bluuebunny/crossref_metadata_2025 huggingface dataset:
Figure S1 from VISTA Targeting of T-cell Quiescence and Myeloid Suppression Overcomes Adaptive Resistance
Authors: Schaafsma Evelien , Croteau Walburga , ElTanbouly Mohamed , Nowak Elizabeth C. , Smits Nicole C. , Deng Jie , Sarde Aurelien , Webber Cecilia A. , Rabadi Dina , Cheng Chao , Noelle Randolph , Lines J. Louise
Published: April 2023

Quality control and cluster identification of CD45-enriched scRNAseq data. In the I/V model, BALB/c mice were administered 100k CT26 cells and treated when tumors reached a size of 40mm3 with isotype (I) or anti-VISTA (V). In the CPV model, BALB/c mice were administered 100k CT26 cells and treated with anti-CTLA-4 and anti-PD-1 plus isotype (CP) or plus anti-VISTA (CPV) when tumors reached a size of 600mm3. In both models, CD45+ cells were isolated from the TME and analyzed by scRNAseq. (A) Histogram of the number of reads (left) and the number of genes (right) detected in all cells within the I/V dataset. (B) Histogram of the number of reads (left) and the number of genes (right) detected in all cells within the CP/CPV dataset. (C) Visual representation of lymphoid and myeloid cell distributions between experimental replicates using UMAP plots. (D) Expected and observed Local Inverse Simpson’s Index (LISI) measuring the degree of mixing among datasets. The LISI score ranges from 1 in an unmixed space to 2 in a perfectly mixed space with equal numbers of cells in each dataset. (E) AUCs calculated based on fivefold cross-validation where cells from each cluster are split into training and test datasets, a logistic regression classifier is trained on the training dataset and applied to the test dataset to measure model accuracy for each cluster. Higher AUC values correspond to better cluster discrimination. (F) Comparison of the number of cells detected per cluster in the I/V and CP/CPV datasets. (G) Comparison of the number of genes detected per cluster. (H) UMAP of all CD45+ cells sorted from I/V- and CP/CPV-treated tumor infiltrates, colored by cell cluster based on Louvain clustering. (I) Dot plots of cluster-defining marker genes for cell type annotations (as in H). Dot size represents fraction of cells expressing a gene in each cluster. Dot color represents scaled average expression by gene column.
Supplementary Data from LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells
Authors: Ngai Ho , Barragan Gabriel A. , Tian Gengwen , Balzeau Julien C. , Zhang Chunchao , Courtney Amy N. , Guo Linjie , Xu Xin , Wood Michael S. , Drabek Janice M. , Demberg Thorsten , Sands Caroline M. , Chauvin-Fleurence Cynthia N. , Di Pierro Erica J. , Rosen Jeffrey M. , Metelitsa Leonid S.
Published: April 2023

Combined supplementary figures file
Table S6 from Low TCR Binding Strength Results in Increased Progenitor-like CD8+ Tumor-Infiltrating Lymphocytes
Authors: Hay Zachary L.Z. , Knapp Jennifer R. , Magallon Roman E. , O'Connor Brian P. , Slansky Jill E.
Published: April 2023

Differential gene expression between Tet-Positive or Tet-Negative clusters
Table S5 from Low TCR Binding Strength Results in Increased Progenitor-like CD8+ Tumor-Infiltrating Lymphocytes
Authors: Hay Zachary L.Z. , Knapp Jennifer R. , Magallon Roman E. , O'Connor Brian P. , Slansky Jill E.
Published: April 2023

Differential gene expression between all clusters
Supplementary Table S3 from High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis
Authors: Cortese Nina , Carriero Roberta , Barbagallo Marialuisa , Putignano Anna Rita , Costa Guido , Giavazzi Fabio , Grizzi Fabio , Pasqualini Fabio , Peano Clelia , Basso Gianluca , Marchini Sergio , Colombo Federico Simone , Soldani Cristiana , Franceschini Barbara , Di Tommaso Luca , Terracciano Luigi , Donadon Matteo , Torzilli Guido , Kunderfranco Paolo , Mantovani Alberto , Marchesi Federica
Published: April 2023

Supplementary Table S3
Supplementary Table S2 from High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis
Authors: Cortese Nina , Carriero Roberta , Barbagallo Marialuisa , Putignano Anna Rita , Costa Guido , Giavazzi Fabio , Grizzi Fabio , Pasqualini Fabio , Peano Clelia , Basso Gianluca , Marchini Sergio , Colombo Federico Simone , Soldani Cristiana , Franceschini Barbara , Di Tommaso Luca , Terracciano Luigi , Donadon Matteo , Torzilli Guido , Kunderfranco Paolo , Mantovani Alberto , Marchesi Federica
Published: April 2023

