bluuebunny/crossref_metadata_2025 huggingface dataset:
Supplementary Figure S1 from Effective Antitumor Immunity Can Be Triggered by Targeting VISTA in Combination with a TLR3-Specific Adjuvant
Authors: Wang Bo , Ou Ziwei , Zhong Wenlong , Huang Lin , Liao Wenjian , Sheng Yiyu , Guo Zhixing , Chen Junyu , Yang Wenjuan , Chen Ke , Huang Xiaodong , Yang Tenghao , Lin Tianxin , Huang Jian
Published: December 2023

Schematic diagram of sODN-dsRNA
Supplementary Tables from Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1− Dysfunctional State
Authors: Roetman Jessica J. , Erwin Megan M. , Rudloff Michael W. , Favret Natalie R. , Detrés Román Carlos R. , Apostolova Minna K.I. , Murray Kristen A. , Lee Ting-Fang , Lee Youngmin A. , Philip Mary
Published: December 2023

Supplementary Tables 1 and 2
Figure S7 from Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1− Dysfunctional State
Authors: Roetman Jessica J. , Erwin Megan M. , Rudloff Michael W. , Favret Natalie R. , Detrés Román Carlos R. , Apostolova Minna K.I. , Murray Kristen A. , Lee Ting-Fang , Lee Youngmin A. , Philip Mary
Published: December 2023

SST and TST have distinct underlying transcriptional programs.
Figure S5 from Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1− Dysfunctional State
Authors: Roetman Jessica J. , Erwin Megan M. , Rudloff Michael W. , Favret Natalie R. , Detrés Román Carlos R. , Apostolova Minna K.I. , Murray Kristen A. , Lee Ting-Fang , Lee Youngmin A. , Philip Mary
Published: December 2023

Persistent SST in spleens of tumor-bearing hosts have memory-like immunophenotype, and a subset of endogenous PD1+ CD8 T cells share the SST-like TCF1+TOX- profile.
Figure S7 from A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer
Authors: Pinioti Sotiria , Sharma Himal , Flerin Nina C. , Yu Qian , Tzoumpa Amalia , Trusso Cafarello Sarah , De Bousser Elien , Callewaert Nico , Oldenhove Guillaume , Schlenner Susan , Thienpont Bernard , Garg Abhishek D. , Di Matteo Mario , Mazzone Massimiliano
Published: December 2023

Additional evidence of FucoLOW Tregs predicting better outcome in cancer patients. Related to figure 7. (A-E) Pearson’s correlation plots for FucoLOW Tregs signature vs. bulk-transcriptome signatures of endothelial cells, fibroblasts, macrophages, melanocytic (cancer) cells or Tregs in TCGA dataset from melanoma cancer patients shown in Fig 7F-H. (F) Gene Set Enrichment score of Golgi-fucosylation signature in Tregs derived from PBMCs, tumor-derived Tregs and Tregs derived from Normal Breast Parenchyma (NBP) in published RNA-seq data derived from breast cancer patients. (G) Graphical abstract of the study: An in vitro metabolic CRISPR/cas9 screen was performed in in vitro-induced Tregs revealing positive and negative regulators of Foxp3 expression. Slc35c1 was revealed as the top positive regulator of Foxp3 and its role in Tregs biology was studied in vitro and in vivo. Slc35c1-deficient Tregs exhibit impaired fucosylation, reduced Foxp3 and PD-1 expression and increased IFNγ production accompanied by reduced suppressive function. In vivo suppressive assay with sgSlc35c1 Tregs transfer in Rag2-/- tumor-bearing mice led to reduced tumor growth, high infiltration of CD4+ and CD8+ T cells and reduced number of tumor-associated macrophages. In colorectal cancer patients, FucoLOW Tregs are highly immunogenic compared to FucoHIGH Tregs. In melanoma cancer patients FucoLOW signature mainly derived by Tregs, is correlated with high overall survival and better response to immunotherapy.
Figure S4 from Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1− Dysfunctional State
Authors: Roetman Jessica J. , Erwin Megan M. , Rudloff Michael W. , Favret Natalie R. , Detrés Román Carlos R. , Apostolova Minna K.I. , Murray Kristen A. , Lee Ting-Fang , Lee Youngmin A. , Philip Mary
Published: December 2023

SST and TST undergo apoptosis at similar rates.
Figure S5 from A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer
Authors: Pinioti Sotiria , Sharma Himal , Flerin Nina C. , Yu Qian , Tzoumpa Amalia , Trusso Cafarello Sarah , De Bousser Elien , Callewaert Nico , Oldenhove Guillaume , Schlenner Susan , Thienpont Bernard , Garg Abhishek D. , Di Matteo Mario , Mazzone Massimiliano
Published: December 2023

The effect of 2FF pretreatment on Tregs number and phenotype is not measurable at tumor end-stage. Related to Figure 5. (A) DMSO and 2FF-treated Tregs were injected in MC38 tumor-bearing mice. Percentage of Tregs out of alive detected in the tumor at the end-stage. (B) MFI Foxp3 in Tregs detected in the tumor at the end-stage. (C) Percentage of Foxp3+ (out of Tregs) detected in the tumor at the end-stage. Statistical analysis was performed using unpaired two-tailed t-test and are represented as mean ± SEM (ns, p > 0.05, * p  0.05, ** p  0.01 and *** p  0.001).
Figure S4 from A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer
Authors: Pinioti Sotiria , Sharma Himal , Flerin Nina C. , Yu Qian , Tzoumpa Amalia , Trusso Cafarello Sarah , De Bousser Elien , Callewaert Nico , Oldenhove Guillaume , Schlenner Susan , Thienpont Bernard , Garg Abhishek D. , Di Matteo Mario , Mazzone Massimiliano
Published: December 2023

