bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Modulation of Endoplasmic Reticulum Stress Controls CD4+ T-cell Activation and Antitumor Function
Authors: Thaxton Jessica E. , Wallace Caroline , Riesenberg Brian , Zhang Yongliang , Paulos Chrystal M. , Beeson Craig C. , Liu Bei , Li Zihai
Published: April 2023

Abstract
The endoplasmic reticulum (ER) is an energy-sensing organelle with intimate ties to programming cell activation and metabolic fate. T-cell receptor (TCR) activation represents a form of acute cell stress and induces mobilization of ER Ca2+ stores. The role of the ER in programming T-cell activation and metabolic fate remains largely undefined. Gp96 is an ER protein with functions as a molecular chaperone and Ca2+ buffering protein. We hypothesized that the ER stress response may be important for CD4+ T-cell activation and that gp96 may be integral to this process. To test our hypothesis, we utilized genetic deletion of the gp96 gene Hsp90b1 in a CD4+ T cell–specific manner. We show that gp96-deficient CD4+ T cells cannot undergo activation-induced glycolysis due to defective Ca2+ mobilization upon TCR engagement. We found that activating naïve CD4+ T cells while inhibiting ER Ca2+ exchange, through pharmacological blockade of the ER Ca2+ channel inositol trisphosphate receptor (IP3R), led to a reduction in cytosolic Ca2+ content and generated a pool of CD62Lhigh/CD44low CD4+ T cells compared with wild-type (WT) matched controls. In vivo IP3R-inhibited CD4+ T cells exhibited elevated tumor control above WT T cells. Together, these data show that ER-modulated cytosolic Ca2+ plays a role in defining CD4+ T-cell phenotype and function. Factors associated with the ER stress response are suitable targets for T cell–based immunotherapies. Cancer Immunol Res; 5(8); 666–75. ©2017 AACR.
Data from Somatic Mutations and Immune Alternation in Rectal Cancer Following Neoadjuvant Chemoradiotherapy
Authors: Ji Dengbo , Yi Haizhao , Zhang Dakui , Zhan Tiancheng , Li Zhaowei , Li Ming , Jia Jinying , Qiao Meng , Xia Jinhong , Zhai Zhiwei , Song Can , Gu Jin
Published: April 2023

Abstract
Checkpoint blockade therapy triggers tumor-specific immune responses in a variety of cancer types. We presumed that rectal cancer patients could have become sensitive to immunotherapy after receiving neoadjuvant chemoradiotherapy (nCRT). In this study, we report immune alternation in post-nCRT patients compared with pretreatment conditions from gene-expression omnibus (GEO) data. Whole-exome sequencing of 14 locally advanced rectal cancer (LARC) patient samples showed that nCRT induced new mutations compared with the paired pretreatment biopsies, evidenced by appearance of a neoantigen landscape. An association was identified between mutation burden and enrichment of immune activation–related pathways. Animal experiment results further demonstrated that radiotherapy enhanced the efficacy of anti–PD-1. Mutation burden and the neoantigens of LARC patients were associated with response to nCRT. The mRNA expression profiling of 66 pretreatment biopsy samples from LARC patients showed that immune activation–related pathways were enriched in response to nCRT. PD-L1 expression was negatively correlated with disease-free survival in the CD8-low expression patient group who received nCRT in a cohort of 296 samples. Thus, nCRT was able to alter immune function in LARC patients, which may be associated with the appearance of neoantigens. Neoantigens could make rectal cancer patients potential candidates to receive checkpoint blockade immunotherapy, and mutation burden could be a useful biomarker to stratify patients into responding and nonresponding groups for immunotherapy. Cancer Immunol Res; 6(11); 1401–16. ©2018 AACR.
Data from Impact of Tumor Purity on Immune Gene Expression and Clustering Analyses across Multiple Cancer Types
Authors: Rhee Je-Keun , Jung Yu Chae , Kim Kyu Ryung , Yoo Jinseon , Kim Jeeyoon , Lee Yong-Jae , Ko Yoon Ho , Lee Han Hong , Cho Byoung Chul , Kim Tae-Min
Published: April 2023

Abstract
Surgical archives of tumor specimens are often impure. The presence of RNA transcripts from nontumor cells, such as immune and stromal cells, can impede analyses of cancer expression profiles. To systematically analyze the impact of tumor purity, the gene expression profiles and tumor purities were obtained for 7,794 tumor specimens across 21 tumor types (available in The Cancer Genome Atlas consortium). First, we observed that genes with roles in immunity and oxidative phosphorylation were significantly inversely correlated and correlated with the tumor purity, respectively. The expression of genes implicated in immunotherapy and specific immune cell genes, along with the abundance of immune cell infiltrates, was substantially inversely correlated with tumor purity. This relationship may explain the correlation between immune gene expression and mutation burden, highlighting the need to account for tumor purity in the evaluation of expression markers obtained from bulk tumor transcriptome data. Second, examination of cluster membership of gene pairs, with or without controlling for tumor purity, revealed that tumor purity may have a substantial impact on gene clustering across tumor types. Third, feature genes for molecular taxonomy were analyzed for correlation with tumor purity, and for some tumor types, feature genes representing the mesenchymal and classical subtypes were inversely correlated and correlated with tumor purity, respectively. Our findings indicate that tumor purity is an important confounder in evaluating the correlation between gene expression and clinicopathologic features such as mutation burden, as well as gene clustering and molecular taxonomy. Cancer Immunol Res; 6(1); 87–97. ©2017 AACR.
Data from Clinical Significance of Pancreatic Atrophy Induced by Immune-Checkpoint Inhibitors: A Case–Control Study
Authors: Eshet Yael , Baruch Erez Nissim , Shapira-Frommer Ronnie , Steinberg-Silman Yael , Kuznetsov Teodor , Ben-Betzalel Guy , Daher Sameh , Gluck Iris , Asher Nethanel , Apter Sara , Schachter Jacob , Bar Jair , Boursi Ben , Markel Gal
Published: April 2023