Supplementary Table S2
Supplementary Figure legends from High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis
Authors: Cortese Nina , Carriero Roberta , Barbagallo Marialuisa , Putignano Anna Rita , Costa Guido , Giavazzi Fabio , Grizzi Fabio , Pasqualini Fabio , Peano Clelia , Basso Gianluca , Marchini Sergio , Colombo Federico Simone , Soldani Cristiana , Franceschini Barbara , Di Tommaso Luca , Terracciano Luigi , Donadon Matteo , Torzilli Guido , Kunderfranco Paolo , Mantovani Alberto , Marchesi Federica
Published: April 2023

Supplementary figure legends
Data from Acute Influenza Infection Promotes Lung Tumor Growth by Reprogramming the Tumor Microenvironment
Authors: Garmendia Irati , Varthaman Aditi , Marmier Solenne , Angrini Mahmud , Matchoua Ingrid , Darbois-Delahousse Aurelie , Josseaume Nathalie , Foy Pierre-Emmanuel , Roumenina Lubka T. , Naouar Naïra , Meylan Maxime , Sibéril Sophie , Russick Jules , Joubert Pierre-Emmanuel , Leroy Karen , Damotte Diane , Mansuet-Lupo Audrey , Wislez Marie , Alifano Marco , Menger Laurie , Garcia-Verdugo Ignacio , Sallenave Jean-Michel , Lantz Olivier , Petitprez Florent , Cremer Isabelle
Published: April 2023

Abstract
One billion people worldwide get flu every year, including patients with non–small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.
Data from iNKT Cell Cytotoxic Responses Control T-Lymphoma Growth In Vitro and In Vivo
Authors: Bassiri Hamid , Das Rupali , Guan Peng , Barrett David M. , Brennan Patrick J. , Banerjee Pinaki P. , Wiener Susan J. , Orange Jordan S. , Brenner Michael B. , Grupp Stephan A. , Nichols Kim E.
Published: April 2023

Abstract
Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. Cancer Immunol Res; 2(1); 59–69. ©2013 AACR.
Data from Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment
Authors: Muroyama Yuki , Nirschl Thomas R. , Kochel Christina M. , Lopez-Bujanda Zoila , Theodros Debebe , Mao Wendy , Carrera-Haro Maria A. , Ghasemzadeh Ali , Marciscano Ariel E. , Velarde Esteban , Tam Ada J. , Thoburn Christopher J. , Uddin Muniza , Meeker Alan K. , Anders Robert A. , Pardoll Drew M. , Drake Charles G.
Published: April 2023

Abstract
Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.
Data from High-Throughput Stability Screening of Neoantigen/HLA Complexes Improves Immunogenicity Predictions
Authors: Blaha Dylan T. , Anderson Scott D. , Yoakum Daniel M. , Hager Marlies V. , Zha Yuanyuan , Gajewski Thomas F. , Kranz David M.
Published: April 2023

Abstract
Mutated peptides (neoantigens) from a patient's cancer genome can serve as targets for T-cell immunity, but identifying which peptides can be presented by an MHC molecule and elicit T cells has been difficult. Although algorithms that predict MHC binding exist, they are not yet able to distinguish experimental differences in half-lives of the complexes (an immunologically relevant parameter, referred to here as kinetic stability). Improvement in determining actual neoantigen peptide/MHC stability could be important, as only a small fraction of peptides in most current vaccines are capable of eliciting CD8+ T-cell responses. Here, we used a rapid, high-throughput method to experimentally determine peptide/HLA thermal stability on a scale that will be necessary for analysis of neoantigens from thousands of patients. The method combined the use of UV-cleavable peptide/HLA class I complexes and differential scanning fluorimetry to determine the Tm values of neoantigen complexes. Measured Tm values were accurate and reproducible and were directly proportional to the half-lives of the complexes. Analysis of known HLA-A2–restricted immunogenic peptides showed that Tm values better correlated with immunogenicity than algorithm-predicted binding affinities. We propose that temperature stability information can be used as a guide for the selection of neoantigens in cancer vaccines in order to focus attention on those mutated peptides with the highest probability of being expressed on the cell surface.
Data from Intravenous Injection of MVA Virus Targets CD8+ Lymphocytes to Tumors to Control Tumor Growth upon Combinatorial Treatment with a TLR9 Agonist
Authors: Fend Laetitia , Gatard-Scheikl Tanja , Kintz Jacqueline , Gantzer Murielle , Schaedler Emmanuelle , Rittner Karola , Cochin Sandrine , Fournel Sylvie , Préville Xavier
Published: April 2023