Unaltered number of antigen-specific CD8+ T cells in tumors treated with sgNT or sgSlc35c1 Tregs. Related to Figure 4. (A) Number of tetramer+ CD8+ T cells per gram of tumor. Results are representative of three independent experiments, ns, p > 0.05, * p  0.05, ** p  0.01 and *** p  0.001.
Figure S3 from A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer
Authors: Pinioti Sotiria , Sharma Himal , Flerin Nina C. , Yu Qian , Tzoumpa Amalia , Trusso Cafarello Sarah , De Bousser Elien , Callewaert Nico , Oldenhove Guillaume , Schlenner Susan , Thienpont Bernard , Garg Abhishek D. , Di Matteo Mario , Mazzone Massimiliano
Published: December 2023

Additional data on the effect of Slc35c1 deficiency on Tregs. Related to Figure 3. (A) Gating strategy of alive, transduced (CD90.1+), Foxp3 and IFNγ Tregs. (B) Flow cytometry to assess the MFI of Foxp3 and IFNγ expression 5 days after transduction for sgSlc35c1 compared to sgNT Tregs. (C) Pdcd1 expression by PCR on sgNT and sgSlc35c1 iTregs. (D Representative histogram of the AAL staining between sgNT and sgSlc35c1 in vivo-derived Tregs. As negative control was used Tregs incubated with fucosidase. (E) Percentage of EdU+ cells out of transduced in vivo-derived Tregs. (F) Representative gating of the 50:50 mix iTregs sorted as alive, CD90.1+ (here reporter Foxp3) used for the migration assay at Fig. 3I. (G) Representative gating of the iTregs derived from different congenic mice CD45.1 and CD45.1/2 injected in mice in Fig. 3J. (H) Percentage of sgNT and sgSlc35c1 iTregs derived from different congenic mice CD45.1 and CD45.1/2 found in the blood of healthy Rag2-/- mice 7 days after ACT. (I) Representative gating of the iTregs derived from different congenic mice CD45.1 and CD45.1/2 in the spleen of mice sacrificed 7 days upon ATC (related to Fig. 3J). In vitro and in vivo data are the pooled data of at least three independent experiments. Statistical analysis was performed using paired or unpaired two-tailed t-test and are represented as mean ± SEM (ns, p > 0.05, * p  0.05, ** p  0.01 and *** p  0.001).
Supplementary Figure Legends from Antigen Priming Induces Functional Reprogramming in iNKT Cells via Metabolic and Epigenetic Regulation: An Insight into iNKT Cell-Based Antitumor Immunotherapy
Authors: Zhang Huimin , Chen Sanwei , Zhang Yuwei , Tian Chenxi , Pan Jun , Wang Yu , Bai Shiyu , Wu Qielan , Su Miya , Xie Di , Fu Sicheng , Li Shuhang , Zhang Jing , Chen Yusheng , Zhu Shasha , Qian Yeben , Bai Li
Published: December 2023

Supplementary Figure Legends
Supplementary Figure 4 from Antigen Priming Induces Functional Reprogramming in iNKT Cells via Metabolic and Epigenetic Regulation: An Insight into iNKT Cell-Based Antitumor Immunotherapy
Authors: Zhang Huimin , Chen Sanwei , Zhang Yuwei , Tian Chenxi , Pan Jun , Wang Yu , Bai Shiyu , Wu Qielan , Su Miya , Xie Di , Fu Sicheng , Li Shuhang , Zhang Jing , Chen Yusheng , Zhu Shasha , Qian Yeben , Bai Li
Published: December 2023

Gating strategy for identifying different lymphocytes in tumors.
Data file CRISPR_cas9 screen from A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer
Authors: Pinioti Sotiria , Sharma Himal , Flerin Nina C. , Yu Qian , Tzoumpa Amalia , Trusso Cafarello Sarah , De Bousser Elien , Callewaert Nico , Oldenhove Guillaume , Schlenner Susan , Thienpont Bernard , Garg Abhishek D. , Di Matteo Mario , Mazzone Massimiliano
Published: December 2023

Data file CRISPR_cas9 screen
Supplementary Figure 3 from Antigen Priming Induces Functional Reprogramming in iNKT Cells via Metabolic and Epigenetic Regulation: An Insight into iNKT Cell-Based Antitumor Immunotherapy
Authors: Zhang Huimin , Chen Sanwei , Zhang Yuwei , Tian Chenxi , Pan Jun , Wang Yu , Bai Shiyu , Wu Qielan , Su Miya , Xie Di , Fu Sicheng , Li Shuhang , Zhang Jing , Chen Yusheng , Zhu Shasha , Qian Yeben , Bai Li
Published: December 2023

