bluuebunny/crossref_metadata_2025_split huggingface dataset:
It takes a village: STATs control IE-CTL pathogenesis in Celiac Disease
Authors: Borregard Megan Elisabeth , Broekema Roeland , Jonkers Iris , Jabri Bana 
Published: August 2023

Abstract
Celiac Disease (CeD) is an autoimmune-like inflammatory disorder triggered by exposure to dietary gluten in genetically predisposed individuals. Tissue destruction in CeD is driven by tissue-resident, cytotoxic CD8+ intraepithelial lymphocytes (IE-CTLs). Upregulation of tissue alarmins and inflammatory cytokines – including IL-15, IL-21, and type I interferon – is evident in biopsies from CeD patients. It is thought that these cytokines may license IE-CTLs to kill stressed intestinal epithelial cells and contribute to their long-term pathogenic reprogramming. We therefore explored the contribution of these cytokines, interferons, and TCR stimulation in IE-CTL activation and pathogenic conversion. We found that both cytokine and TCR stimulation induced overlapping and distinct transcriptional programs. Pathways associated with activation and effector function were most robustly induced by a combination of cytokines. Further, genes identified from CeD-associated GWAS were also more strongly induced by cytokine stimulation. Cytokine signaling resulted in the activation of signal transducer and activator of transcription (STAT) pathways, which subsequently act as key TFs in IE-CTL reprogramming. To link specific transcriptional responses to particular STATs we performed individual and combined knockdowns of the four STATs associated with these cytokines. Our results showed significant redundancy in the STAT signaling pathways following cytokine stimulation, suggesting that convergence in individual STAT signaling nodes in IE-CTLs is integral to CeD pathogenesis.
Supported by grants from NSF GRFP 1746045 and NIH BIBIB 2 T32 EB 9412.
Cytometric analysis authenticates adjuvanticity of interferon-γ and suggests potential synergy with toll-like receptor agonists
Authors: Bian Yuanzhi , Walter Debra , Zhang Chenming 
Published: August 2023

Abstract
Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been investigated as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases. In this study, we aimed to explore the potentiality of IFN-γ and TLR agonists being applied as dual adjuvant systems. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic-polycytidylic acid (poly I:C) or resiquimod (R848). Subsequently, the cells were stained for an activation marker, cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 μg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% positive cells obtained by 10 ng/mL IFN-γ (p = 0.0004). These results suggested that IFN-γ and TLR agonists could be applied as a complementary, dual adjuvant system to promote dendritic cell activation and antigen presentation. Additionally, there might be a synergy between the two classes of adjuvants, and further investigation is warranted to ascertain the interaction of their adjuvant activities.
This work was supported by the NIDA/NIH award UG3DA048775.
Reshaping the anti-tumor CD4 T-cell repertoire contributes to oncolytic virus therapeutic efficacy in malignant glioma
Authors: Leavenworth Jianmei Wu , Grimes Jeffrey M , Ghosh Sadashib , Murchison Charles , New J Stewart , Crossman David , Markert James M 
Published: August 2023

Abstract
Malignant gliomas are deadly brain tumors with no cure. We engineered herpes simplex virus (HSV)-1 and developed oncolytic HSV (oHSV) to treat brain tumors, given its potential to selectively kill tumor cells while sparing normal brain cells, and to boost anti-tumor immunity. The therapy shows great promise based on our Phase I trial results. However, the precise mechanisms for its enhanced anti-tumor immune responses remain poorly understood. Using an oHSV secreting murine IL-12 (M002), murine intracranial glioma models and adoptive transfer approaches, we observed that M002 treatment induced unique CD4+ T-cell populations to acquire cytolytic effector phenotypes, promoting anti-glioma responses in an MHCII-dependent manner. Notably, single-cell RNA sequencing and T-cell receptor (TCR) repertoire analysis revealed that M002 treatment shifted the TCR dominance while reducing the clonal diversity. These results suggest that the capacity of oncolytic virus therapy to reshape the anti-tumor CD4+ T-cell repertoire contributes to its improved efficacy. Insights obtained from this study can facilitate the development of better therapeutic strategies for brain tumor patients.
Supported by DoD W81XH-18-1-0315 and UAB Faculty Start-up Funds (to JWL)
RNA-based Personalized Cancer Vaccine for AML immunotherapy
Authors: Liu Haiyan , Tham Guo Hong , Ren Xi , Yang Henry 
Published: August 2023

