bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from PD-1/PD-L1–Associated Immunoarchitectural Patterns Stratify Pancreatic Cancer Patients into Prognostic/Predictive Subgroups
Authors: Karamitopoulou Eva , Andreou Andreas , Pahud de Mortanges Aurélie , Tinguely Marianne , Gloor Beat , Perren Aurel
Published: April 2023

Abstract
Immunotherapy, including PD-1/PD-L1 agonists, has shown limited efficacy in pancreatic ductal adenocarcinoma (PDAC). We examined the PD-1/PD-L1 expression and immunoarchitectural features by automated morphometric analysis using multiplex immunofluorescence and 118 microsatellite-stable, treatment–naïve, surgically resected PDACs (study cohort). Five microsatellite-instable cases were stained in parallel (MSI cohort). Molecular analysis was additionally performed. An independent PDAC cohort (n = 226) was immunostained for PD-L1 and used as a validation cohort. PD-L1 expression on tumor cells (TC) and/or immune cells (IC) was present in 32% and 30% of the study and validation cohorts, respectively, and assigned into one of four patterns: “adaptive-1” (TC: 0, IC > 1%), “adaptive-2” (TC > 1% to < 25%, IC > 1%), “constitutive” (TC ≥ 25%, IC: 0), and “combined” (TC ≥ 25%, IC > 1%). “Constitutive” tumors were characterized by reduced numbers of all ICs and poor outcome. In contrast, “adaptive-1” tumors exhibited abundant T cells, including high counts of cytotoxic CD3+CD8+ and PD-1+CD3+CD8+ cells, but low counts of PD-L1+CD3+CD8+ cells and associated with the best outcome. “Adaptive-2” tumors displayed higher proportions of PD-L1+CD3+CD8+ T cells and tumor-associated macrophages (CD68+ and CD68+CD206+) compared with “adaptive-1” tumors. In the “combined” pattern, extensive PD-L1 expression on TCs was accompanied by increased numbers of T cells and improved overall survival. ICs were closer to PD-L1− than to PD-L1+ PDAC cells. TP53 and PIK3CA alterations tended to be more frequent in PD-L1+ tumors. The 5 MSI cases were PD-L1−. The distinct PD-1/PD-L1–associated immunoarchitectural patterns underpin the heterogeneity of the immunologic responses and might be used to inform patient outcomes and therapeutic decisions in pancreatic cancer.
Data from BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy
Authors: Salmon Avery J. , Shavkunov Alexander S. , Miao Qi , Jarjour Nicholas N. , Keshari Sunita , Esaulova Ekaterina , Williams Charmelle D. , Ward Jeffrey P. , Highsmith Anna M. , Pineda Josué E. , Taneja Reshma , Chen Ken , Edelson Brian T. , Gubin Matthew M.
Published: April 2023

Abstract
Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell–dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti–PD-1 and anti–CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen–specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti–PD-1 and anti–CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome—tumor outgrowth—several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.
Data from High Mobility Group Protein B1 Decreases Surface Localization of PD-1 to Augment T-cell Activation
Authors: Gao Qun , Wang Shumin , Li Feng , Lian Jingyao , Cheng Shaoyan , Yue Dongli , Zhang Zhen , Liu Shasha , Ren Feifei , Zhang Daiqun , Wang Shengdian , Wang Liping , Zhang Yi
Published: April 2023

Abstract
High-mobility group protein B1 (HMGB1) is a danger signaling molecule that has been found to trigger an effective antitumor immune response. However, the mechanisms underlying its antitumor effects are not fully understood. Here, we found that HMGB1 release induced by chemotherapy in patients with non–small cell lung cancer was negatively correlated with PD-1 expression on CD8+ T cells. In vitro analysis indicated that treatment with HMGB1 led to a significant decrease in the level of expression of PD-1 on CD8+ T cells. Further analysis demonstrated that HMGB1 reduced PD-1 expression by inducing dynamin-mediated internalization of the protein, leading to early endocytosis in the cytoplasm, and subsequently degradation in the lysosomes. In a xenograft model, HER2-targeted chimeric antigen receptor (CAR) T cells had enhanced function in the presence of HMGB1. These data identify a role for HMGB1 as a negative regulator of PD-1 signaling in lung cancer and the observed antitumor effect of HMGB1 on CAR T cells may provide a theoretical foundation for a new immunotherapy combination.
Data from Arginase 1–Based Immune Modulatory Vaccines Induce Anticancer Immunity and Synergize with Anti–PD-1 Checkpoint Blockade
Authors: Aaboe Jørgensen Mia , Ugel Stefano , Linder Hübbe Mie , Carretta Marco , Perez-Penco Maria , Weis-Banke Stine Emilie , Martinenaite Evelina , Kopp Katharina , Chapellier Marion , Adamo Annalisa , De Sanctis Francesco , Frusteri Cristina , Iezzi Manuela , Zocca Mai-Britt , Hargbøll Madsen Daniel , Wakatsuki Pedersen Ayako , Bronte Vincenzo , Andersen Mads Hald
Published: April 2023