A, Procedure for TCR signaling priming of human iNKT cells in vitro. B, The TMRM/MitoTracker ratio (n = 4) in control and TCR signaling-primed human iNKT cells, in the presence of ZLN005 or DMSO as control. C, Frequencies of IFN+ cells and GzmB+ cells in control and TCR signaling-primed human iNKT cells after restimulation with anti-CD3 plus anti-CD28 antibodies in the presence of ZLN005 or DMSO as indicated in (A) (n = 7). Data are represented as mean ± SD. Student’s t test was used to determine statistical significance (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant).
Supplementary Figure 1 from Antigen Priming Induces Functional Reprogramming in iNKT Cells via Metabolic and Epigenetic Regulation: An Insight into iNKT Cell-Based Antitumor Immunotherapy
Authors: Zhang Huimin , Chen Sanwei , Zhang Yuwei , Tian Chenxi , Pan Jun , Wang Yu , Bai Shiyu , Wu Qielan , Su Miya , Xie Di , Fu Sicheng , Li Shuhang , Zhang Jing , Chen Yusheng , Zhu Shasha , Qian Yeben , Bai Li
Published: December 2023

Antigen priming leads to dysfunction in distinct iNKT subsets.
Supplementary Table S3 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

LM22 signature matrix significantly differentially expressed genes.
Supplementary Table S2 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Clinical cohort patient characteristics.
Figure S8 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Validation of YUMMER2.1 and YUMMER2.1 B2M-KO. (A) In vitro and in vivo growth curves in twenty-four clones from the YUMM2.1 UV cell line compared to WT. In vitro growth curves represent the percent confluence of cells (y-axis) over time (x-axis) as measured by IncuCyte continuous live-cell imaging. Error bars reflect the standard error of the mean across six replicates of each subline. YUMM2.1 UV clone 2 had similar proliferation rates and in vitro and in vivo growth curves compared to the parental cell line and was used for in vivo studies (YUMMER2.1). (B) Validation of B2M-KO by CRISPR/Cas9 gene editing from YUMMER2.1 using Western blot.
Figure S7 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

(A) Identification of tumor immune cell infiltration by CyTOF. Percentage of Ly6C+, TAMs, CD11b+, Ly6G+, CD103 mDCs and DCs cells from CD45+ cells. Mean ± SEM, Unpaired t test, n = 3-4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Percentages of TAMs, M1, M2 and index M1/M2 from live cells in MC38 B2M-KO tumors treated with isotype monoclonal antibody, bempegaldesleukin, or bempegaldesleukin plus anti-CD4. Mean ± SEM, Unpaired t test, n = 4. *, P < 0.05; **, P < 0.01.
Figure S4 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Gating strategy in dendritic cell panel.
Figure S2 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

CIBERSORTx p-value distribution. Histograms of the p-value distributions of the samples (n=100) analyzed through CIBERSORTx for (A) fractions (median=0.065) and (B) absolute mode (median=0.075). Red line represents the median.
Figure S11 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Immune cell subsets in responsive human melanoma tumors. CIBERSORTx immune cell deconvolution analysis using bulk RNAseq data showing (A) fraction and (B) absolute scores of immune cell types in responsive (CR/PR) B2M unaltered (n=35) versus B2M LOH (n=6) tumors. Cell types are sorted by greatest difference in median score between groups; Wilcoxon test, ns= not significant, * p<0.05.
Figure S10 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Immune cell subsets in baseline human melanoma tumors. CIBERSORTx immune cell deconvolution analysis using bulk RNAseq data showing (A) absolute scores of immune cell types in B2M unaltered (n=75) versus B2M LOH (n=25) tumors, and (B) absolute scores of immune cell subtypes in B2M LOH progressive (n=19) versus responsive tumors (n=6). Cell types are sorted by greatest difference in median score between groups; Wilcoxon test, ns= not significant, * p<0.05.
Figure S1 from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Clinical dataset and analysis workflow.
Data from Antitumor Immune Responses in B2M-Deficient Cancers
Authors: Torrejon Davis Y. , Galvez Mildred , Abril-Rodriguez Gabriel , Campbell Katie M. , Medina Egmidio , Vega-Crespo Agustin , Kalbasi Anusha , Comin-Anduix Begoña , Ribas Antoni
Published: December 2023

Abstract
β2-microglobulin (B2M) is a critical component of the MHC class I molecule and is required to present tumor antigens to T cells. Its loss results in acquired resistance to immune checkpoint blockade (ICB) therapies. However, there have been well-documented cases of B2M-inactivated tumors responding to ICB, justifying investigation of how an antitumor immune response can be generated to tumors without surface MHC class I. We knocked out B2M in three murine models with varying baseline MHC class I expression and sensitivity to anti-programmed death receptor (PD-1) therapy and analyzed the immune responses. MC38 and YUMMER2.1 without B2M responded to anti–PD-1 alone or with an IL2 agonist, and this was mediated by CD4+ T cells and natural killer (NK) cells. The more aggressive B16 without B2M expression only partially responded to the IL2 agonist, and this was dependent on NK cells. When analyzing nearly 300 pretreatment biopsies from patients with melanoma receiving PD-1 blockade–based therapies, we found infrequent B2M mutations or homozygous loss but more frequent LOH or copy-number gains. B2M LOH was enriched in biopsies from patients without response to therapy, and these biopsies were more frequently infiltrated by activated NK cells. We conclude that in the absence of B2M, activation of CD4+ T cells and NK cells can mediate responses to murine models of PD-1 blockade therapy. In addition, in human melanoma, the intratumoral presence of activated NK cells upon partial B2M loss likely selects against tumor escape through low surface MHC class I expression.
Abstract A002: Analysis of tumor microenvironments in ovarian cancer patients receiving anti-BTN1A1 immunotherapy combined with standard-of-care therapy
Authors: Yoo Stephen S , Kim Youngseung , Lee Seung Hoon , Lee Soohyeon , Shin Sangjoon , LoRusso Patricia , Jung Hyunjin , Hong David
Published: December 2023

