bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Spatial UMAP and Image Cytometry for Topographic Immuno-oncology Biomarker Discovery
Authors: Giraldo Nicolas A. , Berry Sneha , Becht Etienne , Ates Deniz , Schenk Kara M. , Engle Elizabeth L. , Green Benjamin , Nguyen Peter , Soni Abha , Stein Julie E. , Succaria Farah , Ogurtsova Aleksandra , Xu Haiying , Gottardo Raphael , Anders Robert A. , Lipson Evan J. , Danilova Ludmila , Baras Alexander S. , Taube Janis M.
Published: April 2023

Abstract
Multiplex immunofluorescence (mIF) can detail spatial relationships and complex cell phenotypes in the tumor microenvironment (TME). However, the analysis and visualization of mIF data can be complex and time-consuming. Here, we used tumor specimens from 93 patients with metastatic melanoma to develop and validate a mIF data analysis pipeline using established flow cytometry workflows (image cytometry). Unlike flow cytometry, spatial information from the TME was conserved at single-cell resolution. A spatial uniform manifold approximation and projection (UMAP) was constructed using the image cytometry output. Spatial UMAP subtraction analysis (survivors vs. nonsurvivors at 5 years) was used to identify topographic and coexpression signatures with positive or negative prognostic impact. Cell densities and proportions identified by image cytometry showed strong correlations when compared with those obtained using gold-standard, digital pathology software (R2 > 0.8). The associated spatial UMAP highlighted “immune neighborhoods” and associated topographic immunoactive protein expression patterns. We found that PD-L1 and PD-1 expression intensity was spatially encoded—the highest PD-L1 expression intensity was observed on CD163+ cells in neighborhoods with high CD8+ cell density, and the highest PD-1 expression intensity was observed on CD8+ cells in neighborhoods with dense arrangements of tumor cells. Spatial UMAP subtraction analysis revealed numerous spatial clusters associated with clinical outcome. The variables represented in the key clusters from the unsupervised UMAP analysis were validated using established, supervised approaches. In conclusion, image cytometry and the spatial UMAPs presented herein are powerful tools for the visualization and interpretation of single-cell, spatially resolved mIF data and associated topographic biomarker development.
Data from B7-H3 Suppresses Antitumor Immunity via the CCL2–CCR2–M2 Macrophage Axis and Contributes to Ovarian Cancer Progression
Authors: Miyamoto Taito , Murakami Ryusuke , Hamanishi Junzo , Tanigaki Kenji , Hosoe Yuko , Mise Nathan , Takamatsu Shiro , Mise Yuka , Ukita Masayo , Taki Mana , Yamanoi Koji , Horikawa Naoki , Abiko Kaoru , Yamaguchi Ken , Baba Tsukasa , Matsumura Noriomi , Mandai Masaki
Published: April 2023

Abstract
New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study, we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2–CCR2–M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1–low, nonimmunoreactive HGSOC tumors, and its expression negatively correlated with an IFNγ signature, which reflects the tumor immune reactivity. In syngeneic mouse models, B7-H3 (Cd276) knockout (KO) in tumor cells, but not in stromal cells, suppressed tumor progression, with a reduced number of M2 macrophages and an increased number of IFNγ+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2–CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation, and tumor progression. In patients with HGSOC, B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance, and patients with B7-H3–high tumors had fewer tumoral IFNγ+CD8+ T cells and poorer prognosis than patients with B7-H3–low tumors. Thus, B7-H3 expression in tumor cells contributes to CCL2–CCR2–M2 macrophage axis–mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype.
Data from Combination Neoantigen-Based Dendritic Cell Vaccination and Adoptive T-Cell Transfer Induces Antitumor Responses Against Recurrence of Hepatocellular Carcinoma
Authors: Peng Sui , Chen Shuling , Hu Wei , Mei Jie , Zeng Xuezhen , Su Tianhong , Wang Wei , Chen Zebin , Xiao Han , Zhou Qian , Li Bin , Xie Yubin , Hu Huanjing , He Minghui , Han Yanyan , Tang Longqing , Ma Yifan , Li Xiaoshuang , Zhou Xiangjun , Dai Zihao , Liu Zelong , Tan Jiehui , Xu Lixia , Li Shaoqiang , Shen Shunli , Li Dongming , Lai Jiaming , Peng Baogang , Peng Zhenwei , Kuang Ming
Published: April 2023