Abstract
Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti–PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti–PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation.
Data from Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients
Authors: Samson Adel , West Emma J. , Carmichael Jonathan , Scott Karen J. , Turnbull Samantha , Kuszlewicz Bethany , Dave Rajiv V. , Peckham-Cooper Adam , Tidswell Emma , Kingston Jennifer , Johnpulle Michelle , da Silva Barbara , Jennings Victoria A. , Bendjama Kaidre , Stojkowitz Nicolas , Lusky Monika , Prasad K.R. , Toogood Giles J. , Auer Rebecca , Bell John , Twelves Chris J. , Harrington Kevin J. , Vile Richard G. , Pandha Hardev , Errington-Mais Fiona , Ralph Christy , Newton Darren J. , Anthoney Alan , Melcher Alan A. , Collinson Fiona
Published: April 2023

Abstract
Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.
Data from PBRM1 Inactivation Promotes Upregulation of Human Endogenous Retroviruses in a HIF-Dependent Manner
Authors: Zhou Mi , Leung Janet Y. , Gessner Kathryn H. , Hepperla Austin J. , Simon Jeremy M. , Davis Ian J. , Kim William Y.
Published: April 2023

Abstract
Clear cell renal cell carcinoma (ccRCC) is considered an immunotherapy-responsive disease; however, the reasons for this remain unclear. Studies have variably implicated PBRM1 mutations as a predictive biomarker of immune checkpoint blockade (ICB) response, and separate studies demonstrate that expression of human endogenous retroviruses (hERV) might be an important class of tumor-associated antigens. We sought to understand whether specific mutations were associated with hERV expression. Two large, annotated genomic datasets, TCGA KIRC and IMmotion150, were used to correlate mutations and hERV expression. PBRM1 mutations were consistently associated with increased hERV expression in primary tumors. In vitro silencing of PBRM1, HIF1A, and HIF2A followed by RNA sequencing was performed in UMRC2 cells, confirming that PBRM1 regulates hERVs in a HIF1α- and HIF2α-dependent manner and that hERVs of the HERVERI superfamily are enriched in PBRM1-regulated hERVs. Our results uncover a role for PBRM1 in the negative regulation of hERVs in ccRCC. Moreover, the HIF-dependent nature of hERV expression explains the previously reported ccRCC-specific clinical associations of PBRM1-mutant ccRCC with both a good prognosis as well as improved clinical outcomes to ICB.

See related Spotlight by Labaki et al., p. 274.
Data from Circulating Tregs Accumulate in Omental Tumors and Acquire Adipose-Resident Features
Authors: Liu Mingyong , Starenki Dmytro , Scharer Christopher D. , Silva-Sanchez Aaron , Molina Patrick A. , Pollock Jennifer S. , Cooper Sara J. , Arend Rebecca C. , Rosenberg Alexander F. , Randall Troy D. , Meza-Perez Selene
Published: April 2023

Abstract
Tumors that metastasize in the peritoneal cavity typically end up in the omental adipose tissue, a particularly immune-suppressive environment that includes specialized adipose-resident regulatory T cells (Treg). Tregs rapidly accumulate in the omentum after tumor implantation and potently suppress antitumor immunity. However, it is unclear whether these Tregs are recruited from the circulation or derived from preexisting adipose-resident Tregs by clonal expansion. Here we show that Tregs in tumor-bearing omenta predominantly have thymus-derived characteristics. Moreover, naïve tumor antigen-specific CD4+ T cells fail to differentiate into Tregs in tumor-bearing omenta. In fact, Tregs derived from the pretumor repertoire are sufficient to suppress antitumor immunity and promote tumor growth. However, tumor implantation in the omentum does not promote Treg clonal expansion, but instead leads to increased clonal diversity. Parabiosis experiments show that despite tissue-resident (noncirculating) characteristics of omental Tregs in naïve mice, tumor implantation promotes a rapid influx of circulating Tregs, many of which come from the spleen. Finally, we show that newly recruited Tregs rapidly acquire characteristics of adipose-resident Tregs in tumor-bearing omenta. These data demonstrate that most Tregs in omental tumors are recruited from the circulation and adapt to their environment by altering their homing, transcriptional, and metabolic properties.
Data from Metabolic Screening of Cytotoxic T-cell Effector Function Reveals the Role of CRAC Channels in Regulating Lethal Hit Delivery
Authors: Slaats Jeroen , Dieteren Cindy E. , Wagena Esther , Wolf Louis , Raaijmakers Tonke K. , van der Laak Jeroen A. , Figdor Carl G. , Weigelin Bettina , Friedl Peter
Published: April 2023