Abstract
Effector T-cell access to tumor tissue is a limiting step for clinical efficacy of antigen-specific T cell–based immunotherapies. Ectopic mouse tumor models, in which a subcutaneously (s.c.) implanted tumor is treated with s.c. or intramuscular therapeutic immunization, may not be optimal for targeting effector T cells to an organ-borne tumor. We used an orthotopic renal carcinoma model to evaluate the impact of injection routes on therapeutic efficacy of a Modified Vaccinia virus Ankara viral vector expressing the human mucin 1 tumor–associated xeno-antigen (MVA-MUC1). We show that intravenous (i.v.) administration of MVA-MUC1 displayed enhanced efficacy when compared with s.c. injection. Therapeutic efficacy of MVA-MUC1 was further enhanced by i.v. injection of a TLR9 agonist. In all cases, infiltration of tumor-bearing kidney by CD8+ lymphocytes was associated with control of tumor growth. Biodistribution experiments indicate that, following i.v. injection, MVA-encoded antigens are quickly expressed in visceral organs and, in particular, in splenic antigen-presenting cells, compared with those following s.c. injection. This appears to result in a faster generation of MUC1-specific CD8+ T cells. Lymphocytes infiltrating tumor-bearing kidneys are characterized by an effector memory phenotype and express PD-1 and Tim3 immune checkpoint molecules. Therapeutic efficacy was associated with a modification of the tumor microenvironment toward a Th1-type immune response and recruitment of activated lymphocytes. This study supports the clinical evaluation of MVA-based immunotherapies via the i.v. route. Cancer Immunol Res; 2(12); 1163–74. ©2014 AACR.
Data from Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies
Authors: Beatty Gregory L. , Haas Andrew R. , Maus Marcela V. , Torigian Drew A. , Soulen Michael C. , Plesa Gabriela , Chew Anne , Zhao Yangbing , Levine Bruce L. , Albelda Steven M. , Kalos Michael , June Carl H.
Published: April 2023

Abstract
Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB costimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was shown in both patients, and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel antiself antibodies. These data show the potential of using mRNA-engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. Cancer Immunol Res; 2(2); 112–20. ©2013 AACR.
Data from Loss of CXCR4 in Myeloid Cells Enhances Antitumor Immunity and Reduces Melanoma Growth through NK Cell and FASL Mechanisms
Authors: Yang Jinming , Kumar Amrendra , Vilgelm Anna E. , Chen Sheau-Chiann , Ayers Gregory D. , Novitskiy Sergey V. , Joyce Sebastian , Richmond Ann
Published: April 2023

Abstract
The chemokine receptor, CXCR4, is involved in cancer growth, invasion, and metastasis. Several promising CXCR4 antagonists have been shown to halt tumor metastasis in preclinical studies, and clinical trials evaluating the effectiveness of these agents in patients with cancer are ongoing. However, the impact of targeting CXCR4 specifically on immune cells is not clear. Here, we demonstrate that genetic deletion of CXCR4 in myeloid cells (CXCR4MyeΔ/Δ) enhances the antitumor immune response, resulting in significantly reduced melanoma tumor growth. Moreover, CXCR4MyeΔ/Δ mice exhibited slowed tumor progression compared with CXCR4WT mice in an inducible melanocyte BrafV600E/Pten−/− mouse model. The percentage of Fas ligand (FasL)–expressing myeloid cells was reduced in CXCR4MyeΔ/Δ mice as compared with myeloid cells from CXCR4WT mice. In contrast, there was an increased percentage of natural killer (NK) cells expressing FasL in tumors growing in CXCR4MyeΔ/Δ mice. NK cells from CXCR4MyeΔ/Δ mice also exhibited increased tumor cell killing capacity in vivo, based on clearance of NK-sensitive Yac-1 cells. NK cell–mediated killing of Yac-1 cells occurred in a FasL-dependent manner, which was partially dependent upon the presence of CXCR4MyeΔ/Δ neutrophils. Furthermore, enhanced NK cell activity in CXCR4MyeΔ/Δ mice was also associated with increased production of IL18 by specific leukocyte subpopulations. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell–mediated tumor surveillance and thereby enhance tumor growth. Systemic delivery of a peptide antagonist of CXCR4 to tumor-bearing CXCR4WT mice resulted in enhanced NK-cell activation and reduced tumor growth, supporting potential clinical implications for CXCR4 antagonism in some cancers. Cancer Immunol Res; 6(10); 1186–98. ©2018 AACR.
Data from Radretumab Radioimmunotherapy in Patients with Brain Metastasis: A 124I-L19SIP Dosimetric PET Study
Authors: Poli Gian Luca , Bianchi Claudia , Virotta Giorgio , Bettini Anna , Moretti Renzo , Trachsel Eveline , Elia Giuliano , Giovannoni Leonardo , Neri Dario , Bruno Andrea
Published: April 2023