Abstract
A high rate of recurrence after curative therapy is a major challenge for the management of hepatocellular carcinoma (HCC). Currently, no effective adjuvant therapy is available to prevent HCC recurrence. We designed a personalized neoantigen-loaded dendritic cell vaccine and neoantigen-activated T-cell therapy, and used it as adjuvant therapy to treat 10 patients with HCC who had undergone curative resection or radiofrequency ablation in the first stage of a phase II trial (NCT03067493). The primary outcomes were safety and neoantigen-specific immune response. Disease-free survival (DFS) was also evaluated. The immunotherapy was successfully administered to all the patients without unexpected delay and demonstrated a reasonable safety profile with no grade ≥3 treatment-related side effects reported. Seventy percent of patients generated de novo circulating multiclonal neoantigen-specific T-cell responses. Induced neoantigen-specific immunity was maintained over time, and epitope spreading was observed. Patients who generated immune responses to treatment exhibited prolonged DFS compared with nonresponders (P = 0.012), with 71.4% experiencing no relapse for 2 years after curative treatment. High expression of an immune stimulatory signature, enhanced immune-cell infiltration (i.e., CD8+ T cells), and upregulated expression of T-cell inflammatory gene profiles were found in the primary tumors of the responders. In addition, neoantigen depletion (immunoediting) was present in the recurrent tumors compared with the primary tumors (7/9 vs. 1/17, P = 0.014), suggesting that immune evasion occurred under the pressure of immunotherapy. Our study indicates that neoantigen-based combination immunotherapy is feasible, safe, and has the potential to reduce HCC recurrence after curative treatment.
Data from Plexin-A4 Mediates Cytotoxic T-cell Trafficking and Exclusion in Cancer
Authors: Celus Ward , Oliveira Ana I. , Rivis Silvia , Van Acker Heleen H. , Landeloos Ewout , Serneels Jens , Cafarello Sarah Trusso , Van Herck Yannick , Mastrantonio Roberta , Köhler Arnaud , Garg Abhishek D. , Flamand Véronique , Tamagnone Luca , Marine Jean-Christophe , Matteo Mario Di , Costa Bruno M. , Bechter Oliver , Mazzone Massimiliano
Published: April 2023

Abstract
Cytotoxic T cell (CTL) infiltration of the tumor carries the potential to limit cancer progression, but their exclusion by the immunosuppressive tumor microenvironment hampers the efficiency of immunotherapy. Here, we show that expression of the axon guidance molecule Plexin-A4 (Plxna4) in CTLs, especially in effector/memory CD8+ T cells, is induced upon T-cell activation, sustained in the circulation, but reduced when entering the tumor bed. Therefore, we deleted Plxna4 and observed that Plxna4-deficient CTLs acquired improved homing capacity to the lymph nodes and to the tumor, as well as increased proliferation, both achieved through enhanced Rac1 activation. Mice with stromal or hematopoietic Plxna4 deletion exhibited enhanced CTL infiltration and impaired tumor growth. In a melanoma model, adoptive transfer of CTLs lacking Plxna4 prolonged survival and improved therapeutic outcome, which was even stronger when combined with anti–programmed cell death protein 1 (PD-1) treatment. PLXNA4 abundance in circulating CTLs was augmented in melanoma patients versus healthy volunteers but decreased after the first cycle of anti–PD-1, alone or in combination with anti–cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), in those patients showing complete or partial response to the treatment. Altogether, our data suggest that Plxna4 acts as a “checkpoint,” negatively regulating CTL migration and proliferation through cell-autonomous mechanisms independent of the interaction with host-derived Plxna4 ligands, semaphorins. These findings pave the way toward Plxna4-centric immunotherapies and propose Plxna4 detection in circulating CTLs as a potential way to monitor the response to immune checkpoint blockade in patients with metastatic melanoma.
Data from β2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection
Authors: Guenther Carla , Faisal Imrul , Fusciello Manlio , Sokolova Maria , Harjunpää Heidi , Ilander Mette , Tallberg Robert , Vartiainen Maria Kristina , Alon Ronen , Gonzalez-Granado Jose-Maria , Cerullo Vincenzo , Fagerholm Susanna Carola
Published: April 2023

Abstract
Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow–derived DCs (BM-DC) expressing dysfunctional β2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional β2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo–cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, β2-integrin–mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting β2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.
Data from ICBatlas: A Comprehensive Resource for Depicting Immune Checkpoint Blockade Therapy Characteristics from Transcriptome Profiles
Authors: Yang Mei , Miao Ya-Ru , Xie Gui-Yan , Luo Mei , Hu Hui , Kwok Hang Fai , Feng Jian , Guo An-Yuan
Published: April 2023

Abstract
Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. Much work is currently being conducted to investigate the mechanisms of ICB therapy at the transcriptional level. Integrating the data produced by these studies will help us give more insight into the transcriptomic features of ICB therapy. We collected the transcriptome and clinical data of ICB-treated patient samples from the Gene Expression Omnibus, ArrayExpress, The Cancer Genome Atlas, and dbGaP databases. On the basis of the clinical information, all samples are initially classified into response/nonresponse or pretreatment/on-treatment groups. Differential expression, pathway enrichment, and immune cell infiltration analyses are performed between the samples from different groups. We also introduce the Response Score (RS) calculated by integrating the variability degree and the frequency of the dysregulated genes in the responders to evaluate the impact of gene expression on the response. Finally, all the abovementioned contents are integrated into the ICBatlas database. ICBatlas provides the transcriptome features of ICB therapy through the analysis of 1,515 ICB-treated samples from 25 studies across nine cancer types. The data in ICBatlas include clinical outcomes, treatment-related genes, biological pathways, and immune cell infiltration. Users can investigate the abovementioned transcriptome features in the response (R vs. NR) or treatment (Pre vs. On) modules at the data set, cancer type, or immune checkpoint level and compare the degree of gene impact on the response in the RS module. ICBatlas is the first database to show the transcriptome features on ICB therapy in human cancers and freely available at http://bioinfo.life.hust.edu.cn/ICBatlas/.
Data from Checkpoint Blockade–Induced Dermatitis and Colitis Are Dominated by Tissue-Resident Memory T Cells and Th1/Tc1 Cytokines
Authors: Reschke Robin , Shapiro Jason W. , Yu Jovian , Rouhani Sherin J. , Olson Daniel J. , Zha Yuanyuan , Gajewski Thomas F.
Published: April 2023

