bluuebunny/crossref_metadata_2025_split huggingface dataset:
A fast live-cell assay to detect antigen-specific CD8+ T cells based on upregulation of integrin-mediated adhesion
Authors: Dimitrov Stoyan I , Besedovsky Luciana , Jensen Anja Tatiana Ramstedt , Born Jan , Gouttefangeas Cecile , Rammensee Hans-Georg , Lange Tanja 
Published: January 2023

Abstract
Antigen stimulation of T-cell receptors on T cells induces an immediate increase in ligand-binding affinity and clustering in the plasma membrane of beta2-integrins, resulting in a strong binding to intercellular adhesion molecules (ICAMs) on target cells. This firm adhesion, e.g. of cytotoxic T cells to virus-infected or tumor cells with formation of cytotoxic synapses is required for target cell lysis. Therefore, upregulation of integrin-mediated adhesion after stimulation identifies functional effector T cells. Here we present a novel flow cytometry assay to enumerate antigen-specific CD8+ T cells using soluble multimeric ICAM1-Fc-F(ab’)2 fluorescent complexes to identify integrin activation induced after short-term stimulation in vitro. By optimizing and validating this assay we were able to identify pathogen-, tumor- or vaccine-induced CD8+T cells, and show its feasibility for assessing and isolating live, functional antigen-specific CD8+T cells. Performing coexpression analyses, we found that antigen-stimulated cells that expressed cytokines and mobilized CD107a were predominantly highly adherent CD8+T cells. Finally, CD8+ T cells that upregulated integrins were shown to mediate cytolytic activity in an antigen-specific manner. The most striking advantage of this method is the short-time incubation (several minutes) that is necessary to identify and isolate viable antigen-specific cytotoxic T cells by assessing upregulation of T cell adhesiveness, a critical step for elaborating multiple effector functions, including cytotoxicity and cytokine production.
Immune Checkpoint and Immunostimulatory Reporter Bioassays to Advance the Development of Therapeutic Antibodies in Immunotherapy Research Programs
Authors: Somberg Richard , Cong Mei , Cheng Zhi-jie Jey , Grailer Jamison J , Stecha Pete , Wang Jun , Hartnett Jim , Fan Frank 
Published: January 2023

Abstract
Immunomodulatory biologics, which elicit their therapeutic effects by inducing, inhibiting, or modulating immune responses, have shown therapeutic utility in a variety of clinical conditions including cancer and autoimmune disease. As a result, biologics therapeutics, and monoclonal antibodies (mAb) in particular, are a dominant and increasing focus of pharmaceutical pipelines. However, due to their structural and biological complexity, mAb biologics and biosimilars require unique functional and analytical tools for development and characterization.
We have developed a suite of cell and reporter-based bioassays to advance the development and commercialization of mAb biologics. Specifically, we have developed cell-based bioassays in “thaw-and-use” format for the following Immune Checkpoint and Immunostimulatory receptor targets: PD-1/PD-L1, CTLA-4, LAG-3GITR, 4-1BB, CD40, OX40T Cell Activation
These reporter-based bioassays offer high specificity, sensitivity, and reproducibility, and demonstrate the performance required for use in antibody screening, potency determination, and stability studies.
TGF-β-induced regulatory T cells directly suppress B cell responses through a non-cytotoxic mechanism
Authors: Wang Julie , Olsen Nancy J , Zheng Song Guo 
Published: January 2023

Abstract
Foxp3+ regulatory T cells (Treg) playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases consist of thymus-derived naturally-occurring CD4+Foxp3+ Treg cells (nTreg) and those that can be induced ex vivo with TGF-β (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. The role of iTreg in regulating B cells remains unclear so far. The suppression assays of Treg subsets on activation, proliferation and antibodies production of B cells were measured using a Treg and B cell co-culture system in vitro. Transwell and antibody blockade experiments were performed to assess the roles of cell contact and soluble cytokines. Treg were adoptively transferred to lupus mice to assess in vivo effects on B cells. Like nTreg, iTreg subset also directly suppressed activation and proliferation of B cells isolated from C57BL/6. nTreg subset suppressed B cell responses through cytotoxic manner related to expression of Granzyme A, Granzyme B and Perforin; while the role of iTreg subset on B cells did not involve in cytotoxic action using Granzyme B and perforin KO mice, but depending on TGF-β signaling. Furthermore, iTreg subset can significantly suppress antibody produced by lupus B cells in vitro. Comparison experiments using microarray assay on autoantibodies demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo. Our data implicate a role and advantage of iTreg subset in treating B cell-mediated autoimmune diseases, boosting the translational potential of these findings.
IL-17 receptor A signaling impedes NF-κB p50/p50 repressor and subverts B-cell anergy in BXD2 mice
Authors: Hsu Hui-Chen , Hamilton Jennie A , Wu Qi , Yang PingAr , Luo Bao , Xie Shutao , Liu Shanrun , Li Jun , Mountz John D 
Published: January 2023

