bluuebunny/crossref_metadata_2025_split huggingface dataset:
C57BL/6 mice cohoused with pet shop mice phenocopy immune-related adverse events associated with checkpoint inhibition
Authors: Hapak Sophie , Vezys Vaiva 
Published: August 2023

Abstract
Checkpoint inhibition has been successful in the treatment of a number of malignancies. These medications block inhibitory pathways in T cells and allow for their improved function in anti-tumor immune responses. An important factor limiting checkpoint inhibitor therapy is immune-related adverse events (irAE), which can involve any organ system. It is currently not possible to predict which patients will develop irAE or which tissues will be affected. Our understanding of the cellular and molecular factors driving irAE development remains limited, as immune sufficient specific pathogen free (SPF) C57BL/6 mice do not develop irAE when treated with checkpoint inhibition. We have previously reported that C57BL/6 mice cohoused with pet shop mice recapitulate aspects of the human adult immune system, while SPF C57BL/6 mice have immune systems resembling that of human neonates. We hypothesized that checkpoint inhibition in C57BL/6 mice cohoused with pet shop mice will phenocopy irAE development in humans. Here, we report that treating “dirty” C57BL/6 mice with anti-CTLA4 and anti-PD-L1 resulted in the development of irAE, including nephritis, dermatitis, and hepatitis, as evaluated by histology and serum lab values. Ongoing experiments include depletion of CD4 and CD8 T cells to determine whether irAE are mediated by these cells. To our knowledge, this represents the first model of irAE in immune sufficient C57BL/6 mice, enabling further investigation into the mechanistic understanding of irAE and development of treatments targeting irAE while leaving anti-tumor responses intact.
DIABETES T32 RESEARCH TRAINING PROGRAM
Abrogating Regulatory T Cells Overcomes Tumor-Specific T cell Exhaustion and Prevents Metastatic Pancreatic Cancer
Authors: Schmiechen Zoe C , Burrack Adam L , Miller Ebony , Butler Jonah , Cruz Eduardo , Rollins Meagan , Patterson Michael , Wang Iris , Stromnes Ingunn 
Published: August 2023

Abstract
Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 10%. Lethality is attributed to late diagnosis, early metastasis, and therapeutic resistance. We identified pancreatic tumor cells derived from mice which resist immunotherapy (e.g., escape variants, EV) rapidly metastasize upon re-implantation into the pancreas of syngeneic and immunocompetent mice. EVs retain the dominant tumor antigen, and despite a defect in IFNg-inducible MHC class I upregulation, EVs remain sensitive to tumor antigen specific T cell-mediated lysis in vitro, suggesting EVs may confer unique qualities in vivo to resist T cell killing. Using a peptide:MHC tetramer based approach, we identified that tumor-specific CD8 T cells in EV tumors have increased Granzyme B production and a reduction in prototypical exhaustion markers Pd1, Lag3, and Tox. Further, restoring MHC class I upregulation reversed this phenotype. Notably, primary tumors from EVs were significantly enriched with Foxp3+ Tregs compared to parental tumors. Using a genetic model, we identified that Treg depletion resulted in a drastic reduction in tumor burden and metastasis and improved tumor-specific T cell function in EV tumors, expanding a putative anti-metastatic CD8 T cells subset. Investigation of tumor-cell intrinsic changes identified elevated expression of the receptor tyrosine kinase Ddr2in multiple EVs compared to immunotherapy naïve cells. Our data supports the novel hypothesis that Ddr2 overexpression drives intratumoral Treg accumulation and promotes metastasis. In summary, Tregs are key drivers of T cell exhaustion and immunosuppression in pancreatic cancer and may prove a valuable clinical target for tumors that evade immune checkpoint blockade.
Supported by NIH T32 grant – AG029796 – Functional Proteomics of Aging Training Program
T cell-dependent regulatory roles of 18-kDa Translocator protein (TSPO)
Authors: Sendai Yuka , Takeda Kazuyoshi , Ichinohe Tatsuya , Koshika Kyotaro , Okumura Ko , Azuma Toshifumi , Ohno Tatsukuni 
Published: August 2023

Abstract

Objective
TSPO (also called PBR) is a translocator protein that is localized on the outer mitochondrial membrane. TSPO is broadly and constitutively expressed in various types of immune cells including macrophages, monocytes, T cells, and B cells. Although many studies have shown that TSPO negatively regulates innate immune cell, such as macrophage and monocyte, activity, the roles of TSPO in T cell-mediated immune responses remain unclear. In this study, we examine the effects of Ro5-4864, a TSPO agonistic ligand, in an DNFB-induced contact hypersensitivity murine model.