Abstract
Immune-checkpoint inhibitor (ICI)–related diarrhea is attributed to inflammatory colitis, with no other drug-related differential diagnosis. Here, we investigated the occurrence of pancreatic atrophy (PA) in ICI-treated cancer patients and its correlation to exocrine pancreatic insufficiency (EPI). Metastatic melanoma, non–small cell lung carcinoma, and head and neck squamous cell carcinoma patients (n = 403) treated with anti–PD-1 (n = 356) or anti–CTLA-4 (n = 47) were divided into a case group (radiologic evidence of PA); control group matched by age, gender, and previous lines of treatment; and colitis group (ICI-induced colitis). Quantitative pancreatic volumetry was used for calculation of the decrease in pancreatic volume over time (atrophy rate). Thirty-one patients (7.7%) developed PA compared with 41 matched controls (P = 0.006). Four patients developed EPI, all from the anti–PD-1–treated group, which resolved with oral enzyme supplementation. The atrophy rate did not correlate with EPI (P = 0.87). EPI-related diarrhea presented at a median of 9 months, whereas the diarrhea of anti–PD-1–induced colitis patients (n = 22) was presented at a median of 2 months (P = 0.029). ICI-induced PA is irreversible and can result in EPI. EPI should be suspected in cases of late-onset steroid-resistant diarrhea with features of steatorrhea and treated with oral enzyme supplements.
Data from T-cell Receptors Engineered De Novo for Peptide Specificity Can Mediate Optimal T-cell Activity without Self Cross-Reactivity
Authors: Sharma Preeti , Harris Daniel T. , Stone Jennifer D. , Kranz David M.
Published: April 2023

Abstract
Despite progress in adoptive T-cell therapies, the identification of targets remains a challenge. Although chimeric antigen receptors recognize cell-surface antigens, T-cell receptors (TCR) have the advantage that they can target the array of intracellular proteins by binding to peptides associated with major histocompatibility complex (MHC) products (pepMHC). Although hundreds of cancer-associated peptides have been reported, it remains difficult to identify effective TCRs against each pepMHC complex. Conventional approaches require isolation of antigen-specific CD8+ T cells, followed by TCRαβ gene isolation and validation. To bypass this process, we used directed evolution to engineer TCRs with desired peptide specificity. Here, we compared the activity and cross-reactivity of two affinity-matured TCRs (T1 and RD1) with distinct origins. T1-TCR was isolated from a melanoma-reactive T-cell line specific for MART-1/HLA-A2, whereas RD1-TCR was derived de novo against MART-1/HLA-A2 by in vitro engineering. Despite their distinct origins, both TCRs exhibited similar peptide fine specificities, focused on the center of the MART-1 peptide. In CD4+ T cells, both TCRs mediated activity against MART-1 presented by HLA-A2. However, in CD8+ T cells, T1, but not RD1, demonstrated cross-reactivity with endogenous peptide/HLA-A2 complexes. Based on the fine specificity of these and other MART-1 binding TCRs, we conducted bioinformatics scans to identify structurally similar self-peptides in the human proteome. We showed that the T1-TCR cross-reacted with many of these self-peptides, whereas the RD1-TCR was rarely cross-reactive. Thus, TCRs such as RD1, generated de novo against cancer antigens, can serve as an alternative to TCRs generated from T-cell clones.
Data from Enhanced SLAMF7 Homotypic Interactions by Elotuzumab Improves NK Cell Killing of Multiple Myeloma
Authors: Pazina Tatiana , James Ashley M. , Colby Kimberly B. , Yang Yibin , Gale Andrew , Jhatakia Amy , Kearney Alper Y. , Graziano Robert F. , Bezman Natalie A. , Robbins Michael D. , Cohen Adam D. , Campbell Kerry S.
Published: April 2023