Abstract
Cytotoxic T lymphocytes (CTL) mediate cytotoxicity toward tumor cells by multistep cell–cell interactions. However, the tumor microenvironment can metabolically perturb local CTL effector function. CTL activity is typically studied in two-dimensional (2D) liquid coculture, which is limited in recapitulating the mechanisms and efficacy of the multistep CTL effector response. We here developed a microscopy-based, automated three-dimensional (3D) interface coculture model suitable for medium-throughput screening to delineate the steps and CTL effector mechanisms affected by microenvironmental perturbation. CTL effector function was compromised by deregulated redox homeostasis, deficient mitochondrial respiration, as well as dysfunctional Ca2+ release-activated Ca2+ (CRAC) channels. Perturbation of CRAC channel function dampened calcium influx into CTLs, delayed CTL degranulation, and lowered the frequency of sublethal hits (i.e., additive cytotoxicity) delivered to the target cell. Thus, CRAC channel activity controls both individual contact efficacy and CTL cooperativity required for serial killing of target cells. The multistep analysis of CTL effector responses in 3D coculture will facilitate the identification of immune-suppressive mechanisms and guide the rational design of targeted intervention strategies to restore CTL effector function.
Data from Characteristics of Immune Memory and Effector Activity to Cancer-Expressed MHC Class I Phosphopeptides Differ in Healthy Donors and Ovarian Cancer Patients
Authors: Lulu Amanda M. , Cummings Kara L. , Jeffery Erin D. , Myers Paisley T. , Underwood Dennis , Lacy Rachel M. , Chianese-Bullock Kimberly A. , Slingluff Craig L. , Modesitt Susan C. , Engelhard Victor H.
Published: April 2023

Abstract
Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.
Data from IDO Vaccine Ablates Immune-Suppressive Myeloid Populations and Enhances Antitumor Effects Independent of Tumor Cell IDO Status
Authors: Nandre Rahul , Verma Vivek , Gaur Pankaj , Patil Veerupaxagouda , Yang Xingdong , Ramlaoui Zainab , Shobaki Nour , Andersen Mads Hald , Pedersen Ayako Wakatsuki , Zocca Mai-Britt , Mkrtichyan Mikayel , Gupta Seema , Khleif Samir N.
Published: April 2023

Abstract
The immunosuppressive tumor microenvironment (TME) does not allow generation and expansion of antitumor effector cells. One of the potent immunosuppressive factors present in the TME is the indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme, produced mainly by cancer cells and suppressive immune cells of myeloid origin. In fact, IDO+ myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) tend to be more suppressive than their IDO− counterparts. Hence, therapeutic approaches that would target the IDO+ cells in the TME, while sparing the antigen-presenting functions of IDO− myeloid populations, are needed. Using an IDO-specific peptide vaccine (IDO vaccine), we explored the possibility of generating effector cells against IDO and non-IDO tumor-derived antigens. For this, IDO-secreting (B16F10 melanoma) and non–IDO-secreting (TC-1) mouse tumor models were employed. We showed that the IDO vaccine significantly reduced tumor growth and enhanced survival of mice in both the tumor models, which associated with a robust induction of IDO-specific effector cells in the TME. The IDO vaccine significantly enhanced the antitumor efficacy of non-IDO tumor antigen–specific vaccines, leading to an increase in the number of total and antigen-specific activated CD8+ T cells (IFNγ+ and granzyme B+). Treatment with the IDO vaccine significantly reduced the numbers of IDO+ MDSCs and DCs, and immunosuppressive regulatory T cells in both tumor models, resulting in enhanced therapeutic ratios. Together, we showed that vaccination against IDO is a promising therapeutic option for both IDO-producing and non–IDO-producing tumors. The IDO vaccine selectively ablates the IDO+ compartment in the TME, leading to a significant enhancement of the immune responses against other tumor antigen–specific vaccines.
Data from Targeting the PSGL-1 Immune Checkpoint Promotes Immunity to PD-1–Resistant Melanoma
Authors: DeRogatis Julia M. , Viramontes Karla M. , Neubert Emily N. , Henriquez Monique L. , Guerrero-Juarez Christian F. , Tinoco Roberto
Published: April 2023