Abstract
Acute Myeloid Leukemia (AML) is a rapidly progressing malignancy relying primarily on chemotherapy as the frontline treatment option. Success of personalized neoantigen vaccines in melanoma patients have garnered great attention in the field of immunotherapy. We hypothesize that personalized cancer vaccine, in the form of mRNA, can be a viable treatment strategy for AML patients. Next generation sequencing was performed on both C57BL6Jinv mouse’s and C1498 cell line’s nucleic acid materials. Three different neoepitope prediction pipelines were utilized to identify potential immunogenic neoepitopes. Candidate neoepitopes within intron polyadenlyation sites (iPAS), retained intron (RI) sites and somatic mutation (SM) sites were found. A total of 285 SM-derived neoepitopes were identified. Top 38 highly expressed genes were selected for further analysis. Using Sanger sequencing, we confirmed 37 out of the 38 mutations. A total of 5 iPAS-derived neoepitopes were identified. A total of 26 RI-derived neoepitopes were identified in our first RNAseq analysis and a separate 476 RI-derived neoepitopes were identified in our second RNAseq analysis. With these neoepitope candidates, we narrowed down our selection and did a preliminary screen for immunogenic epitopes using a total of 71 synthetic peptides in an ELISpot assay. 10 neoepitope candidates were short-listed for further in-vivo evaluation. mRNA vaccine was synthesized and evaluated in a murine AML model. T cell responses against the 10 neoepitopes were analyzed to provide pre-clinical evidence for patient studies. Neoepitope screening platform with human PBMCs has also been established to validate the prediction pipelines in AML patients.
Supported by grant from NMRC (MOH-CIRG21jun-0008)
Can Engineering IL-2Rα Expression Improve NK Cell Immunotherapy?
Authors: Lee Seung-Hwan , Marr Bryan , Jo Donghyeon , McComb Scott 
Published: August 2023

Abstract
Natural killer (NK) cells are innate lymphocytes central to anti-viral and anti-tumour responses. Activated NK cells upregulate IL-2Rα, promoting the formation of a high-affinity heterotrimeric IL-2Rαβγ that mediates NK cell expansion and enhances cytotoxicity. Several applications of NK cancer immunotherapy, including using chimeric antigen receptors (CAR), have shown early success in clinical studies. Allogeneic NK cells can illicit anti-tumour activity without toxicities, making them attractive for a biomanufactured “off-the-shelf” immunotherapy. Following adoptive transfer, low-dose IL-2 injections are commonly administered to enhance NK cell activity. However, expansion, persistence, and tumour clearance remain largely inadequate. Moreover, regulatory T cells (Tregs) can use their IL-2Rα expression to sequester injected IL-2, leading to Treg expansion and NK cell suppression. We hypothesize that CAR NK cell therapy can be improved by engineering high expression of IL-2Rα alongside the CAR. We predict the resulting CAR-IL2Rα NK cells will be more sensitive to low-dose IL-2 injections. We used lentiviral transduction of a multicistronic transgene to force high IL-2Rα expression alongside a CAR. I found CAR-IL2Rα NK cells were more responsive to low-dose IL-2 in-vitro than conventional CAR NK cells. This novel strategy may improve clinical success by enhancing CAR NK cell expansion, persistence and anti-tumour activity, and limiting Treg suppression in the tumour microenvironment.
NRC Disruptive Technology Solutions Cell and Gene Therapy Challenge Program and the Canadian Institutes of Health Research
Synthetically mannosylated antigens impair the antigen-specific humoral immune response and prolong the therapeutic efficacy of immunogenic protein drugs
Authors: Wallace Rachel P , Tremain Andrew C , Wilson D Scott , Hubbell Jeffrey 
Published: August 2023