Abstract
Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, and CD319), which is highly expressed on multiple myeloma (MM) cells, natural killer (NK) cells, and subsets of other leukocytes. By engaging with FcγRIIIA (CD16), Elo promotes potent NK cell–mediated antibody-dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) toward SLAMF7+ MM tumor cells. Relapsed/refractory MM patients treated with the combination of Elo, lenalidomide, and dexamethasone have improved progression-free survival. We previously showed that Elo enhances NK cell activity via a costimulation mechanism, independent of CD16 binding. Here, we further studied the effect of Elo on cytotoxicity of CD16-negative NK-92 cells. Elo, but not other SLAMF7 antibodies, uniquely enhanced cytotoxicity mediated by CD16-negative NK-92 cells toward SLAMF7+ target cells. Furthermore, this CD16-independent enhancement of cytotoxicity required expression of SLAMF7 containing the full cytoplasmic domain in the NK cells, implicating costimulatory signaling. The CD16-independent costimulation by Elo was associated with increased expression of NKG2D, ICAM-1, and activated LFA-1 on NK cells, and enhanced cytotoxicity was partially reduced by NKG2D blocking antibodies. In addition, an Fc mutant form of Elo that cannot bind CD16 promoted cytotoxicity of SLAMF7+ target cells by NK cells from most healthy donors, especially if previously cultured in IL2. We conclude that in addition to promoting NK cell–mediated ADCC (CD16-dependent) responses, Elo promoted SLAMF7–SLAMF7 interactions in a CD16-independent manner to enhance NK cytotoxicity toward MM cells.
Data from Responsiveness to PD-1 Blockade in End-Stage Colon Cancer with Gene Locus 9p24.1 Copy-Number Gain
Authors: Ree Anne Hansen , Nygaard Vigdis , Russnes Hege G. , Heinrich Daniel , Nygaard Vegard , Johansen Christin , Bergheim Inger Riise , Hovig Eivind , Beiske Klaus , Negård Anne , Børresen-Dale Anne-Lise , Flatmark Kjersti , Mælandsmo Gunhild M.
Published: April 2023

Abstract
Most patients whose large bowel cancer has spread to other organs do not respond to immune therapy. We detected a rare gene mutation, termed 9p24.1 copy-number gain (CNG), in an otherwise incurable colorectal cancer that provoked an immune therapy response. We identified this gene mutation by gene-panel sequencing of DNA from a liver metastasis biopsy from a patient who had disease refractory to standard therapies. Following immune checkpoint blockade (ICB) with pembrolizumab (anti–PD-1), the patient experienced conversion of the tumor phenotype from one with epithelial features to that of an inflamed microenvironment, detected by high-resolution RNA sequencing. Circulating tumor DNA disappeared over the first weeks of therapy. As assessed by standard radiographic measurement, the patient had a partial response that was durable. This patient's response may support the use of histology-agnostic ICB in solid tumors that carry the rare 9p24.1 CNG.
Data from The Impact of High-Dose Glucocorticoids on the Outcome of Immune-Checkpoint Inhibitor–Related Thyroid Disorders
Authors: Ma Chanjuan , Hodi F. Stephen , Giobbie-Hurder Anita , Wang Xiaocheng , Zhou Jing , Zhang Amy , Zhou Ying , Mao Fei , Angell Trevor E. , Andrews Chelsea P. , Hu Jiani , Barroso-Sousa Romualdo , Kaiser Ursula B. , Tolaney Sara M. , Min Le
Published: April 2023

Abstract
Thyroid disorders have emerged as one of the most common immune-related adverse events (irAE), yet optimum management and biomarkers to predict vulnerable individuals remain to be explored. High-dose glucocorticoid (HDG) therapy is routinely recommended for irAEs. However, systematic analysis of the impact of glucocorticoid therapy on the outcome of immune-checkpoint inhibitor (ICI)–induced thyroid disorders is lacking. We analyzed 151 patients with or without ICI-related thyroid disorders. We divided the patients with ICI-related thyroid disorders into two subgroups: those with and without HDG treatment. Our results showed no significant differences between HDG and no HDG groups in terms of the median duration of thyrotoxicosis: 28 (range, 7–85) and 42 (range, 14–273) days, the median time to conversion from thyrotoxicosis to hypothyroidism: 39 days (range, 14–169) and 42 days (range, 14–315) days, the median time to onset of hypothyroidism: 63 (range, 21–190) and 63 (range, 14–489) days, and the median maintenance dose of levothyroxine: 1.5 (range, 0.4–2.3) μg/kg/day, and 1.3 (range, 0.3–2.5) μg/kg/day. The median pretreatment TSH was 2.3 (range, 0.3–5.2) mIU/L and 1.7 (range, 0.5–4.5) mIU/L in patients with and without ICI-related thyroid disorders, respectively. Baseline TSH was significantly higher in patients who developed ICI-related thyroid disorders (P = 0.05). Subgroup analysis revealed significantly higher baseline TSH in male but not in female patients with ICI-induced thyroid dysfunction. Our results show that HDG treatment did not improve the outcome of ICI-related thyroid disorders.
Data from Inhibition of SHP-1 Expands the Repertoire of Antitumor T Cells Available to Respond to Immune Checkpoint Blockade
Authors: Snook Jeremy P. , Soedel Ashleigh J. , Ekiz H. Atakan , O'Connell Ryan M. , Williams Matthew A.
Published: April 2023