Abstract
Immune-checkpoint inhibitors have had impressive efficacy in some patients with cancer, reinvigorating long-term durable immune responses against tumors. Despite the clinical success of these therapies, most patients with cancer continue to be unresponsive to these treatments, highlighting the need for novel therapeutic options. Although P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to inhibit immune responses in a variety of disease models, previous work has yet to address whether PSGL-1 can be targeted therapeutically to promote antitumor immunity. Using an aggressive melanoma tumor model, we targeted PSGL-1 in tumor-bearing mice and found increased effector CD4+ and CD8+ T-cell responses and decreased regulatory T cells (Treg) in tumors. T cells exhibited increased effector function, activation, and proliferation, which delayed tumor growth in mice after anti–PSGL-1 treatment. Targeting PD-1 in PSGL-1–deficient, tumor-bearing mice led to an increased frequency of mice with complete tumor eradication. Targeting both PSGL-1 and PD-1 in wild-type tumor-bearing mice also showed enhanced antitumor immunity and slowed melanoma tumor growth. Our findings showed that therapeutically targeting the PSGL-1 immune checkpoint can reinvigorate antitumor immunity and suggest that targeting PSGL-1 may represent a new therapeutic strategy for cancer treatment.
Data from Single-Cell Analysis of the Pan-Cancer Immune Microenvironment and scTIME Portal
Authors: Hong Fang , Meng Qianqian , Zhang Weiyu , Zheng Ruiqin , Li Xiaoyun , Cheng Tao , Hu Deqing , Gao Xin
Published: April 2023

Abstract
Single-cell sequencing opens a new era for the investigation of tumor immune microenvironments (TIME). However, at single-cell resolution, a pan-cancer analysis that addresses the identity and diversity of TIMEs is lacking. Here, we first built a pan-cancer single-cell reference of TIMEs with refined subcell types and recognized new cell type–specific transcription factors. We then presented a pan-cancer view of the common features of the TIME and compared the variation of each immune cell type across patients and tumor types in the aspects of abundance, cell states, and cell communications. We found that the abundance and the cell states of dysfunctional T cells were most variable, whereas those of regulatory T cells were relatively stable. A subset of tumor-associated macrophages (TAM), PLTP+C1QC+ TAMs, may regulate the abundance of dysfunctional T cells through cytokine/chemokine signaling. The ligand–receptor communication network of TIMEs was tumor-type specific and dominated by the tumor-enriched immune cells. We additionally developed the single-cell TIME (scTIME) portal (http://scTIME.sklehabc.com) with the scTIME-specific analysis modules and a unified cell annotation. In addition to the immune cell compositions and correlation analysis using refined cell type classifications, the portal also provides cell–cell interaction and cell type–specific gene signature analysis. Our single-cell pan-cancer analysis and scTIME portal will provide more insights into the features of TIMEs, as well as the molecular and cellular mechanisms underlying immunotherapies.
Data from Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1
Authors: Ramaswamy Madhu , Kim Taeil , Jones Des C. , Ghadially Hormas , Mahmoud Tamer I. , Garcia Andrew , Browne Gareth , Zenonos Zenon , Puplampu-Dove Yvonne , Riggs Jeffrey M. , Bhat Geetha K. , Herbst Ronald , Schofield Darren J. , Carlesso Gianluca
Published: April 2023

Abstract
Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1–expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1–induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.
Data from Molecular Subgroups of Intrahepatic Cholangiocarcinoma Discovered by Single-Cell RNA Sequencing–Assisted Multiomics Analysis
Authors: Bao Xuanwen , Li Qiong , Chen Jinzhang , Chen Diyu , Ye Chanqi , Dai Xiaomeng , Wang Yanfang , Li Xin , Rong Xiaoxiang , Cheng Fei , Jiang Ming , Zhu Zheng , Ding Yongfeng , Sun Rui , Liu Chuan , Huang Lingling , Jin Yuzhi , Li Bin , Lu Juan , Wu Wei , Guo Yixuan , Fu Wenguang , Langley Sarah Raye , Tano Vincent , Fang Weijia , Guo Tiannan , Sheng Jianpeng , Zhao Peng , Ruan Jian
Published: April 2023