Abstract
Immunogenic protein drugs trigger an anti-drug antibody (ADA) response in patients which reduces drug efficacy and increases adverse reactions. Our lab has previously shown that targeting antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a novel strategy to further increase delivery of an antigen to the tolerogenic microenvironment of the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production which is maintained throughout subsequent administrations of the native biologic. We found that p(Man) treatment impairs the antibody response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten-skewing or control by Tregs. Through transcriptomic analysis we identified increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment, which may inform the underlying mechanism driving the reduction in ADAs. This modular platform may enhance tolerance to current and future biologics, enabling long-term therapeutic solutions for an ever-increasing healthcare problem.
Design and characterization of a tumor-retained interleukin-2 cytokine/antibody fusion protein for cancer immunotherapy
Authors: Silver Aliyah Brianne , Spangler Jamie B 
Published: August 2023

Abstract
Interleukin-2 (IL-2) is a pleiotropic cytokine with clinical utility in stimulating anti-cancer immunity. However, its efficacy is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory and immunosuppressive immune cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor β (IL-2Rβ) and the common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Regulatory T (Treg) cells express high levels of IL-2Rα making them more responsive to IL-2 than natural killer (NK), naïve/resting effector T (Teff), and memory T cells. To extend the half-life and selectively bias the immunostimulatory functions of IL-2 in a unimolecular format, we engineered a single-chain fusion protein, F10 immunocytokine (F10 IC), that intramolecularly fuses IL-2 to an anti-IL-2 antibody that sterically blocks IL-2Rα binding and allosterically enhances IL-2Rβ binding. However, preclinical data suggested a narrow therapeutic index (TI) for F10 IC. To improve the TI of F10 IC we designed a format of F10 IC poised for intratumoral delivery and retention. We fused collagen binding domains (CBD) to F10 IC in various topographies (F10 IC-CBD) to exploit the abundance and exposure of collagen in the tumor microenvironment. With intratumoral delivery, F10 IC-CBD is retained in the tumor and exhibits a therapeutically favorable pharmacokinetic profile. Furthermore, F10 IC-CBD increases and prolongs intratumoral immune cell infiltration, significantly slows tumor progression, minimizes F10 IC toxicity and synergizes with anti-PD-1 blockade in syngeneic mouse tumor models. We have illustrated the potential of F10 IC-CBD as a potent tumor-agnostic cancer therapy.
Supported by grants from the NIH (R01 EB029341, T32 AI007417), Melanoma Research Alliance, Willowcroft Foundation and a Sanofi iAward.
Antigen loaded monocytes prime CD4+ T cell responses through Notch2 dependent cDC2
Authors: Lueck Benjamin D , D’Anniballe Vincent M , Gunn Michael D 
Published: August 2023

Abstract
Cellular vaccines, primarily monocyte-derived dendritic cells (MoDC), have recently been realized as therapeutic cancer vaccines with modest clinical efficacy. Understanding the mechanism by which these vaccines lead to antigen presentation is key to improving their potency. A significant advancement was the discovery that MoDC vaccines do not directly present antigen, but instead rely on endogenous dendritic cells for their antigen presenting cell (APC) function. Subsequent research has also demonstrated that primary monocytes can be loaded with antigen and used as cellular vaccines, outperforming MoDC vaccines in mouse models of melanoma and glioblastoma. In this study, we characterized how monocyte vaccines generate CD4+ T cell responses. We utilized the CD11c-Cre RBPJfl/fl mouse model, which lack a subset of Notch2 dependent cDC2 characterized by expression of Endothelial cell-selective adhesion molecule (ESAMhi). We demonstrate that the ESAMhicDC2 subset is required for effective CD4+ OT-II T cell priming after vaccination with ovalbumin (OVA) loaded monocytes. Interestingly, depletion of the ESAMhi DC population does not affect monocyte vaccine-induced CD8+ T cell priming, a process previously shown to be dependent on cDC1. This indicates that monocyte vaccines generate T cell responses by independently transferring antigen to both ESAMhi and CD8+ DCs, leveraging their APC function to prime CD4+ and CD8+ T cell responses, respectively. This work furthers the mechanistic understanding of monocyte vaccines, and will inform the design of future monocyte vaccines as they enter clinical trials as therapeutic cancer vaccines.
Supported by NCI P50 CA 190991 and funding from the Stead Scholarship Program of the Duke University Department of Medicine.
Synergy between oxaliplatin and adenovirus initiates immunogenic cell death in human colorectal carcinoma cells
Authors: Jessup J Milburn , Hewitt Stephen M , Fuerst Thomas R 
Published: August 2023