Abstract
Radioimmunotherapy (RIT) with 131I-labeled L19SIP (radretumab; a small immunoprotein format antibody directed against the ED-B domain of fibronectin; ∼80 kDa molecular weight) has been investigated in several clinical trials. Here, we describe the use of immuno-PET imaging with iodine-124 (124I)–labeled L19SIP to predict doses delivered to tumor lesions and healthy organs by a subsequent radretumab RIT in patients with brain metastases from solid cancer. Bone marrow doses were evaluated both during the diagnostic phase and posttherapy, measuring activities in blood (germanium detector) and whole body (lanthanum bromide detector). Expected doses for radretumab administration (4,107 MBq/m2) were calculated from data obtained after administration of an average of 167 MBq 124I-L19SIP to 6 patients. To assess lesion average doses, the positron emission tomography (PET) scanner was calibrated for the use of 124I with an International Electrotechnical Commission (IEC) Body Phantom and recovery coefficients were calculated. The average dose to bone red marrow was 0.21 Gy/GBq, with high correlation between provisional and actual posttherapy doses. Although the fraction of injected activity in normal organs was similar in different patients, the antibody uptake in the neoplastic lesions varied by as much as a factor of 60. Immuno-PET with 124I-labeled L19SIP offers significant advantages over conventional 131I imaging, in particular accuracy of dosimetric results. Furthermore, the study indicates that antibody uptake can be highly variable even in different lesions of the same patient and that immuno-PET procedures may guide product development with armed antibodies. Cancer Immunol Res; 1(2); 134–43. ©2013 AACR.
Data from Rapid Construction of Antitumor T-cell Receptor Vectors from Frozen Tumors for Engineered T-cell Therapy
Authors: Tsuji Takemasa , Yoneda Akira , Matsuzaki Junko , Miliotto Anthony , Ryan Courtney , Koya Richard C. , Odunsi Kunle
Published: April 2023

Abstract
T cells genetically engineered with tumor antigen–specific T-cell receptor (TCR) genes have demonstrated therapeutic potential in patients with solid tumors. In order to achieve broader application, an efficient method to identify TCR genes for an array of tumor antigens and HLA restriction elements is required. Here, we have developed a method to construct a TCR-expression library from specimens, including frozen tumor biopsies, that contain antigen-specific T cells. TCR-expressing cassettes were constructed and cloned in a retroviral plasmid vector within 24 hours by unbiased PCR amplification of TCR α and β chain variable regions assembled with TCR constant regions. The method was validated by constructing TCR-expressing vectors from tumor antigen–specific T-cell clones and functionally assessing TCR gene–transduced T cells. We applied this method to frozen ovarian tumor specimens that were infiltrated by tumor antigen–specific T cells. The tumor-derived TCR libraries were expressed in peripheral T cells from healthy volunteers and screened for tumor antigen–specific TCR pairs with the use of an MHC/peptide tetramer reagent. Tumor antigen–specific TCR-expressing transgenes were recovered from isolated tetramer-positive T cells. Peripheral T cells that were engineered with library-derived TCR gene showed potent therapeutic antitumor effect in a tumor xenograft model. Our method can efficiently and rapidly provide tumor-specific TCR-expressing viral vectors for the manufacture of therapeutic and personalized antitumor T-cell products. Cancer Immunol Res; 6(5); 594–604. ©2018 AACR.
Data from Effect of Montanide and Poly-ICLC Adjuvant on Human Self/Tumor Antigen-Specific CD4+ T Cells in Phase I Overlapping Long Peptide Vaccine Trial
Authors: Tsuji Takemasa , Sabbatini Paul , Jungbluth Achim A. , Ritter Erika , Pan Linda , Ritter Gerd , Ferran Luis , Spriggs David , Salazar Andres M. , Gnjatic Sacha
Published: April 2023