Results and Discussion
TSPO expression in draining lymph nodes (dLNs) was elevated after sensitization. Ro5-4864 treatment at sensitization, but not at challenge, significantly suppressed ear swelling. The total cell numbers in the dLNCs were decreased by Ro5-4864 treatment. In the Ro5-4864-treated mice, the ratios of IFN-γ expressing CD4+ T cells and CD8+ T cells in dLNs were decreased. Adoptive transfer of Ro5-4864-treated sensitized T cells into naive mice inhibited ear swelling after ear challenge. Ro5-4864-treated sensitized T cells showed lower proliferative responses against DNFB-pulsed antigen presenting cells. Our results demonstrated that treatment with Ro5-4864 inhibited DNFB-induced contact hypersensitivity, suggesting the regulatory roles of TSPO in T cell-depending immune responses.
Assays for profiling changes in T cell metabolism
Authors: Bach Maggie L , Sylvester Kayla , Leippe Donna , Valley Mike , Cali Jim , Vidugiriene Jolanta 
Published: August 2023

Abstract
Immune cell therapy, including engineered T cells, engineered NK cells, and treatments to enhance native T cell response, is a rapidly advancing field of research with great promise to treat multiple diseases. However, multiple challenges, from isolating the correct cell type, determining its mode of action, manufacturing and establishing appropriate quality control criteria, must be overcome to reach its potential. In recent years, cellular energy metabolism has emerged as an important consideration for efficacy and persistence when developing cell therapies.
Here we evaluate a set of bioluminescent cell-based assays for measuring rate of metabolite uptake, monitoring activity for key metabolic pathways and determining changes in intracellular metabolite levels during T cell transition from naïve to activated to memory phenotypes. Metabolite levels measured include ATP, glucose uptake, malate, branched chain amino acids, and glycogen levels. Depending on the activation state of the cells, the cells respond differently to glycolysis and oxidative phosphorylation inhibitors, as demonstrated by changes to metabolite levels. Energy storage as measured by intracellular glycogen levels also differs depending on the T cell phenotype (naïve, activated, or memory).
Understanding the processes governing dynamic metabolic changes during cell modification and expansion is required for next-generation cell therapy development. The developed bioluminescent cell-based assays provide the sensitivity, throughput and robustness required for rapid evaluation of such changes.
Mitochondrial cristae disruption enables optimal T cell activation and expansion
Authors: Carvalho Edmund , Su Irene , Ecker Christopher , Srivatsa Sriram , Riley James 
Published: August 2023

Abstract
T cell activation is an anabolic process requiring both large amounts of energy and biomaterials to construct daughter T cells. We examined structural changes to the mitochondria during T cell activation, given its predominant role in generating ATP and harboring key building blocks such as NADH. Upon T cell activation, the mitochondria massively disturb their cristae and swell in a process that peaks after 48 hours. As the T cells begin to rest down, we observed that mitochondria begin to revert back to their pre-activation phenotype. Intriguingly environments that limit T cell activation have less cristae disruption, suggesting that this process is beneficial to the immune response. We saw rapid downregulation of electron transport chain subunits required for complexes 1,2 and 4. Moreover, NADH/NADPH, which is largely housed in the mitochondria of a resting T cell leaves the organelle to the cytoplasm, coincident with mitochondria cristae disruption, suggesting that the mitochondrial cristae disturbance causes a shift in the equilibrium towards of these molecules for energy production so that it can be used as a building block for nucleotides. This data suggest that the mitochondria alter their structure and function to promote optimal T cell activation.
Hypoxia promotes inflammatory fibroblast formation in pancreatic cancer
Authors: Mello Ashley M , Ngodup Tenzin , Donahue Katelyn L , Di Magliano Marina Pasca , Lee Kyoung Eun 
Published: August 2023