Abstract
Externalization of Calreticulin (CALR) is the critical first step in the initiation of Immunogenic Cell Death. Since Oxaliplatin (OX) cooperates with an oncolytic chimeric human adenovirus Ad5/3-NP2.ADP (ADP) to induce adaptive immunity as well as regression of established cancer, we sought to determine whether the cooperation was additive or synergistic. Methods include immunohistochemistry (IHC) for Calreticulin expression in mice 11 days after intratumoral injection of the OX-ADP mixture. In vitro assays with limiting dilutions of OX and ADP on human Clone A colorectal carcinoma cell monolayers were cultured for 3 days and cytotoxicity, viral infectivity by GFP, and the expression of externalized CALR (ectoCALR) on Clone A cells measured by static cytometry. Synergy was measured using SynergyFinder 3.0 (Ianevski et al., PMC9252834) that creates 2-D interactive surfaces to measure synergy and antagonism. Areas of synergy have mean scores &gt; 10. IHC revealed strong CALR expression in OX-ADP treated tumors, weak expression in tumors injected with OX or ADP alone and minimal in PBS treated controls. Mean Viral Infectivity synergy score was 27.2 for the area of (Ox μM, ADP MOI): (0.12μM, 4 MOI) to (12.5μM, 100 MOI) while mean synergy score for ectoCALR expression was 29.2 for OX/ADP (0.01μM, 0.16) to (0.12μM, 20 MOI). The role of oxidative and endoplasmic reticulum stress as the basis of synergy is under study. Oxaliplatin and adenovirus are synergistic in their ability to initiate ICD which is important for vaccine development.
Chimeric antigen receptor-T cell efficacy can be evaluated on an Organ-Chip model system
Authors: Woodham Andrew W , Marianne Marianne , Cespedes Adriana V , Heng Anthony R , Frischmann Amanda S , Fortes Gabrielle M , Kajula Ville J , Ewart Lorna , Carman Christopher V 
Published: August 2023

Abstract

Objective
The need for human-centric model systems that can test the efficacy of chimeric antigen receptor (CAR) therapies is expanding rapidly, as these hold great promise for cancer treatment. We recently developed a system for inflammatory immune cell recruitment on the human Colon Intestine-Chip as a model for inflammatory bowel disease (IBD). The goal of the current study was to develop a novel system for measuring the recruitment and killing capacity of CAR-T cells in an Organ-Chip system.

Methods
We seeded a representative human HER2+ non-small cell lung carcinoma cell line (A549) into the top channel and human lung microvascular endothelial cells into the vascular (bottom) channel of Emulate’s S1 Organ-Chips. HER2-specific or CD19-specific (negative control) CAR-T cells were perfused through the vascular channel. Total CAR-T cell recruitment from the vasculature channel to the carcinoma-containing (top) channel and CAR-T-mediated killing (caspase activity, and IFN-g production) were measured over 72 hours. This system is further being used to monitor CAR-T cell exhaustion and efficacy of co-therapies (e.g., that might support CAR-T recruitment and killing efficacy).

Results
HER2 CAR-T cells were recruited to the top channel, which was supported by the addition of known cancer chemokines (e.g. CXCL10), and induced significant killing and response to co-therapies as measured by caspase activity compared to controls.