Abstract
B-cell fate decision checkpoint 2 at the transitional T2 stage represents a critical checkpoint defect in the development of autoimmune disease. We have found that there is a failure of anergy induction in the CD93+IgM+CD23+ transitional T2 B cells in the spleen of lupus prone BXD2 mice, which is associated with B-cell tolerance loss to anti-IgM or TLR7 in vitro. Interestingly, there was an enhanced BXD2-Il17ra−/− T2 B cell anergy phenotype in vivo, which is associated with a strong B cell anergic response to BCR or TLR7 stimulation in vitro. Abnormal survival of T2 B cells is thought to be regulated through a strong BAFF-R signaling. Surprisingly, despite normal expression of BAFF-R, BlyS cannot reverse the anergic response of BXD2-Il17ra−/− B cells to BCR stimulation. Analysis of the expression of IL-17RA in the B-cell subsets in BXD2 mice shows that only T2 and germinal center (PNA+Fas+) B cells, but not T1, T3 or mature (CD93−) B cells express surface IL-17RA. Single cell analysis of T2 B cells reveals the co-expression of Il17ra with B-cell activation gene, Bclxl. The strong anergic phenotype of BXD2-Il17ra−/− mouse B cells is associated with a dramatically enhanced nuclear expression of NF-κB1 (p50) and down-modulation of NF-κB phospho-p65. Our results suggest that, in BXD2-Il17ra−/− B cells, the anergy phenotype is established at the T2 stage. In these B cells, the stimulus-specific transcription repressor p50/p50 homodimer may act as a master transcriptional regulator to counteract pro-activation/survival NF-κB signaling provided by other major B-cell stimulators to enforce B cell anergy. Reagents that can promote the NF-κB p50/p50 repressome complex may be a novel strategy to enhance B-cell tolerance for autoimmunity.
Regulation of transcription factor IRF8 in myeloid progenitors is a critical checkpoint for formation of defective myeloid cells in cancer.
Authors: Messmer Michelle N , Netherby Colleen S , Abrams Scott I 
Published: January 2023

Abstract
Defectivemyeloid cells are commonly observed across a large range of solid cancers. These defects contribute to immune suppression and tumor progression tometastatic disease. Early studies focused on characterizing such myeloid-derivedsuppressor cells (MDSC) in peripheral tissues and the tumor microenvironment, but molecular processes underlying their development remained unclear. Bone marrowis the major site for normal myeloid production and is known to respond to signalsfrom the periphery. Thus, we hypothesizedthat tumors perturb bone marrow myelopoiesis, resulting in production of MDSC. During myelopoiesis, transcription factor IRF8 expression within granulocyte/monocyteprogenitors (GMP) determines differentiation such that high IRF8 favorsmonocytes while low IRF8 favors granulocytes. We previously revealed that IRF8 suppressionby tumor-derived factors leads to accumulation of MDSC, but exactly when andwhere IRF8 is compromised was unknown. Here we utilize a novel mouse model expressingan IRF8-EGFP fusion protein to investigate specific changes in IRF8 during myelopoiesisin both orthotopic and spontaneous mammary tumor progression. Our results show that:1) GMP can be divided into IRF8hi and IRF8lo expressingpopulations corresponding to monocytic and granulocytic progenitor phenotypes,2) IRF8lo GMP preferentially expand with increasing tumor size andincreasing G-CSF serum concentration, and 3) enforced IRF8 expression restrainsthis expansion and reduces the frequency of myeloid suppressors in theperiphery. Altogether, these data identify modulation of IRF8 in myeloidprogenitors as a consequence of tumor development and potential target fortherapeutic intervention.
Inhibition of CREB signaling through antagonism of vasoactive intestinal peptide enhances CD8 T cell function in a murine model of AML
Authors: Petersen Christopher T , Li Jian-Ming , Waller Edmund K 
Published: January 2023