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to all existing therapies. Although immunotherapy has shown great promise against multiple deadly cancers, it has been largely ineffective in PDAC. This lack of response is in part attributed to its extensive, fibroinflammatory stroma and hypoxic microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant stromal cell type in PDAC, and single-cell transcriptomics of human and mouse PDAC has recently revealed a distinct CAF subpopulation, inflammatory CAFs (iCAFs). These inflammatory fibroblasts produce high levels of cytokines and chemokines in PDAC, and have the potential to contribute to its immunosuppressive microenvironment and tumorigenesis. By injecting a hypoxia probe into PDAC mouse models, we recently found that iCAFs predominantly reside in hypoxic tumor regions. We also observed that the hypoxia-related gene signature is positively enriched in iCAFs in human PDAC samples. Importantly, by exposing three-dimensional (3D) co-cultures of pancreatic cancer cells and fibroblasts to either hypoxia or normoxia, we showed that hypoxia induces IL1α from cancer cells and that IL1α is required for hypoxia-mediated iCAF formation. Our data implicate hypoxia as a critical regulator of tumor stroma. Efforts are ongoing to understand the role of hypoxia in the crosstalk between cancer cells, fibroblasts, and immune cells in driving an immunosuppressive tumor microenvironment.
Supported by a grant from the NIH (T32 AI007413) and the University of Michigan Rackham Merit Fellowship.
Melanocyte PDL1 promotes early tumor progression in a novel autochthonous NRAS-mutant melanoma model
Authors: Ontiveros Carlos Oscar , Chen Tiffany , Murray Clare , Kornepati Anand , Deng Yilun , Blinkiewicz Paige , Bai Haiyan , Crossland Grace , Souto-Padron Alvaro , Gupta Harshita B , Turk Mary Jo , Burd Christin , Curiel Tyler J 
Published: August 2023

Abstract
Tumor-intrinsic PDL1 drives melanoma treatment resistance. Melanocytes (melanoma cell-of-origin) do not express PDL1, but PDL1 is expressed in malignant melanomas, suggesting melanocyte PDL1 promotes melanomagenesis. We developed a novel autochthonous mouse model that develops NRASQ61R-mutant melanomas lacking PDL1 only in melanocytes (PDL1KOTNQ61R) and littermate controls (PDL1+/+ TNQ61R). We induced mice with 4-OH tamoxifen ± UV exposure to accelerate melanoma development. Tumor latency significantly increased in PDL1KO vs. PDL1+/+ TNQ61R mice at 0 kJ/m2 and 2 kJ/m2UV(p&lt;0.02), suggesting melanocyte/tumor PDL1 promotes melanomagenesis. Latencies were similar at 4.5 kJ/m2, suggesting immune contributions, e.g., from local immunosuppression. We derived transplantable cell lines from autochthonous tumors. PDL1+/+ TNQ61R cell lines had increased mTORC1 and stemness, and reduced STING and reduced Chk1 inhibitor sensitivity in vitro and in vivo vs. PDL1KOTNQ61R, phenocopied by PDL1KO B16. Tumor PDL1 suppressed ERK signals (NRAS target) in TNQ61R vs. PDL1KO cell lines and promoted resistance to NRAS-targeting small molecule drugs, suggesting tumor PDL1 promotes NRAS signals. Transplanted 4.5 kJ/m2UV-inducedPDL1KO TNQ61R tumors were resistant to αPD1, αPDL1, αPDL2, and CD122-biased IL2 in WT mice vs. B16 melanomas, which are sensitive to biased IL2. Our model allows studies of PDL1 signals in melanomagenesis and progression and distinguishes bona fide cell-intrinsic PDL1 signals.
This research was funded by the NIH T32GM113896 (STX MSTP) Award (C.O.) and the NCI (CA204965, CA054515) supported Curiel.
ST8Sia6 sialylates CD44 in cancer cells to generate ligands for inhibitory Siglecs
Authors: Chen Yin , Friedman David J , Saraswat Mayank , Chen Alex Shuaibo , Shapiro Michael , Wilfahrt Drew , Pandey Akhilesh , Shapiro Virginia Smith 
Published: August 2023