Abstract
Vaccination of patients with ovarian cancer with overlapping long peptides (OLP) from cancer-testis antigen NY-ESO-1 and poly-ICLC in Montanide-ISA-51 (Montanide) was found to consistently induce integrated immune responses (antibody, CD4+, and CD8+ T cells). Using detailed methods, we investigated the respective effects of poly-ICLC and Montanide adjuvant on pre- and postvaccine NY-ESO-1–specific CD4+ T cells, because of their central function for induction and maintenance of both antibody and CD8+ T cells. Polyclonal NY-ESO-1–specific CD4+ T-cell lines were generated from 12 patients using CD154-based selection of precursors before and after vaccination with (i) OLP alone, (ii) OLP in Montanide, or (iii) OLP and poly-ICLC in Montanide. Kinetics, quantification, fine specificity, avidity, and cytokine-producing pattern were analyzed in depth and compared between vaccine cohorts. Vaccination with OLP alone did not elicit CD4+ T-cell responses; it suppressed high-avidity CD4+ T-cell precursors that recognized naturally processed NY-ESO-1 protein before vaccination. Emulsification of OLP in Montanide was required for the expansion of high-avidity NY-ESO-1–specific CD4+ T-cell precursors. Poly-ICLC significantly enhanced CD4+ Th1 responses while suppressing the induction of interleukin (IL)-4–producing Th2 and IL-9–producing Th9 cells. In summary, Montanide and poly-ICLC had distinct and cooperative effects for the induction of NY-ESO-1–specific Th1 cells and integrated immune responses by OLP vaccination. These results support the use of admixing poly-ICLC in Montanide adjuvant to rapidly induce antitumor type I immune responses by OLP from self/tumor antigens in human cancer vaccines. Cancer Immunol Res; 1(5); 340–50. ©2013 AACR.
Data from Mapping the MHC Class I–Spliced Immunopeptidome of Cancer Cells
Authors: Liepe Juliane , Sidney John , Lorenz Felix K.M. , Sette Alessandro , Mishto Michele
Published: April 2023

Abstract
Anticancer immunotherapies demand optimal epitope targets, which could include proteasome-generated spliced peptides if tumor cells were to present them. Here, we show that spliced peptides are widely presented by MHC class I molecules of colon and breast carcinoma cell lines. The peptides derive from hot spots within antigens and enlarge the antigen coverage. Spliced peptides also represent a large number of antigens that would otherwise be neglected by patrolling T cells. These antigens tend to be long, hydrophobic, and basic. Thus, spliced peptides can be a key to identifying targets in an enlarged pool of antigens associated with cancer.
Data from Improving CART-Cell Therapy of Solid Tumors with Oncolytic Virus–Driven Production of a Bispecific T-cell Engager
Authors: Wing Anna , Fajardo Carlos Alberto , Posey Avery D. , Shaw Carolyn , Da Tong , Young Regina M. , Alemany Ramon , June Carl H. , Guedan Sonia
Published: April 2023

Abstract
T cells expressing chimeric antigen receptors (CART) have shown significant promise in clinical trials to treat hematologic malignancies, but their efficacy in solid tumors has been limited. Oncolytic viruses have the potential to act in synergy with immunotherapies due to their immunogenic oncolytic properties and the opportunity of incorporating therapeutic transgenes in their genomes. Here, we hypothesized that an oncolytic adenovirus armed with an EGFR-targeting, bispecific T-cell engager (OAd-BiTE) would improve the outcome of CART-cell therapy in solid tumors. We report that CART cells targeting the folate receptor alpha (FR-α) successfully infiltrated preestablished xenograft tumors but failed to induce complete responses, presumably due to the presence of antigen-negative cancer cells. We demonstrated that OAd-BiTE–mediated oncolysis significantly improved CART-cell activation and proliferation, while increasing cytokine production and cytotoxicity, and showed an in vitro favorable safety profile compared with EGFR-targeting CARTs. BiTEs secreted from infected cells redirected CART cells toward EGFR in the absence of FR-α, thereby addressing tumor heterogeneity. BiTE secretion also redirected CAR-negative, nonspecific T cells found in CART-cell preparations toward tumor cells. The combinatorial approach improved antitumor efficacy and prolonged survival in mouse models of cancer when compared with the monotherapies, and this was the result of an increased BiTE-mediated T-cell activation in tumors. Overall, these results demonstrated that the combination of a BiTE-expressing oncolytic virus with adoptive CART-cell therapy overcomes key limitations of CART cells and BiTEs as monotherapies in solid tumors and encourage its further evaluation in human trials. Cancer Immunol Res; 6(5); 605–16. ©2018 AACR.
Data from Immunosurveillance and Immunoediting of Breast Cancer via Class I MHC Receptors
Authors: Tu Megan M. , Rahim Mir Munir A. , Sayed Céline , Mahmoud Ahmad Bakur , Makrigiannis Andrew P.
Published: April 2023