Background Immune checkpoints have emerged as key regulators of the immune responses against various tumor types. Butyrophilin 1A1 (BTN1A1) has been identified as a novel immune checkpoint protein that has the potential to be targeted for new immunotherapeutic treatment options. We have observed that BTN1A1 and PD-L1 expression are mutually exclusive in various human solid tumors. Recently, Nelmastobart (BTN1A1-targeting antibody, hSTC810) has successfully completed phase 1 clinical trials. To ensure the effectiveness of Nelmastobart, it is crucial to possess a profound comprehension of patients’ tumor microenvironment factors involved. Here, we report Opal multiplex immunofluorescence and Hyperion imaging mass cytometry approach that includes a deeper understanding of the spatial relationships in the tumor microenvironment.

Methods In a first-of-its-kind Phase 1 clinical trial, we are to characterize the safety profile and determine the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D). Nelmastobart is administered as monotherapy at increasing dose levels of 0.3, 1, 3, 6, 10, and 15 mg/kg on a Q2W schedule. To understand the heterogeneity of the interaction mechanisms between immune cells and cancer cells, Patients’ FFPE specimens were stained cancer and immune cells using Opal multiplex immunofluorescence and analyzed Hyperion imaging mass cytometer.

Results We enrolled 45 pts with various cancers in a phase I trial of anti-BTN1A1, a novel immunotherapy. We tested doses from 0.3 to 15 mg/kg and found no DLTs. We report results for ovarian cancer. Opal multiplex immunofluorescence analysis showed that anti-BTN1A1 therapy increased the infiltration and activation of CD8+ T cells and NK cells and decreased the level of regulatory T cells in the tumor microenvironment. We also found the opposite expression of BTN1A1 in PD-L1-expressing cancer cells in KI67-negative tumors from ovarian cancer patients.

Conclusions Nelmastobart treatment activates immune response pathways and enhances anti-tumor activity with anti-PD-L1 combination therapy in vitro, in vivo, and clinical settings. The results of the study suggest that BTN1A1 is a novel immune checkpoint that can be targeted to overcome resistance to conventional therapies in cancer patients.

Citation Format: Stephen S Yoo, Youngseung Kim, Seung Hoon Lee, Soohyeon Lee, Sangjoon Shin, Patricia LoRusso, Hyunjin Jung, David Hong. Analysis of tumor microenvironments in ovarian cancer patients receiving anti-BTN1A1 immunotherapy combined with standard-of-care therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A002.
Abstract A016: Reprogramming of immune-enhancing neutrophils by subclinical low-dose endotoxin for the treatment of cancer
Authors: Zhang Yao , Li Liwu
Published: December 2023

Despite the recent resurgence of the pioneering concept of “Coley’s toxin” in ushering in anti-cancer immune therapies highlight by check-point inhibitors and CAR-T approaches, fundamental mechanisms responsible for the immune-enhancing efficacy of innate low-dose “Coley’s toxin” remain poorly understood. This study aims to reveal the novel reprogramming of immune-enhancing neutrophils by super-low dose endotoxin conducive for innate immunity-based anti-cancer therapies. We perform scRNAseq analyses and reveal that neutrophils trained by super-low dose endotoxin (SL-LPS) adopt a unique immune-enhancing cluster characterized by CD177loCD11BloCD80hiCD40hi. Transfusion of SL-LPS trained neutrophils into recipient mice with AOM/DSS-inducing colorectal tumors exhibit potent efficacy in reducing tumor burden. SL-LPS trained neutrophils show relieved suppression of adaptive T cells as compared to un-trained neutrophils in vivo and in vitro. Mechanistically, SL-LPS enables the generation of immune-enhancing neutrophils through activating STAT5 and reducing innate suppressor IRAK-M. Herein, we present evidence revealing the key principle that underlying the immune-enhancing effects of super-low dose endotoxin, unrevealing the long-held mystery of low-dose “Coley’s toxin” in generically boosting host anti-tumor defense. Our mechanistic and translational observations not only reveal fundamental mechanisms for re-programming immune-enhancing neutrophils, but also provide a proof-of-principle in developing innate neutrophil-based anti-tumor therapeutics.