Abstract
Glycans decorate most proteins and lipids on the cell surface, and glycoproteins serve a critical role in cell-cell communication. The terminal modification of many glycans is a sialic acid. There are twenty different mammalian sialyltransferases, each with a unique specificity. Recently, we demonstrated that the sialyltransferase ST8Sia6, which adds a terminal alpha-2,8 disialic acid to a preexisting sialic acid on cell surface proteins, generates ligands for the inhibitory receptor Siglec-E. In patients, ST8Sia6 overexpression is associated with worse outcome. We have previously shown that ST8sia6 overexpression in a mouse tumor model accelerates tumor growth, dependent upon Siglec-E expression by immune cells. To understand the basis for ST8Sia6 function in tumors, proteomic analysis of murine tumor lines was performed to identify proteins modified by ST8Sia6. Here, we demonstrate that ST8Sia6 sialylates CD44. Siglecs vary significantly between species, with 5 non-conserved Siglecs in mice compared to 10 non-conserved Siglecs in humans. The closest ortholog to Siglec-E in mice is Siglec-7 in humans. We also demonstrate that ST8Sia6 sialylates CD44, generating ligands for Siglec-7. Thus, ST8sia6 has a conserved role in both mice and humans in modifying glycans on CD44 to generate ligands for Siglecs. As CD44 expression on tumors is associated with increased metastasis, we predict that ST8Sia6 may also have a role in this process which is under investigation.
Supported by grants from 2T32AI007425-23 to DJF and NIH Glycobiology Common Fund supplement to R01CA243545-01A1 to VSS.
A shift in the hepatic immunological pattern from predominant adaptive immunity to innate immunity compromises tissue-specific immunity and leads to HCC
Authors: Koelsch Nicholas James , Mirshahi Faridoddin , Olex Amy , Sanyal Arun , Manjili Masoud H 
Published: August 2023

Abstract
The incidence of hepatocellular carcinoma (HCC) associated with non-alcoholic fatty liver disease (NAFLD) is growing because of the global consumption of high fat, high sugar or Western diet (WD). Current data suggests a correlation between the hepatic inflammation and progression of NAFLD to HCC. However, contradictory reports on the identification of specific inflammatory cells or cytokines hinder further progress in the field. Such controversial reports are due to highly reductionist approaches and getting lost in the details. By using flow cytometry, we have demonstrated that the hepatic immunological patterns were associated with the progression or inhibition of HCC, which can be discovered through the detection of the proportion of immune cell types functioning as a network. However, flow cytometry utilizes a limited number of markers hindering a comprehensive detection of the patterns. Here, we perform scRNAseq of the liver collected from the mouse model of NAFLD (DIAMOND) being on a WD prior to or after the development of HCC, as well as control mice being on a chow diet (CD). We demonstrate that a WD alters the hepatic immunological pattern by dominating innate immunity, M1 macrophages, over adaptive immunity, B and T cells, associated with the activation of the hepatic fibroblasts. Each immunological pattern created a distinct collective function beyond the function of its cellular constituents. Also, shifts in the immunological pattern compromised the hepatic immune system, transforming its anti-tumor function to carcinogenesis-promoting process. Our data suggest that the discovery and modulation of the hepatic immunological pattern could offer a comprehensive understanding of and curative treatment for NAFLD and HCC.
This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense.This work was also supported, in part, by CTSA award No. UL1TR002649 from the National Center for Advancing Translational Sciences.
Exploring the effects of tissue structural cell and tumor cell heparan sulfate on immune system trafficking, activation, and intercommunication via amphiregulin
Authors: Loffredo Lucas F , Li Fangda , Ringham Olivia R , Surpur Anmol , Cavero Renzo , Arpaia Nicholas 
Published: August 2023

Abstract
Heparan sulfate (HS), a glycan subtype, is ubiquitously present on the surface of structural tissue cells and tumor cells during homeostasis, disease, and cancer, but how it affects immune cell trafficking, activation, and signal transmission in these tissue contexts is poorly understood. Furthermore, amphiregulin (Areg), a growth factor used by immune cells for intercommunication with structural tissue and tumor cells, contains a HS binding domain. We investigated the downstream signaling modalities of Areg-mediated cell activation in the context of HS inhibition. Areg signaling is qualitatively altered, but still present, upon exogenous inhibition or genetic ablation of HS. We further probed the consequences of HS-dependent vs. -independent Areg signaling at the transcriptional level. Next, we used a panel of CRISPR-Cas9-mediated knockout Lewis Lung Carcinoma (LLC) cell lines to interrogate how various aspects of HS (core proteoglycan, HS chain extension, sulfation status) contribute to Areg signaling. We found that the presence of the glypican family of HS-harboring proteoglycans on the surface of cells is critical for Areg signaling. Lastly, we explored the effects of cancer cell HS on immune system trafficking to and activation within tumors. HS-deficient tumors experience similar levels of lymphoid cell trafficking and activation, but show a significant increase in the presence of certain myeloid cell type (neutrophils, monocytes, and macrophages). Additionally, tumor-associated macrophages within HS-deficient tumors show an altered phenotype, with increased maturation markers. Overall, our work has illuminated previously understudied aspects of the relationships between HS, Areg, and the immune system.
Supported by a grant from NIH (R21-AI149657)
Myocyte enhancer factor-2C is critical for the maintenance of ATLL cells by regulating the activity of retroviral antisense promoter and bZIP
Authors: Jain Pooja , Madugula Kiran , Joseph Julie , DeMarino Catherine , Ginwala Rashida , Teixeira Vanessa , Khan Zafar , Sales Dominic , Wilson Sydney , Kashanchi Fatah , Rushing Amanda , Lemasson Isabelle , Harhaj Edward , Janakiram Murali , Ye B Hilda , Jain Pooja 
Published: August 2023