Abstract
Ly49 receptors, which recognize “self” class I major histocompatibility complex (MHC-I) molecules, enable natural killer (NK) cells to detect loss of MHC-I expression on transformed and virally infected cells. The impact of NK cell–mediated MHC-I surveillance on immunoediting of breast cancer is still not fully understood. This work assesses the impact of Ly49 receptors on tumor development in terms of cancer control and in driving immune-evading cancer mutations. Genetically modified Ly49-deficient mice and those lacking NK cells through antibody depletion were less able to control E0771-derived mammary tumors in an MHC-I–dependent fashion. Similarly, Ly49-deficient MMTV-PyVT–transgenic mice developed spontaneous mammary tumors faster than Ly49-sufficient MMTV-PyVT mice. Fewer CD69+ and granzyme B+ NK cells were detected among the tumor-infiltrating lymphocytes in Ly49-deficient than in Ly49-sufficient MMTV-PyVT mice. Furthermore, tumors from Ly49-deficient mice displayed reduced MHC-I expression, suggesting that tumors growing in these mice lacked an Ly49-derived pressure to maintain MHC-I expression. These same MHC-I-low tumors from Ly49-deficient mice were unable to flourish when transferred to Ly49-sufficient hosts, confirming that this tumor mutation was in response to an Ly49-deficient environment. This work demonstrates a role for Ly49 receptors in the control of mammary cancer, and provides evidence to support a model of tumor immunoediting, in which selective pressures from the immune system drive immune-evasive cancer mutations. Cancer Immunol Res; 5(11); 1016–28. ©2017 AACR.
Data from MEK Inhibition, Alone or in Combination with BRAF Inhibition, Affects Multiple Functions of Isolated Normal Human Lymphocytes and Dendritic Cells
Authors: Vella Laura J. , Pasam Anupama , Dimopoulos Nektaria , Andrews Miles , Knights Ashley , Puaux Anne-Laure , Louahed Jamila , Chen Weisan , Woods Katherine , Cebon Jonathan S.
Published: April 2023

Abstract
Combination therapy with BRAF and MEK inhibition is currently in clinical development for the treatment of BRAF-mutated malignant melanoma. BRAF inhibitors are associated with enhanced antigen-specific T-lymphocyte recognition in vivo. Consequently, BRAF inhibition has been proposed as proimmunogenic and there has been considerable enthusiasm for combining BRAF inhibition with immunotherapy. MEK inhibitors inhibit ERK phosphorylation regardless of BRAF mutational status and have been reported to impair T-lymphocyte and modulate dendritic cell function. In this study, we investigate the effects on isolated T lymphocytes and monocyte-derived dendritic cells (moDC) of a MEK (trametinib) and BRAF (dabrafenib) inhibitor combination currently being evaluated in a randomized controlled clinical trial. The effects of dabrafenib and trametinib, alone and in combination, were studied on isolated normal T lymphocytes and moDCs. Lymphocyte viability, together with functional assays including proliferation, cytokine production, and antigen-specific expansion, were assessed. MoDC phenotype in response to lipopolysaccharide stimulation was evaluated by flow cytometry, as were effects on antigen cross-presentation. Dabrafenib did not have an impact on T lymphocytes or moDCs, whereas trametinib alone or in combination with dabrafenib suppressed T-lymphocyte proliferation, cytokine production, and antigen-specific expansion. However, no significant decrease in CD4+ or CD8+ T-lymphocyte viability was observed following kinase inhibition. MoDC cross-presentation was suppressed in association with enhanced maturation following combined inhibition of MEK and BRAF. The results of this study demonstrate that MEK inhibition, alone or in combination with BRAF inhibition, can modulate immune cell function, and further studies in vivo will be required to evaluate the potential clinical impact of these findings. Cancer Immunol Res; 2(4); 351–60. ©2014 AACR.
Data from Peptide Vaccination in Montanide Adjuvant Induces and GM-CSF Increases CXCR3 and Cutaneous Lymphocyte Antigen Expression by Tumor Antigen–Specific CD8 T Cells
Authors: Clancy-Thompson Eleanor , King Laura K. , Nunnley Lenora D. , Mullins Irene M. , Slingluff Craig L. , Mullins David W.
Published: April 2023

Abstract
T-cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules, CXC chemokine receptor 3 (CXCR3) and cutaneous lymphocyte antigen (CLA), on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T-cell infiltration of melanoma. We show that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3+CLA+ cells. Addition of granulocyte macrophage colony—stimulating factor (GM-CSF) significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of T-bet, IFN-γ, and interleukin-12 receptor (IL-12Rβ1). Collectively, these studies show that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma. Cancer Immunol Res; 1(5); 332–9. ©2013 AACR.
Data from Computational Immune Monitoring Reveals Abnormal Double-Negative T Cells Present across Human Tumor Types
Authors: Greenplate Allison R. , McClanahan Daniel D. , Oberholtzer Brian K. , Doxie Deon B. , Roe Caroline E. , Diggins Kirsten E. , Leelatian Nalin , Rasmussen Megan L. , Kelley Mark C. , Gama Vivian , Siska Peter J. , Rathmell Jeffrey C. , Ferrell P. Brent , Johnson Douglas B. , Irish Jonathan M.
Published: April 2023