Abstract
Intrahepatic cholangiocarcinoma (ICC) is a relatively rare but highly aggressive tumor type that responds poorly to chemotherapy and immunotherapy. Comprehensive molecular characterization of ICC is essential for the development of novel therapeutics. Here, we constructed two independent cohorts from two clinic centers. A comprehensive multiomics analysis of ICC via proteomic, whole-exome sequencing (WES), and single-cell RNA sequencing (scRNA-seq) was performed. Novel ICC tumor subtypes were derived in the training cohort (n = 110) using proteomic signatures and their associated activated pathways, which were further validated in a validation cohort (n = 41). Three molecular subtypes, chromatin remodeling, metabolism, and chronic inflammation, with distinct prognoses in ICC were identified. The chronic inflammation subtype was associated with a poor prognosis. Our random forest algorithm revealed that mutation of lysine methyltransferase 2D (KMT2D) frequently occurred in the metabolism subtype and was associated with lower inflammatory activity. scRNA-seq further identified an APOE+C1QB+ macrophage subtype, which showed the capacity to reshape the chronic inflammation subtype and contribute to a poor prognosis in ICC. Altogether, with single-cell transcriptome-assisted multiomics analysis, we identified novel molecular subtypes of ICC and validated APOE+C1QB+ tumor-associated macrophages as potential immunotherapy targets against ICC.
Data from Superkine IL-2 and IL-33 Armored CAR T Cells Reshape the Tumor Microenvironment and Reduce Growth of Multiple Solid Tumors
Authors: Brog Rachel A. , Ferry Shannon L. , Schiebout Courtney T. , Messier Cameron M. , Cook W. James , Abdullah Leena , Zou Jia , Kumar Prathna , Sentman Charles L. , Frost H. Robert , Huang Yina H.
Published: April 2023

Abstract
Chimeric-antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against hematologic tumors. Yet, CAR T-cell therapy has had little success against solid tumors due to obstacles presented by the tumor microenvironment (TME) of these cancers. Here, we show that CAR T cells armored with the engineered IL-2 superkine Super2 and IL-33 were able to promote tumor control as a single-agent therapy. IFNγ and perforin were dispensable for the effects of Super2- and IL-33-armored CAR T cells. Super2 and IL-33 synergized to shift leukocyte proportions in the TME and to recruit and activate a broad repertoire of endogenous innate and adaptive immune cells including tumor-specific T cells. However, depletion of CD8+ T cells or NK cells did not disrupt tumor control, suggesting that broad immune activation compensated for loss of individual cell subsets. Thus, we have shown that Super2 and IL-33 CAR T cells can promote antitumor immunity in multiple solid tumor models and can potentially overcome antigen loss, highlighting the potential of this universal CAR T-cell platform for the treatment of solid tumors.
Data from Coexisting Alterations of MHC Class I Antigen Presentation and IFNγ Signaling Mediate Acquired Resistance of Melanoma to Post–PD-1 Immunotherapy
Authors: Nielsen Morten , Presti Mario , Sztupinszki Zsofia , Jensen Agnete Witness Præst , Draghi Arianna , Chamberlain Christopher Aled , Schina Aimilia , Yde Christina Westmose , Wojcik John , Szallasi Zoltan , Crowther Michael Douglas , Svane Inge Marie , Donia Marco
Published: April 2023

Abstract
Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti–programmed death 1 (anti–PD-1) who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti–PD-1 plus anti–lymphocyte activation gene 3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus kinase 1 (JAK1) signaling that coexisted in progressing tumor cells. Functional analyses revealed a pan T-cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TIL; B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti–PD-1 setting, indicating that coexisting altered pathways can lead to pan T-cell immune escape.
Data from Long Noncoding RNA MIR4435-2HG Suppresses Colorectal Cancer Initiation and Progression By Reprogramming Neutrophils
Authors: Yu Hongfei , Chen Chaoyi , Han Fengyan , Tang Jinlong , Deng Mengli , Niu Yumiao , Lai Maode , Zhang Honghe
Published: April 2023