Abstract
Signaling pathways that activate CREB may inhibit the potency of T cell responses following stimulation. One of the major mechanisms by which this occurs is the sequestration of CBP, which is also an NF-κB binding partner. One of the major molecules that signals through this pathway is vasoactive intestinal peptide (VIP). VIP is a 28 amino acid peptide that is secreted throughout the body by a variety of cell types including immune cells. VIP has shown to be a potent anti-inflammatory molecule with pleiotropic effects on T cells including cell cycle arrest and inhibition of cytokine secretion. The VIP signaling pathway represents a therapeutic target in disease settings in which a more robust T cell response would be beneficial. Acute myeloid leukemia (AML) is a hematological malignancy that has been explored as a candidate for a variety of T cell-based immunotherapies due to weak CD8 responses to AML blasts. We demonstrate here that administration of a hybrid peptide antagonist that blocks VIP signaling enhances CD8 T cell responses in a murine model of AML. CD8 T cells from treated mice showed enhanced secretion of IFN-γ and TNF-α as well as reduced expression of PD-1. These enhanced effector functions lead to a significant survival enhancement, which was lost when treated mice lacked T cells. Additionally, CD8 T cells from antagonist-treated survivors made up the majority of CD3+ cells following transfer to Rag1 knockout recipients. The phenotype of the cells was consistent with enhanced effector functions as transferred CD8+ T cells were predominately CD44+CD62L−. Taken together, these results demonstrate the potential for VIP antagonism as an immunotherapy strategy in settings of hematological malignancies.
Effector CD8 T cells use different homing receptor/ligand interactions to enter subcutaneous and intraperitoneal B16 melanoma tumors
Authors: Woods Amber N. , Wilson Ashley L. , Srivinasan Nithya , Engelhard Victor H. 
Published: January 2023

Abstract
CD8 T cell infiltration into tumors is critical for controlling cancer and relies on interactions between homing receptors (HR) on CD8 T cells and ligands on the vasculature. However, the ligands expressed on tumor vasculature, and the receptor interactions required for entry into tumors are incompletely characterized. The HR ligands VCAM-1 and E-selectin were highly expressed on the vasculature of B16-F1 melanomas expressing the strong antigen ovalbumin (B16-OVA) and growing subcutaneously but were significantly decreased compared to inflamed skin vasculature. In contrast, the vasculature of intraperitoneal tumors expressed MadCAM-1, negligible E-selectin, and lower VCAM-1. However, MadCAM-1 was not displayed on the luminal surface. B16-F1 parental tumors lacking a strong antigen, and B16-OVA tumors grown in Rag1KO animals, expressed negligible VCAM-1 and MadCAM-1, suggesting that previously infiltrating activated adaptive immune cells drove their expression. VCAM-1 and CXCL9/10 enabled entry of CD8 T cell effectors expressing α4β1+ integrin and CXCR3 into both subcutaneous and peritoneal tumors, while E-Selectin enabled entry of E-Selectin ligand+ effectors preferentially into subcutaneous tumors. MadCAM-1 did not mediate entry of α4β7+ effectors into peritoneal tumors, in keeping with its lack of luminal expression. Disrupting immunosuppression with checkpoint blockade inhibitors may increase HR ligand expression on tumor vasculature and improve CD8 T cell entry into tumors.
Manipulation of IL-2 signals by IL-2/antibody complex and CD25 blockade improves tumor immunity, reprograms regulatory T cells, and augments CD8+ central memory in an ovarian cancer model
Authors: Drerup Justin Michael , Padron Alvaro Souto , Chen Wanjiao , Clark Curtis Anthony , Curiel Tyler Jay 
Published: January 2023

Abstract
We used a defined IL-2/αIL-2 complex that directs IL-2 to stimulate only intermediate-affinity IL-2 receptor (CD122/132) to promote tumor rejection and avoid regulatory T cell (Treg) expansion. However, it is unknown if IL-2/αIL-2 alters Treg function, as Tregs also express CD122/132. After challenge with ID8agg ovarian cancer (OC) cells, IL-2/αIL-2 greatly reduced tumor burden, and increased IFN-γ, TNF-α, CD44, and CD25 in CD4+ non-Tregs and CD8+ T cells, as expected. IL-2/αIL-2 was clinically effective, yet it lowered the ascites CD8+/FoxP3+ Treg ratio and increased per cell FoxP3 levels in Tregs, suggesting a change in Treg function. IL-2/αIL-2 decreased the ratio of CD62L+ central Tregs to CD44+ effector Tregs in ID8agg ascites, indicating modulation of Treg differentiation. Surprisingly, ascites Tregs produced IFN-γ, IL-2, and TNF-α in control-treated OC mice, which was abolished by IL-2/αIL-2, suggesting further alteration of Treg differentiation and function. CD25 (high-affinity IL-2 receptor subunit) expression on Tregs was reduced by IL-2/αIL-2, which could lower Treg IL-2 sequestration and increase IL-2 available to antitumor T cells. CD25 blockade with αCD25 during IL-2/αIL-2 treatment reduced clinical efficacy but greatly increased central memory (CM; CD62L+CD44+) CD8+ T cells in blood and mesenteric (tumor-draining) lymph nodes. Thus, reduction of tumor burden by IL-2/αIL-2 is associated with reprogramming of Treg differentiation and function in an OC model. CD25 blockade after IL-2/αIL-2 could improve response durability by boosting CM CD8+ formation. These data demonstrate novel effects of IL-2-modulating drugs on Tregs and CD8+ memory and will aid development of more effective anticancer immunotherapy.
Impact of hypoxia on IFNγ-dependent responses: implications for radiotherapy and anti-tumor immunity
Authors: Murthy Aditi , Gerber Scott A , Koch Cameron J , Lord Edith M 
Published: January 2023