Abstract
Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.
Data from Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy
Authors: Im Nam Gyu , Guillaumet-Adkins Amy , Wal Megha , Rogers Anna J. , Frede Julia , Havig Claire C. , Yang Jing , Anand Praveen , Stegmann Sarah K. , Waldschmidt Johannes M. , Sotudeh Noori , Niu Leili , Voisine Jordan , Schweiger Michal R. , Grassberger Clemens , Lohr Jens G. , Knoechel Birgit
Published: April 2023

Abstract
Chimeric antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T cell–leukemia cell interface followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing, we demonstrated that a subset of refractory CD19low cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the Bruton's tyrosine kinase inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity.

See related Spotlight by Zhao and Melenhorst, p. 1040
Data from Using CD69 PET Imaging to Monitor Immunotherapy-Induced Immune Activation
Authors: Edwards Kimberly J. , Chang Bryan , Babazada Hasan , Lohith Katheryn , Park Daniel H. , Farwell Michael D. , Sellmyer Mark A.
Published: April 2023

Abstract
Immune checkpoint inhibitors (ICI) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from these therapies. Thus, there is a clinical need for reliable tools that allow for the early assessment of response to ICIs, as well as a preclinical need for imaging tools that aid in the future development and understanding of immunotherapies. Here we demonstrate that CD69, a canonical early-activation marker expressed on a variety of activated immune cells, including cytotoxic T cells and natural killer (NK) cells, is a promising biomarker for the early assessment of response to immunotherapies. We have developed a PET probe by radiolabeling a highly specific CD69 mAb, H1.2F3, with Zirconium-89 (89Zr), [89Zr]-deferoxamine (DFO)-H1.2F3. [89Zr]-DFO-H1.2F3 detected changes in CD69 expression on primary mouse T cells in vitro and detected activated immune cells in a syngeneic tumor immunotherapy model. In vitro uptake studies with [89Zr]-DFO-H1.2F3 showed a 15-fold increase in CD69 expression for activated primary mouse T cells, relative to untreated resting T cells. In vivo PET imaging showed that tumors of ICI-responsive mice had greater uptake than the tumors of nonresponsive and untreated mice. Ex vivo biodistribution, autoradiography, and IHC analyses supported the PET imaging findings. These data suggest that the CD69 PET imaging approach detects CD69 expression with sufficient sensitivity to quantify immune cell activation in a syngeneic mouse immunotherapy model and could allow for the prediction of therapeutic immune responses to novel immunotherapies.
Data from Reciprocal Interactions Between the Gut Microbiome and Mammary Tissue Mast Cells Promote Metastatic Dissemination of HR+ Breast Tumors
Authors: Feng Tzu-Yu , Azar Francesca N. , Dreger Sally A. , Rosean Claire Buchta , McGinty Mitchell T. , Putelo Audrey M. , Kolli Sree H. , Carey Maureen A. , Greenfield Stephanie , Fowler Wesley J. , Robinson Stephen D. , Rutkowski Melanie R.
Published: April 2023

Abstract
Establishing commensal dysbiosis, defined as an inflammatory gut microbiome with low biodiversity, before breast tumor initiation, enhances early dissemination of hormone receptor–positive (HR+) mammary tumor cells. Here, we sought to determine whether cellular changes occurring in normal mammary tissues, before tumor initiation and in response to dysbiosis, enhanced dissemination of HR+ tumors. Commensal dysbiosis increased both the frequency and profibrogenicity of mast cells in normal, non–tumor-bearing mammary tissues, a phenotypic change that persisted after tumor implantation. Pharmacological and adoptive transfer approaches demonstrated that profibrogenic mammary tissue mast cells from dysbiotic animals were sufficient to enhance dissemination of HR+ tumor cells. Using archival HR+ patient samples, we determined that enhanced collagen levels in tumor-adjacent mammary tissue positively correlated with mast cell abundance and HR+ breast cancer recurrence. Together, these data demonstrate that mast cells programmed by commensal dysbiosis activate mammary tissue fibroblasts and orchestrate early dissemination of HR+ breast tumors.
Data from Subcapsular Sinus Macrophages Promote Melanoma Metastasis to the Sentinel Lymph Nodes via an IL1α–STAT3 Axis
Authors: Virgilio Tommaso , Bordini Joy , Cascione Luciano , Sartori Giulio , Latino Irene , Molina Romero Daniel , Leoni Cristina , Akhmedov Murodzhon , Rinaldi Andrea , Arribas Alberto J. , Morone Diego , Seyed Jafari S. Morteza , Bersudsky Marina , Ottolenghi Aner , Kwee Ivo , Chiaravalli Anna Maria , Sessa Fausto , Hunger Robert E. , Bruno Antonino , Mortara Lorenzo , Voronov Elena , Monticelli Silvia , Apte Ron N. , Bertoni Francesco , Gonzalez Santiago F.
Published: April 2023