Abstract
Adult T-cell leukemia and lymphoma (ATLL) is an intractable T-cell neoplasia caused by a retrovirus, namely human T-cell leukemia virus type 1 (HTLV-1). Patients suffering from ATLL present a poor prognosis and have a dearth of treatment options. In contrast to the sporadic expression of viral transactivator protein Tax present at the 5′ promoter region long terminal repeats (LTR), HTLV-1 bZIP gene (HBZ) is encoded by 3′LTR (the antisense promoter) and maintains its constant expression in ATLL cells and patients. The antisense promoter is associated with selective retroviral gene expression and has been an understudied phenomenon. Herein, we delineate the activity of transcription factor MEF (myocyte enhancer factor)-2 family members, which were found to be enriched at the 3′LTR and play an important role in the pathogenesis of ATLL. Of the four MEF isoforms (A to D), MEF-2A and 2C were highly overexpressed in a wide array of ATLL cell lines and in acute ATLL patients. The activity of MEF-2 isoforms were determined by knockdown experiments that led to decreased cell proliferation and regulated cell cycle progression. High enrichment of MEF-2C was observed at the 3′LTR along with cofactors Menin and JunD resulting in binding of MEF-2C to HBZ at this region. Chemical inhibition of MEF-2 proteins resulted in the cytotoxicity of ATLL cells in vitro and reduction of proviral load in a humanized mouse model. Taken together, this study provides a novel mechanism of 3′LTR regulation and establishes MEF-2 signaling a potential target for therapeutic intervention for ATLL.
NIH/NINDS: R01 NS097147 to PJ.
Antagonism of regulatory ISGs enhances the anti-melanoma efficacy of STING agonists
Authors: Filderman Jessica , Storkus Walter J 
Published: August 2023

Abstract
STimulator of INterferon Genes (STING) is a dsDNA sensor that triggers type I inflammatory responses. Recently this pathway has been leveraged as a cancer treatment with the use of STING agonists. Our lab has previously demonstrated that treating mice bearing established tumors with STING agonists leads to an inhibition in tumor growth in association with tumor vascular normalization and immune cell recruitment within the tumor microenvironment. However, STING agonism also results in the upregulated expression of regulatory interferon-stimulated genes (ISGs), including ARG2, enzymes involved in the production of immunosuppressive prostaglandins (i.e., PTGES and PTGS2/COX2), PD-L1, and ISG15, which limit the overall efficacy of this immunotherapeutic paradigm. We hypothesized that combined treatment of melanoma-bearing mice with STING agonists along with targeted inhibitors of ARG2, PTGES/COX2, PD-L1, and ISG15 would result in improved control of tumor growth and more robust anti-tumor immune responses in vivo. In the B16 (BRAFWTPTENWT) melanoma model, we observed improvement in tumor growth control after treatment with STING agonists + blocking anti-PD-L1 or anti-ISG15 antibodies vs. monotherapy. However, in the BPR (BRAFV600EPTEN−/−) melanoma model, tumor growth control over that observed for STING agonist alone was only noted upon combined addition of pharmacologic inhibitors of ARG2, COX2, and NOS2. The underlying therapy-associated changes in the TIME that support improved tumor growth control are currently being evaluated by single cell RNAseq, flow cytometry, and IFM to discern mechanism(s) of action.
Supported by grants from NIH (P01 CA234212) and the AAI Careers in Immunology Fellowship.