Conclusion
These early findings suggest that the human-centric Organ-Chip model can evaluate the efficacy of CAR-T cell therapies, and in particular, provide a system that integrates both the recruitment and killing aspects of CAR-T function.
Fourth generation Glypican-3–specific CAR-NK cells co-secreting IL-15 and IFN-α have increased anti-tumor function versus hepatocellular carcinoma
Authors: Badami Ester , Busà Rosalia , Gallina Anna , Iannolo Gioacchin , Amico Giandomenico , Douradinha Bruno , Conaldi Pier Giulio 
Published: August 2023

Abstract
Hepatocellular carcinoma (HCC) ranks as the third most frequent cause of cancer-related mortality, indicating the urgent need for effective therapies. Glypican-3 (GPC3) is up-regulated in HCC and not expressed in normal liver cells, representing a strategic target. Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are a promising immunotherapeutic approach for cancer treatment. However, their potential applications have not been fully explored in HCC. We have patented a protocol of purification and expansion of clinically relevant numbers of NK cells starting from the liver perfusate from deceased donors. Further, we have demonstrated that activating NK cells with interferon-alpha (IFN-α) potentiates the immune response in vitro and in vivo both in a murine model of hepatocarcinoma and of viral hepatitis. In this study, we engineered primary CAR-NK cells to acquire specificity for GPC3. The construct consists of the intracellular signaling region CD28, 4–1BB, CD3ζ and CD8α hinge domain and of the truncated form of human epidermal growth factor receptor (tEGFR) as suicide gene. The fourth-generation construct is designed to secrete the transgenic cytokine IL-15, essential for NK cells function and survival. A second plasmid was also generated differing for the additional secretion of IFN-α. Here we show that we could achieve a transduction efficiency of above 50%. Exposure of GPC3+ HCC cells to CAR-NK cells resulted in enhanced specific cytotoxicity and cytokine production such al IL-2 and IFN-γ; moreover IFN-α secretion significantly increased anti-GPC3 function. Our data encourage preclinical proof-of-concept studies to clinically translate GPC3-specific NK cells as a treatment option for HCC patients.
This work was supported by Fondazione Ri.MED (Fondo Generale, ID 10019) and ISMETT (ATMP-P A105412 project, ID I00000219).
PD-L1 Expression on Circulating Antigen Presenting Macrophages in Blood Predicts PFS & OS in an Array of Metastatic Cancer Types Treated with PD-L1/PD-1 Immunotherapies
Authors: Adams Daniel L , Lopez Pablo , Lin Steven H , Ho Thai H , Gardner Kirby P , Tang Cha Mei 
Published: August 2023

Abstract
Cancer Associated Macrophage Like cells (CAMLs) are antigen presenting circulating stromal cells found in the blood of cancer patients (pts). Recently, PD-L1 expression on CAMLs was described in breast (BC), lung (LC) and renal cell carcinomas (RCC), appearing to parallel the inflammatory PD-L1 state of the tumor microenvironment, and acting as a possible predictive biomarker for PD-L1/PD-1 immunotherapeutics (IMTs). We monitored CAML PD-L1 from 48 pts in an array of solid tumors, prior PD-L1/PD-1 IMTs induction, to evaluate CAML’s ability to predict response by progression free survival (PFS) and overall survival (OS).
Three single blind multi-year prospective studies were run to test PD-L1 CAML expression against PFS & OS, prior to induction of therapy in metastatic (m) LC (n=24), mBC (n=16) or mRCC (n=8). We recruited pts with pathologically confirmed progressing disease after at least one failed prior line of therapy. Blood samples (7.5 mL) were taken prior to start of new therapeutic regimes, i.e. pembrolizumab (n=36) or nivolumab (n=12). Whole blood was filtered by CellSieve microfilters to collect CAMLs, stain for PD-L1, and then scored as a binary high or low, to evaluate PFS & OS hazard ratios (HRs) by censored univariate & multivariate analysis at 24 months.
CAMLs were found in 98% of all tested samples. Overall, pts with high CAML PD-L1 expression treated with IMT had significantly better PFS (HR=2.9, 95%CI=1.4–6.1, p=0.0067) and better OS (HR=2.7, 95%CI=1.2–6.1, p=0.0317) vs patients with low CAML PD-L1.
These data suggests that PD-L1 expression in circulating CAMLs appears to predict pts with increased benefit to PD-L1/PD-1 IMTs in an array of tumor subtypes, though larger studies are needed to validate these findings.
Private Funding by Creatv MicroTech, Inc. and supported grants by NCI-NIH (R43CA206840), and the Defense Advanced Research Projects Agency (DARPA)(W911NF-14-C-0098).