Abstract
During melanoma metastasis, tumor cells originating in the skin migrate via lymphatic vessels to the sentinel lymph node (sLN). This process facilitates tumor cell spread across the body. Here, we characterized the innate inflammatory response to melanoma in the metastatic microenvironment of the sLN. We found that macrophages located in the subcapsular sinus (SS) produced protumoral IL1α after recognition of tumoral antigens. Moreover, we confirmed that the elimination of LN macrophages or the administration of an IL1α-specific blocking antibody reduced metastatic spread. To understand the mechanism of action of IL1α in the context of the sLN microenvironment, we applied single-cell RNA sequencing to microdissected metastases obtained from animals treated with the IL1α-specific blocking antibody. Among the different pathways affected, we identified STAT3 as one of the main targets of IL1α signaling in metastatic tumor cells. Moreover, we found that the antitumoral effect of the anti-IL1α was not mediated by lymphocytes because Il1r1 knockout mice did not show significant differences in metastasis growth. Finally, we found a synergistic antimetastatic effect of the combination of IL1α blockade and STAT3 inhibition with stattic, highlighting a new immunotherapy approach to preventing melanoma metastasis.
Data from A Novel Engineered AAV-Based Neoantigen Vaccine in Combination with Radiotherapy Eradicates Tumors
Authors: Huang Kevin Chih-Yang , Lai Chia-Ying , Hung Wei-Ze , Chang Hsin-Yu , Lin Pei-Chun , Chiang Shu-Fen , Ke Tao-Wei , Liang Ji-An , Shiau An-Cheng , Yang Pei-Chen , Chen William Tzu-Liang , Chao K.S. Clifford
Published: April 2023

Abstract
The potency of tumor-specific antigen (TSA) vaccines, such as neoantigen (neoAg)-based cancer vaccines, can be compromised by host immune checkpoint inhibitory mechanisms, such as programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1), that attenuate neoAg presentation on dendritic cells (DC) and hinder T cell–mediated cytotoxicity. To overcome PD-1/PD-L1 inhibition in DCs, we developed a novel adeno-associated virus (meAAV) neoAg vaccine, modified with TLR9 inhibitory fragments, PD-1 trap, and PD-L1 miRNA, which extend the persistence of meAAV and activate neoAg-specific T-cell responses in immune-competent colorectal and breast cancer murine models. Moreover, we found that in combination with radiotherapy, the meAAV-based neoAg cancer vaccine not only elicited higher antigen presentation ability, but also maintained neoAg-specific cytotoxic T lymphocyte (CTL) responses. These functional PD-1 traps and PD-L1 miRNAs overcome host PD-1/PD-L1 inhibitory mechanisms and boost the therapeutic efficacy of radiotherapy. More importantly, combined radiotherapy and meAAV neoAg cancer vaccines significantly enhanced neoAg-specific CTL responses, increased CTL infiltration in tumor microenvironment, and decreased tumor-associated immunosuppression. This process led to the complete elimination of colorectal cancer and delayed tumor growth of breast cancer in tumor-bearing mice. Taken together, our results demonstrated a novel strategy that combines neoAg cancer vaccine and radiotherapy to increase the therapeutic efficacy against colorectal and breast cancers.
Data from Rational Protein Design Yields a CD20 CAR with Superior Antitumor Efficacy Compared with CD19 CAR
Authors: Chen Ximin , Chen Laurence C. , Khericha Mobina , Meng Xiangzhi , Salvestrini Emma , Shafer Amanda , Iyer Neha , Alag Anya S. , Ding Yunfeng , Nicolaou Demetri M. , Chen Yvonne Y.
Published: April 2023

Abstract
Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens.

See related Spotlight by Scheller and Hudecek, p. 142
Data from P. gingivalis Infection Upregulates PD-L1 Expression on Dendritic Cells, Suppresses CD8+ T-cell Responses, and Aggravates Oral Cancer
Authors: Ren Junling , Han Xiao , Lohner Hannah , Hoyle Rosalie G. , Li Jiong , Liang Shuang , Wang Huizhi
Published: April 2023

Abstract
Accumulating evidence shows that PD-L1 expression on dendritic cells (DC) is critical for cancer immunotherapy and that Porphyromonas gingivalis (Pg) colonization aggravates the progression of upper gastrointestinal cancers. However, the effects of Pg infection on PD-L1 expression on DCs and related immune consequences in the infection milieu of oral cancer remain unexplored. Here, we found that Pg infection robustly enhanced PD-L1 expression on DCs in a gingipain-dependent manner in cultured cell and systemic infection assays. Pg infection suppressed antigen-specific CD8+ T cells through upregulation of PD-L1 expression on ovalbumin (OVA)-pulsed DCs. This suppression was manifested by decreased IFNγ, perforin, granzyme B, and CD107a. Further analysis showed that Pg drastically reduced CD8+ T cells’ ability to lyse OVA-pulsed target cells. Additionally, Pg infection increased the phosphorylation of Akt and STAT3, leading to a significant increase in PD-L1 expression. This was substantiated by using siRNA, overexpression plasmids, and pharmacologic inhibitors. Consistent with the in vitro observations, in a syngeneic mouse oral cancer model, Pg infection significantly enhanced PD-L1 expression on DCs from intratumoral tissues and cervical lymph nodes and exacerbated oral cancer progression, whereas a Pg lysine-specific, gingipain-defective mutant failed to do so. These influences of Pg were largely diminished when tumor cells were pretreated with antibiotics or a STAT3 inhibitor. Therefore, we demonstrated that Pg infection upregulates PD-L1 expression on DCs through Akt-STAT3 signaling, suppresses CD8+ T-cell cytotoxicity, and aggravates oral cancer growth, suggesting targeting Pg, and/or its mediated signaling, could be a therapeutic strategy to improve the efficacy of checkpoint blockade immunotherapy.
Data from PHD2 Constrains Antitumor CD8+ T-cell Activity
Authors: Bisilliat Donnet Charlotte , Acolty Valérie , Azouz Abdulkader , Taquin Anaëlle , Henin Coralie , Trusso Cafarello Sarah , Denanglaire Sébastien , Mazzone Massimiliano , Oldenhove Guillaume , Leo Oberdan , Goriely Stanislas , Moser Muriel
Published: April 2023