Abstract
Advances in single-cell biology have enabled measurements of >40 protein features on millions of immune cells within clinical samples. However, the data analysis steps following cell population identification are susceptible to bias, time-consuming, and challenging to compare across studies. Here, an ensemble of unsupervised tools was developed to evaluate four essential types of immune cell information, incorporate changes over time, and address diverse immune monitoring challenges. The four complementary properties characterized were (i) systemic plasticity, (ii) change in population abundance, (iii) change in signature population features, and (iv) novelty of cellular phenotype. Three systems immune monitoring studies were selected to challenge this ensemble approach. In serial biopsies of melanoma tumors undergoing targeted therapy, the ensemble approach revealed enrichment of double-negative (DN) T cells. Melanoma tumor-resident DN T cells were abnormal and phenotypically distinct from those found in nonmalignant lymphoid tissues, but similar to those found in glioblastoma and renal cell carcinoma. Overall, ensemble systems immune monitoring provided a robust, quantitative view of changes in both the system and cell subsets, allowed for transparent review by human experts, and revealed abnormal immune cells present across multiple human tumor types.
Data from Downregulation of MHC-I Expression Is Prevalent but Reversible in Merkel Cell Carcinoma
Authors: Paulson Kelly G. , Tegeder Andrew , Willmes Christoph , Iyer Jayasri G. , Afanasiev Olga K. , Schrama David , Koba Shinichi , Thibodeau Renee , Nagase Kotaro , Simonson William T. , Seo Aaron , Koelle David M. , Madeleine Margaret , Bhatia Shailender , Nakajima Hideki , Sano Shigetoshi , Hardwick James S. , Disis Mary L. , Cleary Michele A. , Becker Jürgen C. , Nghiem Paul
Published: April 2023

Abstract
Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P < 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC. Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR.
Data from TGFβ Inhibition Prior to Hypofractionated Radiation Enhances Efficacy in Preclinical Models
Authors: Young Kristina H. , Newell Pippa , Cottam Benjamin , Friedman David , Savage Talicia , Baird Jason R. , Akporiaye Emmanuel , Gough Michael J. , Crittenden Marka
Published: April 2023

Abstract
The immune infiltrate in colorectal cancer has been correlated with outcome, such that individuals with higher infiltrations of T cells have increased survival independent of the disease stage. For patients with lower immune infiltrates, overall survival is limited. Because the patients with colorectal cancer studied have received conventional cancer therapies, these data may indicate that the pretreatment tumor environment increases the efficacy of treatments such as chemotherapy, surgery, and radiotherapy. This study was designed to test the hypothesis that an improved immune environment in the tumor at the time of treatment will increase the efficacy of radiotherapy. We demonstrate that inhibition of TGFβ using the orally available small-molecule inhibitor SM16 improved the immune environment of tumors in mice and significantly improved the efficacy of subsequent radiotherapy. This effect was not due to changes in radiosensitivity, epithelial–mesenchymal transition, or changes in vascular function in the tumor; rather, this effect was dependent on adaptive immunity and resulted in long-term protective immunity in cured mice. These data demonstrate that immunotherapy is an option to improve the immune status of patients with poor tumor infiltrates and that pretreatment improves the efficacy of radiotherapy. Cancer Immunol Res; 2(10); 1011–22. ©2014 AACR.
Data from Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes
Authors: Simon Petra , Omokoko Tana A. , Breitkreuz Andrea , Hebich Lisa , Kreiter Sebastian , Attig Sebastian , Konur Abdo , Britten Cedrik M. , Paret Claudia , Dhaene Karl , Türeci Özlem , Sahin Ugur
Published: April 2023

Abstract
The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8+ or CD4+ T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro–transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8+ and CD4+ T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes. Cancer Immunol Res; 2(12); 1230–44. ©2014 AACR.
Data from Influenza Virus Infection Elicits Protective Antibodies and T Cells Specific for Host Cell Antigens Also Expressed as Tumor-Associated Antigens: A New View of Cancer Immunosurveillance
Authors: Iheagwara Uzoma K. , Beatty Pamela L. , Van Phu T. , Ross Ted M. , Minden Jonathan S. , Finn Olivera J.
Published: April 2023

Abstract
Most tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and become targets of antitumor immune responses. Antibodies and T cells specific for some TAAs have been found in healthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer has also been associated with a history of febrile viral diseases. We hypothesized that virus infections could lead to transient expression of abnormal forms of self-molecules, some of which are TAAs; facilitated by the adjuvant effects of infection and inflammation, these molecules could elicit specific antibodies, T cells, and lasting immune memory simultaneously with immunity against viral antigens. Such infection-induced immune memory for TAA would be expected to provide life-long immune surveillance of cancer. Using influenza virus infection in mice as a model system, we tested this hypothesis and demonstrated that influenza-experienced mice control 3LL mouse lung tumor challenge better than infection-naive control mice. Using 2D-difference gel electrophoresis and mass spectrometry, we identified numerous molecules, some of which are known TAAs, on the 3LL tumor cells recognized by antibodies elicited by two successive influenza infections. We studied in detail immune responses against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H4, HSP90, malate dehydrogenase 2, and annexin A2, all of which were overexpressed in influenza-infected lungs and in tumor cells. Finally, we show that immune responses generated through vaccination against peptides derived from these antigens correlated with improved tumor control. Cancer Immunol Res; 2(3); 263–73. ©2013 AACR.
Data from Myeloid-Derived Suppressor Cells Attenuate TH1 Development through IL-6 Production to Promote Tumor Progression
Authors: Tsukamoto Hirotake , Nishikata Ryutaro , Senju Satoru , Nishimura Yasuharu
Published: April 2023