Abstract
MIR4435-2HG, also known as LINC00978, has previously been described as an oncogenic long noncoding RNA (lncRNA). However, we show here that Mir4435-2hg depletion promoted colorectal tumorigenesis and progression in in vivo models of colitis-associated colorectal cancer, spontaneous intestinal adenomatous polyposis, and subcutaneous tumors. Alteration of MIR4435-2HG in colorectal cancer cells did not change the potential for cell proliferation, migration, or invasion in vitro. RNAscope assays showed that most MIR4435-2HG was located in the tumor stroma, which caused high expression of MIR4435-2HG in colorectal cancer tumor tissue. Transcriptome analysis of colorectal cancer tissues from wild-type and Mir4435-2hg–deficient mice revealed Mir4435-2hg as a tumor suppressor gene that regulated the immune microenvironment. Loss of Mir4435-2hg led to a decline in neutrophils and elevation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). In tissue-specific Mir4435-2hg knockout mice, we confirmed that Mir4435-2hg depletion in neutrophils, but not in intestinal epithelial cells, promoted colorectal cancer progression. Mechanistically, Mir4435-2hg depletion enhanced the immunosuppressive ability of PMN-MDSCs by disturbing their fatty acid metabolism. These findings suggest that MIR4435-2HG is a tumor-suppressing lncRNA whose deficiency could increase tumor-infiltrating PMN-MDSCs and enhance the immunosuppressive potential of PMN-MDSCs to promote colorectal cancer development. This provides a theoretical basis for further illustrating the pathogenesis of colorectal cancer and a potential antitumor immunotherapy target.
Data from Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma
Authors: Welters Carlotta , Lammoglia Cobo María Fernanda , Stein Christian Alexander , Hsu Meng-Tung , Ben Hamza Amin , Penter Livius , Chen Xiaojing , Buccitelli Christopher , Popp Oliver , Mertins Philipp , Dietze Kerstin , Bullinger Lars , Moosmann Andreas , Blanc Eric , Beule Dieter , Gerbitz Armin , Strobel Julian , Hackstein Holger , Rahn Hans-Peter , Dornmair Klaus , Blankenstein Thomas , Hansmann Leo
Published: April 2023

Abstract
Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow–infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non–multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C–restricted, self-peptide–specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell–dependent therapeutic strategies.
Data from KDM5A Inhibits Antitumor Immune Responses Through Downregulation of the Antigen-Presentation Pathway in Ovarian Cancer
Authors: Liu Heng , Lin Jianhuang , Zhou Wei , Moses Renyta , Dai Zhongping , Kossenkov Andrew V. , Drapkin Ronny , Bitler Benjamin G. , Karakashev Sergey , Zhang Rugang
Published: April 2023

Abstract
The extent to which effector CD8+ T cells infiltrate into tumors is one of the major predictors of clinical outcome for patients with epithelial ovarian cancer (EOC). Immune cell infiltration into EOC is a complex process that could be affected by the epigenetic makeup of the tumor. Here, we have demonstrated that a lysine 4 histone H3 (H3K4) demethylase, (lysine-specific demethylase 5A; KDM5A) impairs EOC infiltration by immune cells and inhibits antitumor immune responses. Mechanistically, we found that KDM5A silenced genes involved in the antigen processing and presentation pathway. KDM5A inhibition restored the expression of genes involved in the antigen-presentation pathway in vitro and promoted antitumor immune responses mediated by CD8+ T cells in vivo in a syngeneic EOC mouse model. A negative correlation between expression of KDM5A and genes involved in the antigen processing and presentation pathway such as HLA-A and HLA-B was observed in the majority of cancer types. In summary, our results establish KDM5A as a regulator of CD8+ T-cell infiltration of tumors and demonstrate that KDM5A inhibition may provide a novel therapeutic strategy to boost antitumor immune responses.
Data from Treg-Dominant Tumor Microenvironment Is Responsible for Hyperprogressive Disease after PD-1 Blockade Therapy
Authors: Wakiyama Hiroaki , Kato Takuya , Furusawa Aki , Okada Ryuhei , Inagaki Fuyuki , Furumoto Hideyuki , Fukushima Hiroshi , Okuyama Shuhei , Choyke Peter L. , Kobayashi Hisataka
Published: April 2023

Abstract
Programmed cell death 1 (PD-1) blockade therapy can result in dramatic responses in some patients with cancer. However, about 15% of patients receiving PD-1 blockade therapy experience rapid tumor progression, a phenomenon termed “hyperprogressive disease” (HPD). The mechanism(s) underlying HPD has been difficult to uncover because HPD is challenging to reproduce in animal models. Near-infrared photoimmunotherapy (NIR-PIT) is a method by which specific cells in the tumor microenvironment (TME) can be selectively depleted without disturbing other cells in the TME. In this study, we partially depleted CD8+ T cells with NIR-PIT by targeting the CD8β antigen thereby temporarily changing the balance of T-cell subsets in two different syngeneic tumor models. PD-1 blockade in these models led to rapid tumor progression compared with controls. CD3ε+CD8α+/CD3ε+CD4+FoxP3+ (Teff/Treg) ratios in the PD-1 and NIR-PIT groups were lower than in controls. Moreover, in a bilateral tumor model, low-dose CD8β-targeted NIR-PIT with anti–PD-1 blockade showed rapid tumor progression only in the tumor exposed to NIR light. In this experiment CD8β-targeted NIR-PIT in the exposed tumor reduced local CD8+ T cells resulting in a regulatory T-cell (Treg)–dominant TME. In conclusion, this reports an animal model to simulate the Treg-dominant TME, and the data generated using the model suggest that HPD after PD-1 blockade therapy can be attributed, at least in part, to imbalances between effector T cells and Tregs in the TME.
Data from Extracellular Vesicle–Packaged circATP2B4 Mediates M2 Macrophage Polarization via miR-532-3p/SREBF1 Axis to Promote Epithelial Ovarian Cancer Metastasis
Authors: Wang Fang , Niu Yuequn , Chen Kelie , Yuan Xiaoyu , Qin Yuheng , Zheng Fang , Cui Zhenyan , Lu Weiguo , Wu Yihua , Xia Dajing
Published: April 2023