Abstract
The prolyl hydroxylase domain/hypoxia-inducible factor (PHD/HIF) pathway has been implicated in a wide range of immune and inflammatory processes, including in the oxygen-deprived tumor microenvironment. To examine the effect of HIF stabilization in antitumor immunity, we deleted Phd2 selectively in T lymphocytes using the cre/lox system. We show that the deletion of PHD2 in lymphocytes resulted in enhanced regression of EG7-OVA tumors, in a HIF-1α–dependent manner. The enhanced control of neoplastic growth correlated with increased polyfunctionality of CD8+ tumor-infiltrating lymphocytes, as indicated by enhanced expression of IFNγ, TNFα, and granzyme B. Phenotypic and transcriptomic analyses pointed to a key role of glycolysis in sustaining CTL activity in the tumor bed and identified the PHD2/HIF-1 pathway as a potential target for cancer immunotherapy.
Supplementary Appendix from Dissecting Clonal Hematopoiesis in Tissues of Patients with Classic Hodgkin Lymphoma
Authors: Venanzi Alessandra , Marra Andrea , Schiavoni Gianluca , Milner Sara G. , Limongello Roberto , Santi Alessia , Pettirossi Valentina , Ultimo Simona , Tasselli Luisa , Pucciarini Alessandra , Falini Lorenza , Sciabolacci Sofia , Martelli Maria Paola , Sportoletti Paolo , Ascani Stefano , Falini Brunangelo , Tiacci Enrico
Published: April 2023

Supplementary Appendix includes Supplementary Data and Discussion.
Supplementary Table S1 from Bortezomib Induces Anti–Multiple Myeloma Immune Response Mediated by cGAS/STING Pathway Activation
Authors: Gulla Annamaria , Morelli Eugenio , Samur Mehmet K. , Botta Cirino , Hideshima Teru , Bianchi Giada , Fulciniti Mariateresa , Malvestiti Stefano , Prabhala Rao H. , Talluri Srikanth , Wen Kenneth , Tai Yu-Tzu , Richardson Paul G. , Chauhan Dharminder , Sewastianik Tomasz , Carrasco Ruben D. , Munshi Nikhil C. , Anderson Kenneth C.
Published: April 2023

Supp. table S1 shows immune genes regulated after treatment with bortezomib in vivo.
Supplementary Figure from BCOR and BCORL1 Mutations Drive Epigenetic Reprogramming and Oncogenic Signaling by Unlinking PRC1.1 from Target Genes
Authors: Schaefer Eva J. , Wang Helen C. , Karp Hannah Q. , Meyer Clifford A. , Cejas Paloma , Gearhart Micah D. , Adelman Emmalee R. , Fares Iman , Apffel Annie , Lim Klothilda , Xie Yingtian , Gibson Christopher J. , Schenone Monica , Murdock H. Moses , Wang Eunice S. , Gondek Lukasz P. , Carroll Martin P. , Vedula Rahul S. , Winer Eric S. , Garcia Jacqueline S. , Stone Richard M. , Luskin Marlise R. , Carr Steven A. , Long Henry W. , Bardwell Vivian J. , Figueroa Maria E. , Lindsley R. Coleman
Published: April 2023

Supplementary Figure from BCOR and BCORL1 Mutations Drive Epigenetic Reprogramming and Oncogenic Signaling by Unlinking PRC1.1 from Target Genes
Supplementary Figure from Txnip Enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells via p21
Authors: Zhang Christine R. , Ostrander Elizabeth L. , Kukhar Ostap , Mallaney Cates , Sun Jiameng , Haussler Emily , Celik Hamza , Koh Won Kyun , King Katherine Y. , Gontarz Paul , Challen Grant A.
Published: April 2023

Supplementary Figure from Txnip Enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells via p21
Supplementary Data from Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression
Authors: Menssen Andrew J. , Khanna Ajay , Miller Christopher A. , Nonavinkere Srivatsan Sridhar , Chang Gue Su , Shao Jin , Robinson Joshua , O'Laughlin Michele , Fronick Catrina C. , Fulton Robert S. , Brendel Kimberly , Heath Sharon E. , Saba Raya , Welch John S. , Spencer David H. , Payton Jacqueline E. , Westervelt Peter , DiPersio John F. , Link Daniel C. , Schuelke Matthew J. , Jacoby Meagan A. , Duncavage Eric J. , Ley Timothy J. , Walter Matthew J.
Published: April 2023

