Turbo (iTurbo) cytokine signaling domain. iTurbo domains consist of modular components that are customizable to a variety of activating inputs, as well as cytokine signaling outputs multiplexable for combinatorial signaling outcomes. Unlike most canonical cytokine receptors that are heterodimeric, iTurbo domains leverage a compact, homodimeric design that minimizes viral vector cargo. Using an iTurbo domain activated by the clinically validated dimerizer, AP1903, homodimeric iTurbo domains instigated signaling that mimicked the endogenous heterodimeric cytokine receptor. Different iTurbo domains programmed iTurboCAR T cells toward divergent phenotypes and resulted in improved antitumor efficacy. iTurbo domains, therefore, offer the flexibility for user-programmable signaling outputs, permitting control over cellular phenotype and function while minimizing viral cargo footprint.
Data from TCR-Independent Metabolic Reprogramming Precedes Lymphoma-Driven Changes in T-cell Fate
Authors: Hesterberg Rebecca S. , Liu Min , Elmarsafawi Aya G. , Koomen John M. , Welsh Eric A. , Hesterberg Stephen G. , Ranatunga Sujeewa , Yang Chunying , Li Weimin , Lawrence Harshani R. , Rodriguez Paulo C. , Berglund Anders E. , Cleveland John L.
Published: April 2023

Abstract
Chronic T-cell receptor (TCR) signaling in the tumor microenvironment is known to promote T-cell dysfunction. However, we reasoned that poorly immunogenic tumors may also compromise T cells by impairing their metabolism. To address this, we assessed temporal changes in T-cell metabolism, fate, and function in models of B-cell lymphoma driven by Myc, a promoter of energetics and repressor of immunogenicity. Increases in lymphoma burden most significantly impaired CD4+ T-cell function and promoted regulatory T cell (Treg) and Th1-cell differentiation. Metabolomic analyses revealed early reprogramming of CD4+ T-cell metabolism, reduced glucose uptake, and impaired mitochondrial function, which preceded changes in T-cell fate. In contrast, B-cell lymphoma metabolism remained robust during tumor progression. Finally, mitochondrial functions were impaired in CD4+ and CD8+ T cells in lymphoma-transplanted OT-II and OT-I transgenic mice, respectively. These findings support a model, whereby early, TCR-independent, metabolic interactions with developing lymphomas limits T cell–mediated immune surveillance.
Data from A Population of TIM4+FOLR2+ Macrophages Localized in Tertiary Lymphoid Structures Correlates to an Active Immune Infiltrate Across Several Cancer Types
Authors: Bugatti Mattia , Bergamini Marco , Missale Francesco , Monti Matilde , Ardighieri Laura , Pezzali Irene , Picinoli Sara , Caronni Nicoletta , Missolo-Koussou Yoann , Helft Julie , Benvenuti Federica , Vermi William
Published: April 2023

Abstract
TIM4 has previously been associated with antitumor immunity, yet the pattern of expression and the function of this receptor across human cancer tissues remain poorly explored. Here we combined extensive immunolabeling of human tissues with in silico analysis of pan-cancer transcriptomic data sets to explore the clinical significance of TIM4 expression. Our results unveil that TIM4 is expressed on a fraction of cavity macrophages (CATIM4+MΦ) of carcinoma patients. Moreover, we uncover a high expression of TIM4 on macrophages of the T-cell zone of the carcinoma-associated tertiary lymphoid structures (TLSTIM4+MΦ). In silico analysis of a pan-cancer data set revealed a positive correlation between TIM4 expression and markers of B cells, effector CD8+ T cells, and a 12-chemokine signature defining tertiary lymphoid structure. In addition, TLSTIM4+MΦ were enriched in cancers displaying microsatellite instability and high CD8+ T-cell infiltration, confirming their association with immune-reactive tumors. Both CATIM4+MΦ and TLSTIM4+MΦ express FOLR2, a marker of tissue-resident MΦ. However, CATIM4+MΦ had a higher expression of the immunosuppressive molecules TREM2, IL10, and TGFβ as compared with TLSTIM4+MΦ. By analyzing a scRNA sequence data set of tumor-associated myeloid cells, we identified two TIM4+FOLR2+ clusters coherent with CATIM4+MΦ and TLSTIM4+MΦ. We defined specific gene signatures for each subset and found that the CATIM4+ MΦ signature was associated with worse patient survival. In contrast, TLSTIM4+MΦ gene signature positively correlates with a better prognosis. Together, these data illustrate that TIM4 marks two distinct macrophage populations with distinct phenotypes and tissue localization and that may have opposing roles in tumor immunity.
Data from Gastric Microbiome Alterations Are Associated with Decreased CD8+ Tissue-Resident Memory T Cells in the Tumor Microenvironment of Gastric Cancer
Authors: Peng Rui , Liu Shuai , You Wenhua , Huang Yedi , Hu Chupeng , Gao Ye , Jia Xuemei , Li Gang , Xu Zekuan , Chen Yun
Published: April 2023

Abstract
The host microbiota is closely associated with tumor initiation and progression in multiple solid tumors including gastric cancer. The aim of this study was to investigate in patients with gastric cancer whether there are alterations in gastric microbiota and any potential association these may have with immune dysregulation. 16S rRNA gene sequencing was used to analyze tumor microbiota of 53 patients with gastric cancer and gastric mucosal tissue microbiota of 30 patients with chronic gastritis. The effect of microbiota on the tumor microenvironment (TME) was studied by single-cell sequencing, immunohistochemistry, multiplex immunofluorescence staining, and flow cytometry, as well as in a mouse model of primary gastric cancer. The gastric cancer microbiota was characterized by reduced microbial diversity and enrichment of the Oceanobacter, Methylobacterium, and Syntrophomonas genera. Intratumoral Methylobacterium was significantly associated with poor prognoses in patients with gastric cancer. It also was inversely correlated with the frequency of CD8+ tissue-resident memory T (TRM) cells in the TME. TGFβ was significantly reduced in gastric cancer samples with higher abundance of Methylobacterium. Finally, we verified that Methylobacterium can decrease TGFβ expression and CD8+ TRM cells in the tumor by establishing a mouse model of primary gastric cancer. The results suggest that tumor microbiota and exhausted CD8+ TRM cells in the TME of gastric cancer are significantly correlated, and that Methylobacterium may play a role in gastric carcinogenesis.