Abstract
The presence and activity of CD8+ T cells within the tumor microenvironment are essential for the control of tumor growth. Utilizing B16-F10 melanoma tumors that express altered peptide ligands of chicken ovalbumin, OVA257-264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-I CD8+ T cell into mice subsequently challenged with tumors. T-cell receptor (TCR) affinity positively correlated with the frequency of OT-I tumor-infiltrating lymphocytes (TIL). Differences in TCR affinity inversely corresponded to in vivo tumor growth rate. Blockade of the PD-1 and CTLA-4 checkpoints preferentially increased the frequency and antitumor function of TIL responding to high-affinity antigens, while failing to enhance the antitumor activity of low-affinity T cells. To determine whether lowering the TCR activation threshold could enhance the breadth and magnitude of the antitumor T-cell response, we inhibited Src homology region 2 domain–containing phosphatase 1 (SHP-1) in OT-I T cells prior to tumor antigen exposure. SHP-1 knockdown increased the cytokine-producing potential of high- and low-affinity T cells but failed to enhance control of tumor growth. In contrast, when SHP-1 knockdown of OT-I T cells was combined with immunotherapy, we observed a significant and long-lasting suppression of tumor growth mediated by low-affinity T cells. We conclude that lowering the TCR activation threshold by targeting SHP-1 expands the repertoire of T cells available to respond to conventional checkpoint blockade, leading to enhanced control of tumor growth.
Data from A Gene Signature Predicting Natural Killer Cell Infiltration and Improved Survival in Melanoma Patients
Authors: Cursons Joseph , Souza-Fonseca-Guimaraes Fernando , Foroutan Momeneh , Anderson Ashley , Hollande Frédéric , Hediyeh-Zadeh Soroor , Behren Andreas , Huntington Nicholas D. , Davis Melissa J.
Published: April 2023

Abstract
Natural killer (NK) cell activity is essential for initiating antitumor responses and may be linked to immunotherapy success. NK cells and other innate immune components could be exploitable for cancer treatment, which drives the need for tools and methods that identify therapeutic avenues. Here, we extend our gene-set scoring method singscore to investigate NK cell infiltration by applying RNA-seq analysis to samples from bulk tumors. Computational methods have been developed for the deconvolution of immune cell types within solid tumors. We have taken the NK cell gene signatures from several such tools, then curated the gene list using a comparative analysis of tumors and immune cell types. Using a gene-set scoring method to investigate RNA-seq data from The Cancer Genome Atlas (TCGA), we show that patients with metastatic cutaneous melanoma have an improved survival rate if their tumor shows evidence of NK cell infiltration. Furthermore, these survival effects are enhanced in tumors that show higher expression of genes that encode NK cell stimuli such as the cytokine IL15. Using this signature, we then examine transcriptomic data to identify tumor and stromal components that may influence the penetrance of NK cells into solid tumors. Our results provide evidence that NK cells play a role in the regulation of human tumors and highlight potential survival effects associated with increased NK cell activity. Our computational analysis identifies putative gene targets that may be of therapeutic value for boosting NK cell antitumor immunity.
Data from Combined Low Densities of FoxP3+ and CD3+ Tumor-Infiltrating Lymphocytes Identify Stage II Colorectal Cancer at High Risk of Progression
Authors: Cavalleri Tommaso , Bianchi Paolo , Basso Gianluca , Celesti Giuseppe , Grizzi Fabio , Bossi Paola , Greco Luana , Pitrone Calogero , Valtorta Emanuele , Mauri Gianluca , Truini Mauro , Dall'Olio Filippo Gustavo , Brandi Giovanni , Sartore-Bianchi Andrea , Ricciardiello Luigi , Torri Valter , Rimassa Lorenza , Siena Salvatore , Mantovani Alberto , Malesci Alberto , Laghi Luigi
Published: April 2023

Abstract
The densities of CD3+ and CD8+ tumor-infiltrating lymphocytes (TILs), combined with tumor–node–metastasis (TNM) staging, have prognostic value for patients with nonmetastatic colorectal cancer. We compared the prognostic value of CD3+ and FoxP3+ TILs at the invasive front, TNM classifiers, and microsatellite (MS) status in a trial set of patients with stage II and III colorectal cancer (n = 413), by recursive partitioning with a classification and regression tree (CART). Significant prognostic factors and interactions were reassessed by logistic regression and Cox proportional-hazards modeling in the trial and a validation set (n = 215) of patients with stage II colorectal cancer. In the trial set, CART indicated that TIL numbers were of value only in predicting recurrence risk for stage II cancers, where low densities of FoxP3+ TILs ranked first and low densities of CD3+ TILs further stratifying risk. Multivariate analysis showed that TILs interacted with tumor stage (FoxP3+, P = 0.06; CD3+, P = 0.02) and MS instability (MSI; FoxP3+; P = 0.02). In stage II MS-stable cancers, concomitant low densities of both FoxP3+ and CD3+ TILs identified patients with the highest progression risk in the trial [HR 7.24; 95% confidence interval (CI), 3.41–15.4; P < 0.001] and the validation (HR 15.16; 95% CI, 3.43–66.9; P < 0.001) sets. FoxP3+ and CD3+ TIL load in colorectal cancer was more informative than other prognostic factors before the cancer progressed to lymph nodes. This prognostic information about TILs, including FoxP3+ cells, suggests that randomized controlled trials might be refined to include interactions between TNM status, molecular classifiers, and postsurgical treatments.
Data from Dysregulated NF-κB–Dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Patients with Melanoma
Authors: Maurer Deena M. , Adamik Juraj , Santos Patricia M. , Shi Jian , Shurin Michael R. , Kirkwood John M. , Storkus Walter J. , Butterfield Lisa H.
Published: April 2023