Supplementary Data from Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression
Supplementary Data from Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms
Authors: Ibarra Juan , Elbanna Yassmin A. , Kurylowicz Katarzyna , Ciboddo Michele , Greenbaum Harrison S. , Arellano Nicole S. , Rodriguez Deborah , Evers Maria , Bock-Hughes Althea , Liu Chenyu , Smith Quinn , Lutze Julian , Baumeister Julian , Kalmer Milena , Olschok Kathrin , Nicholson Benjamin , Silva Diane , Maxwell Luke , Dowgielewicz Jonathan , Rumi Elisa , Pietra Daniela , Casetti Ilaria Carola , Catricala Silvia , Koschmieder Steffen , Gurbuxani Sandeep , Schneider Rebekka K. , Oakes Scott A. , Elf Shannon E.
Published: April 2023

Supplementary Data from Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms
Supplementary Table from RETRACTED: In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target
Authors: Murakami Shohei , Barroca Vilma , Perié Leïla , Bravard Anne , Bernardino-Sgherri Jacqueline , Tisserand Amandine , Devanand Caroline , Edmond Valérie , Magniez Aurélie , Tenreira Bento Sabrina , Torres Claire , Pasquier Florence , Plo Isabelle , Vainchenker William , Villeval Jean-Luc , Roméo Paul-Henri , Lewandowski Daniel
Published: April 2023

Supplementary Table from RETRACTED: In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target
Supplementary Data from Targeting the EIF2AK1 Signaling Pathway Rescues Red Blood Cell Production in SF3B1-Mutant Myelodysplastic Syndromes With Ringed Sideroblasts
Authors: Adema Vera , Ma Feiyang , Kanagal-Shamanna Rashmi , Thongon Natthakan , Montalban-Bravo Guillermo , Yang Hui , Peslak Scott A. , Wang Feng , Acha Pamela , Sole Francesc , Lockyer Pamela , Cassari Margherita , Maciejewski Jaroslaw P. , Visconte Valeria , Gañán-Gómez Irene , Song Yuanbin , Bueso-Ramos Carlos , Pellegrini Matteo , Tan Tuyet M. , Bejar Rafael , Carew Jennifer S. , Halene Stephanie , Santini Valeria , Al-Atrash Gheath , Clise-Dwyer Karen , Garcia-Manero Guillermo , Blobel Gerd A. , Colla Simona
Published: April 2023

Supplementary Data from Targeting the EIF2AK1 Signaling Pathway Rescues Red Blood Cell Production in SF3B1-Mutant Myelodysplastic Syndromes With Ringed Sideroblasts
Supplementary Data from Deep Morphology Learning Enhances Ex Vivo Drug Profiling-Based Precision Medicine
Authors: Heinemann Tim , Kornauth Christoph , Severin Yannik , Vladimer Gregory I. , Pemovska Tea , Hadzijusufovic Emir , Agis Hermine , Krauth Maria-Theresa , Sperr Wolfgang R. , Valent Peter , Jäger Ulrich , Simonitsch-Klupp Ingrid , Superti-Furga Giulio , Staber Philipp B. , Snijder Berend
Published: April 2023

Supplementary Data from Deep Morphology Learning Enhances Ex Vivo Drug Profiling-Based Precision Medicine
Supplementary Table S1 from A Therapeutically Targetable NOTCH1–SIRT1–KAT7 Axis in T-cell Leukemia
Authors: Lancho Olga , Singh Amartya , da Silva-Diz Victoria , Aleksandrova Maya , Khatun Jesminara , Tottone Luca , Nunes Patricia Renck , Luo Shirley , Zhao Caifeng , Zheng Haiyan , Chiles Eric , Zuo Zhenyu , Rocha Pedro P. , Su Xiaoyang , Khiabanian Hossein , Herranz Daniel
Published: April 2023

Acetyl-proteomic results
Supplementary Table S1 from Impaired SARS-CoV-2 Variant Neutralization and CD8+ T-cell Responses Following 3 Doses of mRNA Vaccines in Myeloma: Correlation with Breakthrough Infections
Authors: Azeem Maryam I. , Nooka Ajay K. , Shanmugasundaram Uma , Cheedarla Narayanaiah , Potdar Sayalee , Manalo Renee Julia , Moreno Alberto , Switchenko Jeffrey M. , Cheedarla Suneethamma , Doxie Deon Bryant , Radzievski Roman , Ellis Madison Leigh , Manning Kelly E. , Wali Bushra , Valanparambil Rajesh M. , Maples Kathryn T. , Baymon Essence , Kaufman Jonathan L. , Hofmeister Craig C. , Joseph Nisha S. , Lonial Sagar , Roback John D. , Sette Alessandro , Ahmed Rafi , Suthar Mehul S. , Neish Andrew S. , Dhodapkar Madhav V. , Dhodapkar Kavita M.
Published: April 2023

Suppl Table 1. Correlative analysis of breakthrough SARS CoV-2 infection following booster vaccination.
Data from T-cell Acute Lymphoblastic Leukemia: A Roadmap to Targeted Therapies
Authors: Cordo' Valentina , van der Zwet Jordy C.G. , Canté-Barrett Kirsten , Pieters Rob , Meijerink Jules P.P.
Published: April 2023