Abstract
Ovarian cancer is one of the most common gynecologic malignancies with a highly immunosuppressive tumor microenvironment (TME) and poor prognosis. Circular RNA (circRNA) is a type of noncoding RNA with high stability, which has been shown to play an important role in biological processes and TME reprogramming in a variety of tumors. The biological function of a novel circRNA, circATP2B4, in epithelial ovarian cancer (EOC) was detected and evaluated. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of extracellular vesicles (EV)-packaged circATP2B4. Macrophage uptake of circATP2B4 was determined by EVs tracing. Dual luciferase reporter, FISH, Western blotting, and flow cytometry assays were used to investigate the interactions between circATP2B4 and miR-532-3p as well as sterol regulatory element-binding factor 1 (SREBF1) expression in macrophages. CircATP2B4 was upregulated in EOC tissues and positively correlated with ovarian cancer progression. Functionally, circATP2B4 promoted carcinogenic progression and metastasis of EOC both in vitro and in vivo. Mechanistically, EV-packaged circATP2B4 in EOC could be transmitted to infiltrated macrophages and acted as competing endogenous RNA of miR-532-3p to relieve the repressive effect of miR-532-3p on its target SREBF1. Furthermore, circATP2B4 induced macrophage M2 polarization by regulating the miR-532-3p/SREBF1/PI3Kα/AKT axis, thereby leading to immunosuppression and ovarian cancer metastasis. Collectively, these data indicate that circATP2B4-containing EVs generated by EOC cells promoted M2 macrophages polarization and malignant behaviors of EOC cells. Thus, targeting EVs-packaged circATP2B4 may provide a potential diagnosis and treatment strategy for ovarian cancer.
Data from Enhancing the Antitumor Immunity of T Cells by Engineering the Lipid-Regulatory Site of the TCR/CD3 Complex
Authors: Liang Wenhua , Yi Ruirong , Wang Weifang , Shi Yiwei , Zhang Jiqin , Xu Xiang , Wang Qingcan , Liu Mingyao , Wang Feng
Published: April 2023

Abstract
The engagement of the T-cell receptor (TCR) by a specific peptide–MHC ligand initiates transmembrane signaling to induce T-cell activation, a key step in most adaptive immune responses. Previous studies have indicated that TCR signaling is tightly regulated by cholesterol and its sulfate metabolite, cholesterol sulfate (CS), on the membrane. Here, we report a novel mechanism by which CS modulates TCR signaling through a conformational change of CD3 subunits. We found that the negatively charged CS interacted with the positively charged cytoplasmic domain of CD3ε (CD3εCD) to enhance its binding to the cell membrane and induce a stable secondary structure. This secondary structure suppressed the release of CD3εCD from the membrane in the presence of Ca2+, which in turn inhibited TCR phosphorylation and signaling. When a point mutation (I/A) was introduced to the intracellular immunoreceptor tyrosine-based activation motifs (YxxI-x6–8-YxxL) of CD3ε subunit, it reduced the stability of the secondary structure and regained sensitivity to Ca2+, which abolished CS-mediated inhibition and enhanced the signaling of the TCR complex. Notably, the I/A mutation could be applied to both murine and human TCR-T cell therapy to improve the antitumor efficacy. Our study reveals insights into the regulatory mechanism of TCR signaling and provides a strategy to functionally engineer the TCR/CD3 complex for T cell–based cancer immunotherapy.
Data from Efficacy and Synergy with Cisplatin of an Adenovirus Vectored Therapeutic E1E2E6E7 Vaccine against HPV Genome–Positive C3 Cancers in Mice
Authors: Boilesen Ditte Rahbæk , Neckermann Patrick , Willert Torsten , Müller Mikkel Dons , Schrödel Silke , Pertl Cordula , Thirion Christian , Asbach Benedikt , Wagner Ralf , Holst Peter Johannes
Published: April 2023