Abstract
Therapeutic cancer vaccines targeting melanoma-associated antigens are commonly immunogenic but are rarely effective in promoting objective clinical responses. To identify critical molecules for activation of effective antitumor immunity, we have profiled autologous dendritic cell (DC) vaccines used to treat 35 patients with melanoma. We showed that checkpoint molecules induced by ex vivo maturation correlated with in vivo DC vaccine activity. Melanoma patient DCs had reduced expression of cell surface inducible T-cell costimulator ligand (ICOSL) and had defective intrinsic NF-κB signaling. Chromatin immunoprecipitation assays revealed NF-κB–dependent transcriptional regulation of ICOSL expression by DCs. Blockade of ICOSL on DCs reduced priming of antigen-specific CD8+ and CD4+ T cells from naïve donors in vitro. Concentration of extracellular/soluble ICOSL released from vaccine DCs positively correlated with patient clinical outcomes, which we showed to be partially regulated by ADAM10/17 sheddase activity. These data point to the critical role of canonical NF-κB signaling, the regulation of matrix metalloproteinases, and DC-derived ICOSL in the specific priming of cognate T-cell responses in the cancer setting. This study supports the implementation of targeted strategies to augment these pathways for improved immunotherapeutic outcomes in patients with cancer.
Data from Pathogen-Boosted Adoptive Cell Transfer Therapy Induces Endogenous Antitumor Immunity through Antigen Spreading
Authors: Xin Gang , Khatun Achia , Topchyan Paytsar , Zander Ryan , Volberding Peter J. , Chen Yao , Shen Jian , Fu Chunmei , Jiang Aimin , See William A. , Cui Weiguo
Published: April 2023

Abstract
Loss of target antigens in tumor cells has become one of the major hurdles limiting the efficacy of adoptive cell therapy (ACT)–based immunotherapies. The optimal approach to overcome this challenge includes broadening the immune response from the initially targeted tumor-associated antigen (TAA) to other TAAs expressed in the tumor. To induce a more broadly targeted antitumor response, we utilized our previously developed Re-energized ACT (ReACT), which capitalizes on the synergistic effect of pathogen-based immunotherapy and ACT. In this study, we showed that ReACT induced a sufficient endogenous CD8+ T-cell response beyond the initial target to prevent the outgrowth of antigen loss variants in a B16-F10 melanoma model. Sequentially, selective depletion experiments revealed that Batf3-driven cDC1s were essential for the activation of endogenous tumor-specific CD8+ T cells. In ReACT-treated mice that eradicated tumors, we observed that endogenous CD8+ T cells differentiated into memory cells and facilitated the rejection of local and distal tumor rechallenge. By targeting one TAA with ReACT, we provided broader TAA coverage to counter antigen escape and generate a durable memory response against local relapse and metastasis.

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Data from Inhibition of the SRC Kinase HCK Impairs STAT3-Dependent Gastric Tumor Growth in Mice
Authors: Poh Ashleigh R. , Dwyer Amy R. , Eissmann Moritz F. , Chand Ashwini L. , Baloyan David , Boon Louis , Murrey Michael W. , Whitehead Lachlan , O'Brien Megan , Lowell Clifford A. , Putoczki Tracy L. , Pixley Fiona J. , O'Donoghue Robert J.J. , Ernst Matthias
Published: April 2023

Abstract
Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow–derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.
Data from Direct Detection and Quantification of Neoantigens
Authors: Wang Qing , Douglass Jacqueline , Hwang Michael S. , Hsiue Emily Han-Chung , Mog Brian J. , Zhang Ming , Papadopoulos Nickolas , Kinzler Kenneth W. , Zhou Shibin , Vogelstein Bert
Published: April 2023

Abstract
Many immunotherapeutic approaches under development rely on T-cell recognition of cancer-derived peptides bound to human leukocyte antigen molecules on the cell surface. Direct experimental demonstration that such peptides are processed and bound is currently challenging. Here, we describe a method that meets this challenge. The method entailed an optimized immunoprecipitation protocol coupled with two-dimensional chromatography and mass spectrometry. The ability to detect and quantify minute amounts of predefined antigens should be useful both for basic research in tumor immunology and for the development of rationally designed cancer vaccines.
Data from High Numbers of Circulating CD57+ NK Cells Associate with Resistance to HER2-Specific Therapeutic Antibodies in HER2+ Primary Breast Cancer
Authors: Muntasell Aura , Servitja Sònia , Cabo Mariona , Bermejo Begoña , Pérez-Buira Sandra , Rojo Federico , Costa-García Marcel , Arpí Oriol , Moraru Manuela , Serrano Laia , Tusquets Ignasi , Martínez María Teresa , Heredia Gemma , Vera Andrea , Martínez-García María , Soria Laura , Comerma Laura , Santana-Hernández Sara , Eroles Pilar , Rovira Ana , Vilches Carlos , Lluch Ana , Albanell Joan , López-Botet Miguel
Published: April 2023