Abstract
Collaborative action between tumor cells and host-derived suppressor cells leads to peripheral tolerance of T cells to tumor antigens. Here, we showed that in tumor-bearing mice, generation of tumor antigen-specific effector T-helper cells (TH1) was significantly attenuated, and impaired TH1 differentiation was restored by the temporal blockade of interleukin (IL)-6 activity at the T-cell priming phase. Furthermore, we found that Gr-1+ myeloid-derived suppressor cells (MDSC) served as a source of IL-6 in tumor-bearing mice. Adoptive transfer of effector CD4+ T cells revealed that MDSC-sensitized effector CD4+ T cells were less potent in mounting antitumor immune responses, although effector T cells generated together with Gr-1+ cells from tumor-free mice eradicated established tumors. CD8+ T cells, IFN-γ, and MHC-class II expression in host mice were indispensable for the antitumor activity initiated by effector CD4+ T cells. Despite comparable suppressive activity of IL-6+/+ and IL-6−/− MDSC on primary T-cell activation, transfer of IL-6+/+ MDSC, but not IL-6−/− MDSC, dampened the efficient induction of effector TH1 cells and counteracted CD4+ T cell–mediated antitumor immunity including cognate help for CD8+ T cells in vivo. These findings suggest that, apart from the inhibitory effects on primary T-cell activation, MDSC promote tumor progression by attenuating functional differentiation of tumor-specific CD4+ T cells into effector TH1 cells through IL-6 production to promote tumor progression. This novel mode of MDSC-induced tolerance of effector CD4+ T cells should be considered as the basis for the rational design of effective T cell–mediated antitumor therapies. Cancer Immunol Res; 1(1); 64–76. ©2013 AACR.
Data from Enhancement of Tumor-Reactive Cytotoxic CD4+ T-cell Responses after Ipilimumab Treatment in Four Advanced Melanoma Patients
Authors: Kitano Shigehisa , Tsuji Takemasa , Liu Caillian , Hirschhorn-Cymerman Daniel , Kyi Chrisann , Mu Zhenyu , Allison James P. , Gnjatic Sacha , Yuan Jianda D. , Wolchok Jedd D.
Published: April 2023

Abstract
CD4+ T cells provide help to enhance and sustain cytotoxic CD8+ T-cell responses. A direct lytic role for this cell population in mouse models further supports the use of tumor-reactive CD4+ T cells for cancer immunotherapy. CTLA-4 blockade has been shown to expand antigen-specific cytotoxic CD4+ T cells in mouse models. We took advantage of spontaneous immunity to the NY-ESO-1 cancer-testis antigen to investigate quantitative and qualitative changes in antigen-specific CD4+ T-cell responses after ipilimumab (anti-CTLA-4 monoclonal antibody) treatment in patients with advanced melanoma. Four patients with NY-ESO-1 seropositive melanoma were chosen upon the availability of suitable blood specimens for characterizing the functions of NY-ESO-1 antigen-specific CD4+ T-cell response by enzyme-linked immunospot (ELISPOT), intracellular cytokine staining (ICS), and cytotoxicity assays. Multiple NY-ESO-1 antigen-specific CD4+ T-cell responses with TH1 dominance were induced or enhanced after ipilimumab treatment in peripheral blood in all four patients. NY-ESO-1 antigen–specific CD4+ T-cell lines established from all four patients after ipilimumab treatment recognized naturally processed NY-ESO-1 protein in antigen-presenting cells, expressed master transcription factor Eomesodermin (Eomes), and secreted perforin and Granzyme B. Finally, we showed that these NY-ESO-1 antigen-specific CD4+ T-cell lines directly lysed autologous melanoma cell lines expressing NY-ESO-1 in an MHC class II restricted manner. Our results show that antigen-specific cytotoxic CD4+ T-cell responses are induced after ipilimumab therapy in human cancer patients. Ipilimumab may induce the expression of lytic granules on antigen-specific cytotoxic CD4+ T cells via Eomes, revealing a novel consequence of immunologic checkpoint blockade. Cancer Immunol Res; 1(4); 235–44. ©2013 AACR.