Abstract
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by aberrant proliferation of immature thymocytes. Despite an overall survival of 80% in the pediatric setting, 20% of patients with T-ALL ultimately die from relapsed or refractory disease. Therefore, there is an urgent need for novel therapies. Molecular genetic analyses and sequencing studies have led to the identification of recurrent T-ALL genetic drivers. This review summarizes the main genetic drivers and targetable lesions of T-ALL and gives a comprehensive overview of the novel treatments for patients with T-ALL that are currently under clinical investigation or that are emerging from preclinical research.
Significance:
T-ALL is driven by oncogenic transcription factors that act along with secondary acquired mutations. These lesions, together with active signaling pathways, may be targeted by therapeutic agents. Bridging research and clinical practice can accelerate the testing of novel treatments in clinical trials, offering an opportunity for patients with poor outcome.
Data from Aging of Preleukemic Thymocytes Drives CpG Island Hypermethylation in T-cell Acute Lymphoblastic Leukemia
Authors: Roels Juliette , Thénoz Morgan , Szarzyńska Bronisława , Landfors Mattias , De Coninck Stien , Demoen Lisa , Provez Lien , Kuchmiy Anna , Strubbe Steven , Reunes Lindy , Pieters Tim , Matthijssens Filip , Van Loocke Wouter , Erarslan-Uysal Büşra , Richter-Pechańska Paulina , Declerck Ken , Lammens Tim , De Moerloose Barbara , Deforce Dieter , Van Nieuwerburgh Filip , Cheung Laurence C. , Kotecha Rishi S. , Mansour Marc R. , Ghesquière Bart , Van Camp Guy , Berghe Wim Vanden , Kowalczyk Jerzy R. , Szczepański Tomasz , Davé Utpal P. , Kulozik Andreas E. , Goossens Steven , Curtis David J. , Taghon Tom , Dawidowska Małgorzata , Degerman Sofie , Van Vlierberghe Pieter
Published: April 2023

Abstract
Cancer cells display DNA hypermethylation at specific CpG islands in comparison with their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. Here, we show that CpG island methylation in human T-cell acute lymphoblastic leukemia (T-ALL) mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T cells and that display a reciprocal association with H3K27me3 binding. In addition, we reveal that this aberrant methylation profile reflects the epigenetic history of T-ALL and is established already in preleukemic, self-renewing thymocytes that precede T-ALL development. Finally, we unexpectedly uncover that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent decitabine. Altogether, we provide conceptual evidence for the involvement of a preleukemic phase characterized by self-renewing thymocytes in the pathogenesis of human T-ALL.
Significance:
We developed a DNA methylation signature that reveals the epigenetic history of thymocytes during T-cell transformation. This human signature was recapitulated by murine self-renewing preleukemic thymocytes that build an age-related CpG island hypermethylation phenotype, providing conceptual evidence for the involvement of a preleukemic thymic phase in human T-cell leukemia.

This article is highlighted in the In This Issue feature, p. 215
Data from COVID-19 Infections and Clinical Outcomes in Patients with Multiple Myeloma in New York City: A Cohort Study from Five Academic Centers
Authors: Hultcrantz Malin , Richter Joshua , Rosenbaum Cara A. , Patel Dhwani , Smith Eric L. , Korde Neha , Lu Sydney X. , Mailankody Sham , Shah Urvi A. , Lesokhin Alexander M. , Hassoun Hani , Tan Carlyn , Maura Francesco , Derkach Andriy , Diamond Benjamin , Rossi Adriana , Pearse Roger N. , Madduri Deepu , Chari Ajai , Kaminetzky David , Braunstein Marc J. , Gordillo Christian , Reshef Ran , Taur Ying , Davies Faith E. , Jagannath Sundar , Niesvizky Ruben , Lentzsch Suzanne , Morgan Gareth J. , Landgren Ola
Published: April 2023

Abstract
Patients with multiple myeloma have a compromised immune system, due to both the disease and antimyeloma therapies, and may therefore be particularly susceptible to COVID-19. Here, we report outcomes and risk factors for serious disease in patients with multiple myeloma treated at five large academic centers in New York City in the spring of 2020, during which it was a global epicenter of the SARS-CoV-2 pandemic. Of 100 patients with multiple myeloma (male 58%; median age 68) diagnosed with COVID-19, 75 were admitted; of these, 13 patients (17%) were placed on invasive mechanical ventilation, and 22 patients (29%) expired. Of the 25 nonadmitted patients, 4 were asymptomatic. There was a higher risk of adverse outcome (intensive care unit admission, mechanical ventilation, or death) in Hispanics/Latinos (n = 21), OR = 4.7 (95% confidence interval, 1.3–16.7), and African American Blacks (n = 33), OR = 3.5 (1.1–11.5), as compared with White patients (n = 36). Patients who met the adverse combined endpoint had overall higher levels of inflammatory markers and cytokine activation. None of the other studied risk factors were significantly associated (P > 0.05) with adverse outcome: hypertension (n = 56), OR = 2.2 (0.9–5.4); diabetes (n = 18), OR = 0.9 (0.3–2.9); age >65 years (n = 63), OR = 1.8 (0.7–4.6); high-dose melphalan with autologous stem cell transplant <12 months (n = 7), OR = 0.9 (0.2–5.4); and immunoglobulin G <650 mg/dL (n = 42), OR = 0.9 (0.3–2.2). In this largest cohort to date of patients with multiple myeloma and COVID-19, we found the case fatality rate to be 29% among hospitalized patients and that race/ethnicity was the most significant risk factor for adverse outcome.
Significance:
Patients with multiple myeloma are immunocompromised, raising the question whether they are at higher risk of severe COVID-19 disease. In this large case series on COVID-19 in patients with multiple myeloma, we report 29% mortality rates among hospitalized patients and identify race/ethnicity as the most significant risk factor for severe outcome.