Abstract
Natural killer (NK) cells can orchestrate effective antitumor immunity. The presence of tumor-infiltrating NK cells in diagnostic biopsies predicts pathologic complete response (pCR) to HER2-specific therapeutic antibodies in patients with primary breast cancer. Here, we analyzed whether diversity in circulating NK cells might influence tumor infiltration and HER2-specific therapeutic antibody efficacy. We found that numbers of circulating CD57+ NK cells inversely correlated with pCR to HER2-specific antibody treatment in patients with primary breast cancer independently of age, traditional clinicopathologic factors, and CD16A 158F/V genotype. This association was uncoupled from the expression of other NK-cell receptors, the presence of adaptive NK cells, or changes in major T-cell subsets, reminiscent of cytomegalovirus-induced immunomodulation. NK-cell activation against trastuzumab-coated HER2+ breast cancer cells was comparable in patients with high and low proportions of CD57+ NK cells. However, circulating CD57+ NK cells displayed decreased CXCR3 expression and CD16A-induced IL2-dependent proliferation in vitro. Presence of CD57+ NK cells was reduced in breast tumor–associated infiltrates as compared with paired peripheral blood samples, suggesting deficient homing, proliferation, and/or survival of NK cells in the tumor niche. Indeed, numbers of circulating CD57+ were inversely related to tumor-infiltrating NK-cell numbers. Our data reveal that NK-cell differentiation influences their antitumor potential and that CD57+ NK cells may be a biomarker useful for tailoring HER2 antibody–based therapeutic strategies in breast cancer.
Data from PD-1 and LAG-3 Dominate Checkpoint Receptor–Mediated T-cell Inhibition in Renal Cell Carcinoma
Authors: Zelba Henning , Bedke Jens , Hennenlotter Jörg , Mostböck Sven , Zettl Markus , Zichner Thomas , Chandran Anoop , Stenzl Arnulf , Rammensee Hans-Georg , Gouttefangeas Cécile
Published: April 2023

Abstract
Drugs targeting the programmed cell death protein 1 (PD-1) pathway are approved as therapies for an increasing number of cancer entities, including renal cell carcinoma. Despite a significant increase in overall survival, most treated patients do not show durable clinical responses. A combination of checkpoint inhibitors could provide a promising improvement. The aim of the study was to determine the most promising checkpoint blockade combination for renal cell carcinoma patients. Tumor-infiltrating lymphocytes (TIL) and autologous peripheral blood mononuclear cells (PBMC) were isolated from patients undergoing surgery for primary tumors. Cells were stained for multicolor flow cytometry to determine the (co)expression of five inhibitory receptors (iR), PD-1, LAG-3, Tim-3, BTLA, and CTLA-4, on T-cell populations. The function of these TILs was assessed by intracellular cytokine staining after in vitro stimulation in the presence or absence of PD-1 ± LAG-3 or Tim-3–specific antibodies. Although the percentage of iR+ T cells was low in PBMCs, both CD4+ and CD8+ T cells showed increased frequencies of PD-1+, LAG-3+, and Tim-3+ cells on TILs. The most frequent iR combination was PD-1 and LAG-3 on both CD4+ and CD8+ TILs. Blockade of PD-1 resulted in significant LAG-3, but not Tim-3, upregulation. The dual blockade of PD-1 and LAG-3, but not PD-1 and Tim-3, led to increased IFNγ release upon in vitro stimulation. Together, these data suggest that dual blockade of PD-1 and LAG-3 is a promising checkpoint blockade combination for renal cell carcinoma.
Data from A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy
Authors: Liu Si-Qi , Grantham Alyssa , Landry Casey , Granda Brian , Chopra Rajiv , Chakravarthy Srinivas , Deutsch Sabine , Vogel Markus , Russo Katie , Seiss Katherine , Tschantz William R. , Rejtar Tomas , Ruddy David A. , Hu Tiancen , Aardalen Kimberly , Wagner Joel P. , Dranoff Glenn , D'Alessio Joseph A.
Published: April 2023

Abstract
CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing–related molecules in a cell type–specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell–engaging CD3-bispecific antibody therapies.
Data from Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti–PD-1 Therapy Efficacy
Authors: Montfort Anne , Bertrand Florie , Rochotte Julia , Gilhodes Julia , Filleron Thomas , Milhès Jean , Dufau Carine , Imbert Caroline , Riond Joëlle , Tosolini Marie , Clarke Christopher J. , Dufour Florent , Constantinescu Andrei A. , Junior Nilton De França , Garcia Virginie , Record Michel , Cordelier Pierre , Brousset Pierre , Rochaix Philippe , Silvente-Poirot Sandrine , Therville Nicole , Andrieu-Abadie Nathalie , Levade Thierry , Hannun Yusuf A. , Benoist Hervé , Meyer Nicolas , Micheau Olivier , Colacios Céline , Ségui Bruno
Published: April 2023

Abstract
Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow–derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti–PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell–dependent immune responses and overcome resistance to anti–PD-1.
Data from MHC Class II Antigen Presentation by Lymphatic Endothelial Cells in Tumors Promotes Intratumoral Regulatory T cell–Suppressive Functions
Authors: Gkountidi Anastasia O. , Garnier Laure , Dubrot Juan , Angelillo Julien , Harlé Guillaume , Brighouse Dale , Wrobel Ludovic J. , Pick Robert , Scheiermann Christoph , Swartz Melody A. , Hugues Stéphanie
Published: April 2023