Citation Format: Yao Zhang, Liwu Li. Reprogramming of immune-enhancing neutrophils by subclinical low-dose endotoxin for the treatment of cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A016.
Abstract A021: Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer
Authors: Mehla Kamiya
Published: December 2023

Tumor cells present constant nutritional demand of macronutrients and micronutrients for their rapid growth needs. Often this nutrient limitation hampers the function of anti-tumor immune cells, which need basal energy levels to execute cytotoxic effects against malignant cells. Hence, we investigated nutrient limitation-induced metabolic alterations in NK cells that regulate the anti-tumor activity of NK cells. Using 2D cultures and PDAC organoid models, we demonstrate that pancreatic tumor cells exhaust vitamin B6 (VB6) in the co-culture milieu, causing reduced killing activity of NK cells. Additionally, we demonstrate that PDAC patients have significantly reduced plasma VB6 as compared to the healthy counterparts. This data aligns with the epidemiological studies showing that VB6 intake reduces the risk of pancreatic cancer incidence. Moreover, we noted a significant reduction in circulating VB6 in the pancreatic tumor-bearing mice as compared to healthy counterparts. Utilizing mass spectrometry-based metabolomics we noted that limitation of the VB6 prevents glycogen breakdown in NK cells. This was validated by follow up electron microscopy-based studies that demonstrated glycogen depots in NK cells that were very prominent upon inhibiting glycogenolysis. Metabolic tracing studies demonstrated that NK cells require VB6 for intracellular glycogen breakdown. Correspondingly, knocking down glycogen phosphorylase, a key enzyme involved in glycogen breakdown abrogated anti-tumoral NK cell function. Notably, supplementation of co-cultures in 2D and organoid models and in vivo orthotopic mouse models, with VB6 restored NK cell function against pancreatic tumor cells. In parallel, we observed dependence of tumor cell metabolism on VB6 for sustaining growth. Accordingly, we observed that tumor cells actively deplete VB6 in tumor microenvironments, as observed by comparing VB6 levels in circulation and in tumor interstitial fluids. Finally, we observed that VB6 supplementation in combination with inhibitors targeting VB6-driven metabolic pathway dependencies in tumor cells effectively enhances NK cell frequency and diminishes tumor burden in vivo. These studies demonstrate a novel role of glycogen breakdown as a critical energy source for activated NK cells and demonstrate, for the first time a key role of glycogenolysis in NK cell cytotoxic activities. Our results expand the understanding of the critical role of micronutrients (vitamin B6) in regulating cancer progression and anti-tumor immunity and open new avenues for developing NK cell-based immunotherapies for PDAC patients.

Citation Format: Kamiya Mehla. Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A021.
Abstract A045: Development of automated deep learning-based off-target distribution prediction system for CRISPR-Cas13 system
Authors: Chuai Guohui , Zhu Yanjing
Published: December 2023

CRISPR-Cas13 RNA editing system is a new member of the CRISPR-Cas gene editing system family, which can perform guided editing on RNA sequence. CRISPR-Cas gene editing system is currently one of the breakthrough technologies in life science, which is easy to operate, has a high editing efficiency and theoretically can perform guided editing on genome of any species. CRISPR-Cas9 system is the most commonly used CRISPR-Cas system, but it is mainly applied to DNA. Hence, fornon-destructive gene therapy and RNA virus detection, CRISPR-Cas13 system has its unique advantage. However, there are severe issues limiting its application, the most important one of which is off-target effect, namely the guide RNA points system to the wrong position causing non-targeted region edited, which leading to wrong expression spectrum of the cell. Therefore, based on automated deep learning and transfer learning techniques, this project aims to establishing an off-target distribution prediction system of CRISPR-Cas13 RNA editing system, in order to make high-specificity RNA editing possible.

Citation Format: Guohui Chuai, Yanjing Zhu. Development of automated deep learning-based off-target distribution prediction system for CRISPR-Cas13 system [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A045.
Abstract A053: Notch completes CD4 T cell help in CD8 T cells and ensures timely departure
Authors: Laurent Adrien Remi Gabriel , Karrich Julien , Babala Nikolina , Amsen Derk
Published: December 2023

Efficient activation, expansion and differentiation of CD8 T cells depend on help from CD4+ T cells. Such help is especially critical for CD8 T cell responses against tumor antigens. Priming of CD8 T cells in lymph nodes (LN) involves two steps that are geographically and temporally separate. Naïve CD8 T cells are first preactivated by a migratory dendritic cell (DC) carrying antigen from tissues. Preactivated CD8 T cells must then find and interact with lymph node-resident XCR1+ DC that have been licensed for optimal CD8 T cell activation via CD4 T cell-mediated activation of CD40. The efferent signals provided by licensed CD8+XCR1+ DC to install the CD4 T cell help program in CD8 T cells are incompletely known. Moreover, it is not clear whether and, if so, how premature lymph node exit by preactivated CD8 T cells before receipt of help is avoided. Here, we show that completion of CD4 help delivery is coupled to the ability to leave lymph nodes through activation of Notch receptors on CD8 T cells. Expression of Notch receptors is induced on naïve CD8 T cells during the first step of priming, while CD40 ligation elicits expression of Notch ligands on XCR1+ lymph node resident DCs. Using a DNA vaccination model that allows induction of CD8 T cell responses in the presence or absence of CD4 T cell help, we show that the Notch1/2 receptors are necessary in CD8+ T cells for CD4+ T cell help-dependent proliferation and acquisition of effector capability. Correspondingly, RNA sequencing revealed that more than half of the genetic CD4 T cell help program in CD8 T cells depends on Notch. Strikingly, activated Notch1/2 deficient and unhelped CD8 T cells remain largely stuck in lymph nodes owing to a lack of expression of the lymph node exit receptor S1PR5. Control of expression of S1PR5 by Notch therefore ensures that preactivated CD8 T cells remain in lymph nodes until completion of their activation process by receiving the CD4 T cell help signal.