Abstract
Human papillomavirus (HPV) infections are the main cause of cervical and oropharyngeal cancers. As prophylactic vaccines have no curative effect, an efficient therapy would be highly desired. Most therapeutic vaccine candidates target only a small subset of HPV regulatory proteins, namely, E6 and E7, and are therefore restricted in the breadth of their immune response. However, research has suggested E1 and E2 as promising targets to fight HPV+ cancer. Here, we report the design of adenoviral vectors efficiently expressing HPV16 E1 and E2 in addition to transformation-deficient E6 and E7. Vaccination elicited vigorous CD4+ and CD8+ T-cell responses against all encoded HPV16 proteins in outbred mice and against E1 and E7 in C57BL/6 mice. Therapeutic vaccination of C3 tumor-bearing mice led to significantly reduced tumor growth and enhanced survival for both small and established tumors. Tumor biopsies revealed increased numbers of tumor-infiltrating CD8+ T cells in treated mice. Cisplatin enhanced the effect of therapeutic vaccination, accompanied by enhanced infiltration of dendritic cells into the tumor. CD8+ T cells were identified as effector cells in T-cell depletion assays, seemingly under regulation by FoxP3+CD4+ regulatory T cells. Finally, therapeutic vaccination with Ad-Ii-E1E2E6E7 exhibited significantly enhanced survival compared with vaccination with two peptides each harboring a known E6/E7 epitope. We hypothesize that this difference could be due to the induction of additional T-cell responses against E1. These results support the use of this novel vaccine candidate targeting an extended set of antigens (Ad-Ii-E1E2E6E7), in combination with cisplatin, as an advanced strategy to combat HPV+ cancers.
Data from Extracellular Galectin 4 Drives Immune Evasion and Promotes T-cell Apoptosis in Pancreatic Cancer
Authors: Lidström Tommy , Cumming Joshua , Gaur Rahul , Frängsmyr Lars , Pateras Ioannis S. , Mickert Matthias J. , Franklin Oskar , Forsell Mattias N.E. , Arnberg Niklas , Dongre Mitesh , Patthey Cedric , Öhlund Daniel
Published: April 2023

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell–produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1−/− mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.
Data 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

Abstract
T-cell receptor (TCR) binding strength to peptide-MHC antigen complex influences numerous T-cell functions. However, the vast diversity of a polyclonal T-cell repertoire for even a single antigen greatly increases the complexity of studying the impact of TCR affinity on T-cell function. Here, we determined how TCR binding strength affected the protein and transcriptional profile of an endogenous, polyclonal T-cell response to a known tumor-associated antigen (TAA) within the tumor microenvironment (TME). We confirmed that the staining intensity by flow cytometry and the counts by sequencing from MHC-tetramer labeling were reliable surrogates for the TCR-peptide-MHC steady-state binding affinity. We further demonstrated by single-cell RNA sequencing that tumor-infiltrating lymphocytes (TIL) with high and low binding affinity for a TAA can differentiate into cells with many antigen-specific transcriptional profiles within an established TME. However, more progenitor-like phenotypes were significantly biased towards lower affinity T cells, and proliferating phenotypes showed significant bias towards high-affinity TILs. In addition, we found that higher affinity T cells advanced more rapidly to terminal phases of T-cell exhaustion and exhibited better tumor control. We confirmed the polyclonal TIL results using a TCR transgenic mouse possessing a single low-affinity TCR targeting the same TAA. These T cells maintained a progenitor-exhausted phenotype and exhibited impaired tumor control. We propose that high-affinity TCR interactions drive T-cell fate decisions more rapidly than low-affinity interactions and that these cells differentiate faster. These findings illustrate divergent forms of T-cell dysfunction based on TCR affinity which may impact TIL therapies and antitumor responses.
Supplementary Data from Targeting MEF2D-fusion Oncogenic Transcriptional Circuitries in B-cell Precursor Acute Lymphoblastic Leukemia
Authors: Tsuzuki Shinobu , Yasuda Takahiko , Kojima Shinya , Kawazu Masahito , Akahane Koshi , Inukai Takeshi , Imaizumi Masue , Morishita Takanobu , Miyamura Koichi , Ueno Toshihide , Karnan Sivasundaram , Ota Akinobu , Hyodo Toshinori , Konishi Hiroyuki , Sanada Masashi , Nagai Hirokazu , Horibe Keizo , Tomita Akihiro , Suzuki Kyogo , Muramatsu Hideki , Takahashi Yoshiyuki , Miyazaki Yasushi , Matsumura Itaru , Kiyoi Hitoshi , Hosokawa Yoshitaka , Mano Hiroyuki , Hayakawa Fumihiko
Published: April 2023

SupplementaryTables