Abstract
Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II–restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo. In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II–restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II–restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell–mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.
Data from Targeting CMTM6 Suppresses Stem Cell–Like Properties and Enhances Antitumor Immunity in Head and Neck Squamous Cell Carcinoma
Authors: Chen Lei , Yang Qi-Chao , Li Yi-Cun , Yang Lei-Lei , Liu Jian-Feng , Li Hao , Xiao Yao , Bu Lin-Lin , Zhang Wen-Feng , Sun Zhi-Jun
Published: April 2023

Abstract
CMTM6, a regulator of PD-L1 expression, also modulates tumor immunity. Little is known about the function of CMTM6 and its mechanism of action in head and neck squamous cell carcinoma (HNSCC). In this study, we found by IHC analysis that CMTM6 overexpression predicted a poor prognosis for patients with HNSCC. We discovered that CMTM6 expression was correlated with increased activity through the Wnt/β-catenin signaling pathway, which is essential for tumorigenesis, maintenance of cancer stem cells (CSC), and the epithelial-to-mesenchymal transition (EMT) characteristic of multiple cancers. We used short hairpin RNA to eliminate expression of CMTM6, which led, in HNSCC cells, to reduced expression of nuclear β-catenin as well as inhibition of stem cell–like properties, TGFβ-induced EMT, and cell proliferation. Consistent with these results, we identified a significant positive correlation between expression of CMTM6 and EMT- and CSC-related genes in The Cancer Genome Atlas (TCGA). We found positive correlations for both RNA and protein between expression of CMTM6 and immune checkpoint components. CMTM6 silencing–induced PD-L1 downregulation delayed SCC7 tumor growth and increased CD8+ and CD4+ T-cell infiltration. The proportions of PD-1+, TIM-3+, VISTA+, LAG-3+, and B7-H3+ exhausted T cells were decreased significantly in the CMTM6 knockdown group. CMTM6 thus regulates stemness, EMT, and T-cell dysfunction and may be a promising therapeutic target in the treatment of HNSCC.
Data from Impact of TCR Diversity on the Development of Transplanted or Chemically Induced Tumors
Authors: Schreiber Karin , Karrison Theodore G. , Wolf Steven P. , Kiyotani Kazuma , Steiner Madeline , Littmann Eric R. , Pamer Eric G. , Kammertoens Thomas , Schreiber Hans , Leisegang Matthias
Published: April 2023

Abstract
Burnet postulated that the diversity of T-cell receptors (TCR) allows T cells to protect against the development of cancers that display antigens with a similar, seemingly endless diversity. To test this hypothesis, we developed a strategy in which a single breeding pair of mice gives rise to four groups of sibling mice. Three of the four groups had a similar number of CD8+ T cells, but TCR diversity was either broad, significantly reduced, or absent when expressing only one type of TCR. The fourth group had no T cells. All mice shared the same housing, and, therefore, their microbial environment was similar. Only slight differences in the intestinal flora were observed under these conditions. An undisturbed broad TCR repertoire was required for the rejection of inoculated cancers displaying the natural antigenic heterogeneity of primary tumors, whereas even one type of TCR was sufficient to protect against artificial cancers stably expressing cognate antigens. The three groups of mice with limited or no TCR repertoire showed an increased risk of developing primary tumors after chemical induction. However, the risk of early death or morbidity in these cohorts of mice was significantly higher than in mice with a diverse TCR repertoire, and it remains unknown whether mice with reduced TCR diversity, who died early without cancer, would have developed tumors with higher, lower, or equal probability after induction. Together, TCR diversity seems crucial to overcome the natural genetic instability of cancers and their antigenic heterogeneity, which impacts the design of cellular therapies.
Data from Transfer of MicroRNA via Macrophage-Derived Extracellular Vesicles Promotes Proneural-to-Mesenchymal Transition in Glioma Stem Cells
Authors: Zhang Zongpu , Xu Jianye , Chen Zihang , Wang Huizhi , Xue Hao , Yang Chunlei , Guo Qindong , Qi Yanhua , Guo Xiaofan , Qian Mingyu , Wang Shaobo , Qiu Wei , Gao Xiao , Zhao Rongrong , Guo Xing , Li Gang
Published: April 2023

Abstract
Proneural-to-mesenchymal transition (PMT) is a common process in glioblastoma (GBM) progression that leads to increased radiotherapy resistance. However, the mechanism underlying PMT is poorly understood. Here, we found that tumor-associated macrophages triggered PMT in glioma stem cells (GSC) via small extracellular vesicles (sEV). sEVs from monocyte-derived macrophages transferred miR-27a-3p, miR-22-3p, and miR-221-3p to GSCs, and these miRNAs promoted several mesenchymal phenotypes in proneural (PN) GSCs by simultaneously targeting CHD7. We found that CHD7 played a critical role in the maintenance of the PN phenotype, and CHD7 knockdown significantly promoted PMT in GSCs via the RelB/P50 and p-STAT3 pathways. The induction of PMT by sEVs containing miR-27a-3p, miR-22-3p, and miR-221-3p in a xenograft nude mouse model exacerbated radiotherapy resistance and thus decreased the benefits of radiotherapy. Collectively, these findings identified macrophage-derived sEVs as key regulators of PMT in GSCs and demonstrated that CHD7 is a novel inhibitor of PMT.
Data from Overproduction of Gastrointestinal 5-HT Promotes Colitis-Associated Colorectal Cancer Progression via Enhancing NLRP3 Inflammasome Activation
Authors: Li Tao , Fu Bin , Zhang Xin , Zhou Yunjiang , Yang Mengdi , Cao Mengran , Chen Yaxin , Tan Yingying , Hu Rong
Published: April 2023