Citation Format: Adrien Remi Gabriel Laurent, Julien Karrich, Nikolina Babala, Derk Amsen. Notch completes CD4 T cell help in CD8 T cells and ensures timely departure [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A053.
Abstract B012: Transcriptional activation of mouse LINE-1 promoter by transcription factor YY1
Authors: Saha Karani , Nielsen Grace , An Wenfeng
Published: December 2023

Long interspersed element type 1 (LINE-1) are highly abundant non-LTR retrotransposons in mammalian genomes. LINE-1 hypomethylation and overexpression has been considered as a hallmark of many human cancers. Moreover, active movement of LINE-1 results in insertional mutagenesis and may contribute to cancer initiation or progression. Transcription is the initial and critical regulatory step for active LINE-1 movement. Therefore, investigating LINE-1 transcriptional regulation is important for our understanding of LINE-1’s role in cancer. In human LINE-1, an intact YY1 transcription factor binding motif is required for accurate transcription initiation. Two subfamilies of mouse LINE-1, Tf_I and Gf_I, have predicted YY1 binding motifs in their promoter region but their role in mouse LINE-1 transcription has not been characterized. In this study, we aim to investigate the role of these putative YY1 motifs in Tf_I and Gf_I subfamilies. Promoter sequences carrying the wild-type or mutant YY1 motifs were cloned into a dual-luciferase reporter system. Reporter assays showed transcription inactivation upon mutation of the YY1 motif for the Tf_I subfamily. The electrophoretic mobility shift assay (EMSA) demonstrated specific interaction of the Tf_I promoter sequence with the YY1 protein. Knockdown of YY1 protein level in cells reduced Tf_I promoter activity by 2-fold compared to the wild-type control. These results indicate that YY1 is a transcriptional activator of mouse LINE-1 Tf_I subfamily. On the other hand, our data suggest that the predicted YY1 binding site in the promoter of Gf_I subfamily is not functional due to one nucleotide deviation from the consensus YY1 motif. Indeed, restoring it to the conserved YY1 motif sequence enhanced the promoter activity by 6-fold. In EMSA, YY1 protein did not interact with the putative YY1 motif of the Gf_I subfamily but did so when the motif was restored to consensus YY1 sequence. Findings from our study increase our understanding of the transcriptional regulation of different mouse LINE-1 subfamilies, highlight the differing roles of YY1 in regulating human and mouse L1 transcription, and have important implications in the role of LINE-1s during cancer development.

Citation Format: Karani Saha, Grace Nielsen, Wenfeng An. Transcriptional activation of mouse LINE-1 promoter by transcription factor YY1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B012.
Abstract B027: ATR inhibition upregulates PD-L1 and potentiates the antitumor immune response to chemoimmunotherapy in small-cell lung cancer
Authors: Sen Triparna
Published: December 2023

Introduction: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Recent clinical trials of immune checkpoint blockade (ICB) combined with chemotherapy delivered only very modest benefit. Here, we identified that ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, is highly enriched in SCLC and inhibition of ATR induced DNA damage and apoptosis in multiple SCLC models.

Methods: In this study, we performed genetic and pharmacological inhibition of ATR in a panel of human and murine SCLC models. Furthermore, we investigated the effect of ATR inhibition either alone or in combination with PD-L1 blockade either as a first-line or a second-line treatment in multiple immunocompetent mouse models of SCLC. The downstream effects of ATR inhibition was assessed by single-cell RNA sequencing, multicolor flow cytometry, western blot analysis, IHC, and genomic analysis.

Results: In multiple immunocompetent SCLC mouse models, ATR inhibition (ATRi) remarkably enhanced the anti-tumor effect of PD-L1 blockade. We next tested the ATR inhibition either alone or in combination with PD-L1 in the second-line regimen for SCLC. We observed that ATR inhibition in combination with PD-L1 blockade significantly reduced tumor volume and prolonged survival of aggressive mice models when compared to PD-L1 alone. Targeting ATR enhanced the expression of PD-L1, activated the cGAS/STING pathway, induced the expression of Type I and II interferon pathways, and caused significant infiltration of cytotoxic and memory/effector T-cells into tumors. Interestingly, ATRi also led to significant induction of MHC class I in SCLC in vitro and in vivo models. Analysis of pre- and post-treatment clinical samples from a proof-of-concept study of a first-in-class ATR inhibitor, M6620 (VX970, berzosertib), and TOP1 inhibitor topotecan, in patients with relapsed SCLCs validated the induction of MHC class I and interferon pathway genes, for the first time in this disease.

Conclusion: Our findings highlight ATRi as a potentially transformative vulnerability of SCLC, paving the way for combination clinical trials with anti-PD-L1. Given the increasing importance of immunotherapy for the management of SCLC and that ATR inhibitors are already in clinical trials, combining an ATR inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic.