See related video: https://vimeo.com/486246183/559a80cfae

See related commentary by Munshi and Anderson, p. 218.

This article is highlighted in the In This Issue feature, p. 215
Data from Monocytic Differentiation and AHR Signaling as Primary Nodes of BET Inhibitor Response in Acute Myeloid Leukemia
Authors: Romine Kyle A. , Nechiporuk Tamilla , Bottomly Daniel , Jeng Sophia , McWeeney Shannon K. , Kaempf Andy , Corces M. Ryan , Majeti Ravindra , Tyner Jeffrey W.
Published: April 2023

Abstract
To understand mechanisms of response to BET inhibitors (BETi), we mined the Beat AML functional genomic data set and performed genome-wide CRISPR screens on BETi-sensitive and BETi-resistant acute myeloid leukemia (AML) cells. Both strategies revealed regulators of monocytic differentiation—SPI1, JUNB, FOS, and aryl-hydrocarbon receptor signaling (AHR/ARNT)—as determinants of BETi response. AHR activation synergized with BETi, whereas inhibition antagonized BETi-mediated cytotoxicity. Consistent with BETi sensitivity dependence on monocytic differentiation, ex vivo sensitivity to BETi in primary AML patient samples correlated with higher expression of the monocytic markers CSF1R, LILRs, and VCAN. In addition, HL-60 cell line differentiation enhanced its sensitivity to BETi. Further, screens to rescue BETi sensitivity identified BCL2 and CDK6 as druggable vulnerabilities. Finally, monocytic AML patient samples refractory to venetoclax ex vivo were significantly more sensitive to combined BETi + venetoclax. Together, our work highlights mechanisms that could predict BETi response and identifies combination strategies to overcome resistance.
Significance:
Drug resistance remains a challenge for AML, and new therapies, such as BETi, will require combination approaches to boost single-agent responses. We conducted genome-wide CRISPR screens and functional genomics on AML patient samples to identify leukemic differentiation state and AHR signaling as primary mediators of BETi response.

This article is highlighted in the In This Issue feature, p. 403
Data from SIRT5 Is a Druggable Metabolic Vulnerability in Acute Myeloid Leukemia
Authors: Yan Dongqing , Franzini Anca , Pomicter Anthony D. , Halverson Brayden J. , Antelope Orlando , Mason Clinton C. , Ahmann Jonathan M. , Senina Anna V. , Vellore Nadeem A. , Jones Courtney L. , Zabriskie Matthew S. , Than Hein , Xiao Michael J. , van Scoyk Alexandria , Patel Ami B. , Clair Phillip M. , Heaton William L. , Owen Shawn C. , Andersen Joshua L. , Egbert Christina M. , Reisz Julie A. , D'Alessandro Angelo , Cox James E. , Gantz Kevin C. , Redwine Hannah M. , Iyer Siddharth M. , Khorashad Jamshid S. , Rajabi Nima , Olsen Christian A. , O'Hare Thomas , Deininger Michael W.
Published: April 2023

Abstract
We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.
Significance:
Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.

See related commentary by Li and Melnick, p. 198.
Data from Machine Learning of Bone Marrow Histopathology Identifies Genetic and Clinical Determinants in Patients with MDS
Authors: Brück Oscar E. , Lallukka-Brück Susanna E. , Hohtari Helena R. , Ianevski Aleksandr , Ebeling Freja T. , Kovanen Panu E. , Kytölä Soili I. , Aittokallio Tero A. , Ramos Pedro M. , Porkka Kimmo V. , Mustjoki Satu M.
Published: April 2023

Abstract
In myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN), bone marrow (BM) histopathology is assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, other morphologic findings may elude the human eye. We used convolutional neural networks to extract morphologic features from 236 MDS, 87 MDS/MPN, and 11 control BM biopsies. These features predicted genetic and cytogenetic aberrations, prognosis, age, and gender in multivariate regression models. Highest prediction accuracy was found for TET2 [area under the receiver operating curve (AUROC) = 0.94] and spliceosome mutations (0.89) and chromosome 7 monosomy (0.89). Mutation prediction probability correlated with variant allele frequency and number of affected genes per pathway, demonstrating the algorithms' ability to identify relevant morphologic patterns. By converting regression models to texture and cellular composition, we reproduced the classical del(5q) MDS morphology consisting of hypolobulated megakaryocytes. In summary, this study highlights the potential of linking deep BM histopathology with genetics and clinical variables.
Significance:
Histopathology is elementary in the diagnostics of patients with MDS, but its high-dimensional data are underused. By elucidating the association of morphologic features with clinical variables and molecular genetics, this study highlights the vast potential of convolutional neural networks in understanding MDS pathology and how genetics is reflected in BM morphology.

See related commentary by Elemento, p. 195.