Abstract
Chronic inflammation is a key driver for colitis-associated colorectal cancer. 5-hydroxytryptamine (5-HT), a neurotransmitter, has been reported to promote inflammation in the gastrointestinal tract. However, the mechanism behind this remains unclear. In this study, we found that 5-HT levels, as well as the expression of tryptophan hydroxylase 1 (TPH1), the 5-HT biosynthesis rate-limiting enzyme, were significantly upregulated in colorectal tumor tissues from patients with colorectal cancer, colorectal cancer mouse models, and colorectal cancer cell lines when compared with normal colorectal tissues or epithelial cell lines. Colorectal cancer cell–originated 5-HT enhanced NLRP3 inflammasome activation in THP-1 cells and immortalized bone marrow–derived macrophages (iBMDM) via its ion channel receptor, HTR3A. Mechanistically, HTR3A activation led to Ca2+ influx, followed by CaMKIIα phosphorylation (Thr286) and activation, which then induced NLRP3 phosphorylation at Ser198 (mouse: Ser194) and inflammasome assembling. The NLRP3 inflammasome mediated IL1β maturation, and release upregulated 5-HT biosynthesis in colorectal cancer cells by inducing TPH1 transcription, revealing a positive feedback loop between 5-HT and NLRP3 signaling. Silencing TPH1 or HTR3A by short hairpin RNA slowed down tumor growth in an established CT26 and iBMDM coimplanted subcutaneous allograft colorectal cancer mouse model, whereas treatment with TPH1 inhibitor 4-chloro-DL-phenylalanine or HTR3A antagonist tropisetron alleviated tumor progression in an azoxymethane/dextran sodium sulfate–induced colorectal cancer mouse model. Addressing the positive feedback loop between 5-HT and NLRP3 signaling could provide potential therapeutic targets for colorectal cancer.
Data from Alternative Splicing of the Inhibitory Immune Checkpoint Receptor SLAMF6 Generates a Dominant Positive Form, Boosting T-cell Effector Functions
Authors: Hajaj Emma , Zisman Elad , Tzaban Shay , Merims Sharon , Cohen Jonathan , Klein Shiri , Frankenburg Shoshana , Sade-Feldman Moshe , Tabach Yuval , Yizhak Keren , Navon Ami , Stepensky Polina , Hacohen Nir , Peretz Tamar , Veillette André , Karni Rotem , Eisenberg Galit , Lotem Michal
Published: April 2023

Abstract
SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor-exhausted T cells. Here we show that in humans, SLAMF6 has three splice isoforms involving its V-domain. Although the canonical receptor inhibited T-cell activation through SAP recruitment, the short isoform SLAMF6Δ17–65 had a strong agonistic effect. The costimulatory action depended on protein phosphatase SHP1 and led to a cytotoxic molecular profile mediated by the expression of TBX21 and RUNX3. Patients treated with immune checkpoint blockade showed a shift toward SLAMF6Δ17–65 in peripheral blood T cells. We developed splice-switching antisense oligonucleotides (ASO) designed to target the relevant SLAMF6 splice junction. Our ASOs enhanced SLAMF6Δ17–65 expression in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The yin-yang relationship of SLAMF6 splice isoforms may represent a balancing mechanism that could be exploited to improve cancer immunotherapy.
Data from Targeting PIM1-Mediated Metabolism in Myeloid Suppressor Cells to Treat Cancer
Authors: Xin Gang , Chen Yao , Topchyan Paytsar , Kasmani Moujtaba Y. , Burns Robert , Volberding Peter J. , Wu Xiaopeng , Cohn Alexandra , Chen Yiliang , Lin Chien-Wei , Ho Ping-Chih , Silverstein Roy , Dwinell Michael B. , Cui Weiguo
Published: April 2023

Abstract
There is a strong correlation between myeloid-derived suppressor cells (MDSC) and resistance to immune checkpoint blockade (ICB), but the detailed mechanisms underlying this correlation are largely unknown. Using single-cell RNA sequencing analysis in a bilateral tumor model, we found that immunosuppressive myeloid cells with characteristics of fatty acid oxidative metabolism dominate the immune-cell landscape in ICB-resistant subjects. In addition, we uncovered a previously underappreciated role of a serine/threonine kinase, PIM1, in regulating lipid oxidative metabolism via PPARγ-mediated activities. Enforced PPARγ expression sufficiently rescued metabolic and functional defects of Pim1−/− MDSCs. Consistent with this, pharmacologic inhibition of PIM kinase by AZD1208 treatment significantly disrupted the myeloid cell–mediated immunosuppressive microenvironment and unleashed CD8+ T-cell–mediated antitumor immunity, which enhanced PD-L1 blockade in preclinical cancer models. PIM kinase inhibition also sensitized nonresponders to PD-L1 blockade by selectively targeting suppressive myeloid cells. Overall, we have identified PIM1 as a metabolic modulator in MDSCs that is associated with ICB resistance and can be therapeutically targeted to overcome ICB resistance.