Citation Format: Triparna Sen. ATR inhibition upregulates PD-L1 and potentiates the antitumor immune response to chemoimmunotherapy in small-cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B027.
Abstract B030: Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models
Authors: Wong Stephanie WY , Herbozo Carolina de Amat , Vukosich-Pennell Adriana , Wang Ben X , Ohashi Pamela S , Sacher Adrian G
Published: December 2023

Background: Mucosal-associated invariant T cells (MAIT) are unconventional αβT cells with a semi-invariant TCR that recognizes microbially-derived riboflavin metabolites presented by MHC-related protein 1 (MR1). Upon TCR engagement or cytokine stimulation, MAIT cells can rapidly secrete pro-inflammatory cytokines and cytotoxic molecules. The precise role of MAIT cells in the tumor-immune microenvironment remains unclear, as different reports have described them as either beneficial or detrimental to patient prognosis. Considering that MAIT cells constitute a notable proportion (2-4%) of the lung T cell repertoire in both humans and mice, the lung serves as an appropriate setting for ex vivo and in vivo MAIT cell characterization.

Objective: To characterize the phenotype of MAIT cells in lung tumor and uninvolved tissue from non-small cell lung cancer (NSCLC) specimens and orthotopic murine models. Results: Surgical resections of primary tumor and normal adjacent tissue from 29 treatment-naïve NSCLC patients were profiled using a comprehensive 31-marker spectral flow cytometry panel. MAIT cells expressing activating and inhibitory receptors including CD69, PD-1, CTLA-4, and CD39, were enriched in tumor tissue. Conversely, cytotoxic MAIT cells expressing granzyme B, granulysin, and CD57, were enriched in normal adjacent tissue. For in vivo studies, lungs of B6-MAITCAST mice, which harbor higher frequencies of MAIT cells compared to conventional mouse strains, were orthotopically injected with CMT167 tumor cells or vehicle control. MAIT cells from lungs injected with CMT167 tumors also expressed higher levels of activating and inhibitory receptors, and decreased levels of granzyme B. Additionally, there was a slight decrease in IFNγ and IL-17A production upon stimulation in vitro with PMA/ionomycin compared to vehicle controls. Conclusion: In the context of lung cancer, MAIT cells are activated but may lose functionality within the tumor-immune microenvironment.

Rationale: There is growing interest in the immunotherapeutic potential of unconventional T cells due to their rapid effector functions and non-MHC-restricted nature. By investigating the role of MAIT cells in the tumor microenvironment, we can comprehensively identify their biological advantages and limitations.

Citation Format: Stephanie WY Wong, Carolina de Amat Herbozo, Adriana Vukosich-Pennell, Ben X Wang, Pamela S Ohashi, Adrian G Sacher. Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B030.
Abstract B038: Targeting a Treg-specific deubiquitinase module for antitumor immune therapy
Authors: Fang Deyu
Published: December 2023

Checkpoint immunotherapies have transformed the treatment landscape for multiple human malignancies, but still with very limited success in treatment of many human cancers. A major hurdle is mediated by regulatory T (Treg) cells, which suppress the function of antitumor effector T cells. However, current approaches in targeting Treg to activate antitumor immune responses have shown only transient efficacy and are highly unspecific. Importantly, our recent studies, through unbiased CRISPR screening, identified the death from cancer cell signature gene USP22 required for optimal FoxP3 expression. USP22 genetic inhibition resulted in an approximately 15-25% reduction of FoxP3 expression in Tregs and partially diminishes Treg suppressive functions, which consequently results in elevated antitumor immunity against a broad spectrum of cancer types in mice. Therefore, a 20% reduction in FoxP3 protein expression, in this case, by USP22 inhibition, creates an ideal therapeutic window to boost antitumor immunity without triggering extensive autoimmune inflammatory responses (Nature, 2020). More recently, we further demonstrated that tumor microenvironment factors, including TGF-β, hypoxia, and tumor metabolites, induce Treg fitness/adaptation through selectively upregulating a deubiquitinase module, including USP21 and USP22 (Science Advances, 2022). Our ongoing study has identified USP22-specific small molecule inhibitors that largely diminish Treg fitness and dramatically inhibit tumor growth at a clinically relevant setting. In addition, our recent unpublished single-cell RNA-seq characterization revealed a novel function of the Treg-specific deubiquitination module in Treg terminal differentiation into a unique immune suppressive subpopulation, specifically in the tumor microenvironment. Collectively, our study defines a novel Treg deubiquitination module in tumor immunosurveillance and provides a rationale for the first Treg-specific targeting in antitumor immune therapy.

Citation Format: Deyu Fang. Targeting a Treg-specific deubiquitinase module for antitumor immune therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B038.
Abstract B039: T cell recruitment dynamics at different stages of primary and metastatic colorectal cancer
Authors: Saad Marwa A , Bilate Angelina , Mucida Daniel
Published: December 2023

Colorectal cancer (CRC) is the fourth deadliest cancer worldwide, primarily due to metastasis. The current standard therapy for patients with CRC includes neoadjuvant therapies combined with surgery, often resulting in severe complications that affect patients’ lifestyles. Hence, there is a critical need to improve our understanding of the tumor microenvironment to improve the current therapeutics for metastatic CRC. We characterized T cell dynamics in response to tumors at early (1-2 weeks) versus late stages (6-7 weeks) of CRC development in different colon compartments (tumor, epithelium, lamina propria), as well as in colon-draining lymph nodes and the liver as potential metastasis sites. To achieve this, we injected by colonoscopy tumor AKP mouse organoids (mutations in APC, Kras, and P53 genes) or metastatic AKP mouse organoids in the colon walls of a novel fate-mapping strain (iSell-tomato). This model allows for the labelling of peripheral T cells that are recruited to the tissue or tumor sites. Our initial data showed an enhanced recruitment of CD4 and CD8ab T cells to the colon epithelium in early stage AKP (non-metastatic) tumor models compared to late-stage AKP and metastatic-AKP models at both stages. These recruited cells exhibited an enhanced anti-tumorigenic profile characterized by the production of IFNg and reduced frequency of FoxP3+ CD4 regulatory T cells (Tregs). In contrast, T cells recruited to early stage metastatic-AKP tumors and late-stage tumors of the colon and liver exhibited enhanced exhaustion characterized by an increased frequency of PD1+ CD4 and CD8ab T cells. These data suggest a time- and aggressiveness-dependent effect of tumors on immune cell infiltrates that, in turn, affect tumor progression and metastasis.

Citation Format: Marwa A Saad, Angelina Bilate, Daniel Mucida. T cell recruitment dynamics at different stages of primary and metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B039.
Abstract IA015: Obesity and immunometabolism in cancer immunotherapy
Authors: Rathmell Jeffrey
Published: December 2023

Obesity is increasing in prevalence and contributes to approximately 1 million new cancer cases annually. The effects of obesity and altered metabolism on cancers and anti-tumor immunity, remain poorly understood. The metabolism of T cells and other immune cells is dynamically regulated through growth signals and microenvironmental cues to influence biosynthesis, signaling, and cell fate. We have shown that CD4 T cell subsets are metabolically distinct and that each requires a specific metabolic program for their function. To address how cell-extrinsic factors in obesity regulate immunotherapy responses we have studied obesity and fever temperatures as regulators of immune cell metabolism and fate. Obesity is associated with increased risk of cancer but can paradoxically enhance the efficacy of immune therapy in some cases. We found this effect is in part mediated by the adipokine Leptin, which is elevated in obesity and can stimulate T cells. In addition, tumors in obese individuals and animals have increased frequencies of PD-1+ macrophages. We tested the role of PD-1 on macrophages and show with inhibitors and genetic knockouts that it is a direct regulator of macrophage metabolism and suppresses ability to of macrophages to phagocytose and present antigen and stimulate T cells. Macrophages induced PD-1 in response to multiple inflammatory cytokines, but also upon exposure to lipids or with Leptin or Insulin. PD-1-deficient macrophages had increased glycolysis and ability to stimulate T cells. These data show that obesity both promotes inflammation through adipokines such as Leptin, while simultaneously inhibiting anti-tumor immunity through induction of PD-1 on macrophages.

Citation Format: Jeffrey Rathmell. Obesity and immunometabolism in cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr IA015.
Abstract PR06: Hallmarks of CD8 T cell dysfunction are established within hours of tumor antigen encounter prior to cell division
Authors: Philip Mary , Rudloff Michael W. , Zumbo Paul , Betel Doron , Favret Natalie R. , Roetman Jessica J. , Roman Carlos R. Detres , Erwin Megan M. , Murray Kristen A. , Jonnakuti Sriya T. , Dundar Friederike
Published: December 2023

Tumor-specific CD8 T cells (TST) found in patients with cancer are dysfunctional and unable to halt cancer progressions. TST dysfunction, also known as T cell exhaustion, is thought to be driven by chronic T cell receptor/antigen stimulation and immunosuppressive factors within the tumor microenvironment. However, we know little about the interplay between CD8 T cell function, cell division, and epigenetic remodeling during the first hours following activation and tumor encounter. Here, we assessed TST differentiation, cell division, chromatin accessibility, and transcription in two clinically-relevant cancer mouse models (autochthonous liver cancer and metastatic melanoma), and we compared TST fate trajectories to those of CD8 T cells differentiating during acute infection. Surprisingly, despite robust activation and proliferation, TST had near complete effector function impairment even prior to undergoing cell division, and had acquired hallmark chromatin accessibility features previously observed in late dysfunctional/exhausted T cells. Moreover, continued tumor/antigen exposure drove progressive epigenetic remodeling, “imprinting” the dysfunctional state. Our study defines for the first time the regulation and kinetics underlying T cell fate choice prior to cell division in the context of tumors versus infection.

Citation Format: Mary Philip, Michael W. Rudloff, Paul Zumbo, Doron Betel, Natalie R. Favret, Jessica J. Roetman, Carlos R. Detres Roman, Megan M. Erwin, Kristen A. Murray, Sriya T. Jonnakuti, Friederike Dundar. Hallmarks of CD8 T cell dysfunction are established within hours of tumor antigen encounter prior to cell division [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR06.
Supplementary Figure 7 from CAR T-cell Design-dependent Remodeling of the Brain Tumor Immune Microenvironment Modulates Tumor-associated Macrophages and Anti-glioma Activity
Authors: Haydar Dalia , Ibañez-Vega Jorge , Crawford Jeremy Chase , Chou Ching-Heng , Guy Clifford S. , Meehl Michaela , Yi Zhongzhen , Perry Scott , Laxton Jonathan , Cunningham Trevor , Langfitt Deanna , Vogel Peter , DeRenzo Christopher , Gottschalk Stephen , Roussel Martine F. , Thomas Paul G. , Krenciute Giedre
Published: December 2023

Supplementary Figure S7 shows initial analysis of scRNAseq data.