Abstract
Tumor hypoxia occurs due to an increase in demand for oxygen by the rapidly growing tumor cells together with reduction in the supply of oxygen due to malformed and non-functional tumor vasculature. Tumor hypoxia offers resistance to radiotherapy (RT). Interestingly, a new paradigm has emerged suggesting that hypoxia may also suppress immunotherapy, however the mechanisms behind this observation remain undetermined. Our laboratory and others have shown that IFNγ conditions the tumor milieu and is important for the efficacy of RT. As a result, we hypothesized that hypoxia could inhibit IFNγ mediated anti-tumor responses resulting in decreased RT efficacy. Hypoxia could be modulating the production of intratumoral IFNγ, and/or reducing the ability of cells to respond to IFNγ. To test this we utilized Colon38, a murine tumor model, and measured intratumoral hypoxia using the hypoxia marker EF5 and demonstrated a time dependent increase of hypoxia within untreated tumors. Irradiation resulted in a decrease in total tumor hypoxia. We also detected various sub-populations of hypoxic immune cells in both the untreated and irradiated tumors, with macrophages being the majority population. Importantly, CD8+ T cells, which are an important source of IFNγ and mediate effector anti-tumor responses, were present in these tumors but were not co-localized with hypoxic regions. This deficiency of effector T cells in the hypoxic regions indicates that tumor cells in these areas may also escape from immunotherapy. To alleviate this, we are examining if local IL-12 delivery, which normalizes tumor vasculature, can decrease hypoxia thus increasing IFNγ levels and responsiveness and ultimately enhancing tumor control. Funded by NIH R01 CA28332.
Dicer regulates tumor immunogenicity in melanoma
Authors: Hoffend Nicholas C , Magner William J , Tomasi Thomas B 
Published: January 2023

Abstract
Immunotherapeutic strategies in cancer rely upon tumor immunogenicity for their efficacy. However, the regulation of immune effector molecules in melanoma is poorly understood. High Dicer expression in melanoma correlates with more aggressive tumors and poor prognosis/patient survival. Dicer regulates gene expression through processing of microRNAs, but its potential role in regulating the immunogenicity of melanoma has not been described. Here, we demonstrate that Dicer suppresses MHC class I and II cell surface expression, as well as CD40 and PD-L1 in melanoma. By using a genetic approach wherein we challenged C57BL/6 mice with melanoma cells stably expressing Dicer shRNA, we showed that Dicer knockdown decreased tumor progression and extended overall survival. Interestingly, Dicer knockdown had no effect on melanoma cell proliferation or survival in vitro, suggesting that the delay in tumor growth may be due to an augmented immune response. Tumor control of Dicer knockdown melanoma cells was dependent upon an intact immune system, as revealed by studies using NSG mice. Mechanistically, this anti-tumor immune response was dependent upon CD8+ T cells. Subsequent studies demonstrated that melanoma Dicer expression was able to suppress CD8+ T cell killing in vitro. Taken together, this suggests a model wherein Dicer negatively regulates melanoma immunogenicity and protects against CD8+ T cell anti-tumor immunity. Using this insight, we may better stratify patients for immunotherapies such as checkpoint inhibition and cell-based vaccination strategies. Dicer also makes an attractive target for modulating baseline tumor cell immunogenicity, subsequently enhancing melanoma immunotherapy and patient survival.
Development of a Novel Preclinical Model Based on HLA-A2 Transgenic Mice to Evaluate T-Cell Receptor Mimic Monoclonal Antibodies as Therapeutic Agent against Brain Metastases of Human Breast Cancer
Authors: Park Jee Hyun , Yang Li , Alqahtani Faleh , Bickel Ulrich 
Published: January 2023

Abstract
The HLA-A2 restricted T-cell receptor mimic (TCRm) antibody RL6A has been shown to have antitumor effects in a breast cancer orthotopic xenograft model with MDA-MB-231 cells. Transgenic mice expressing HLA-A2 also take up RL6A into brain across the blood-brain barrier (BBB). The present study evaluates different approaches to obtain a reliable brain tumor model using HLA-A2 transgenic mice, in which pharmacokinetics and antitumor efficacy of RL6A on brain metastasis could be studied. First, available immunocompromised HLA-A2 transgenic strains, such as the HLA-A2 transgenic NSG mouse (Jackson Labs), were utilized and various tumor injection methods were tested. As NSG mice are the most immunocompromised and vulnerable strains, intracardiac injection of brain-seeking human breast cancer cells invariably resulted in tumors of multiple peripheral organs even when using a brain seeking subclone of MDA-MB-231. More targeted approaches (common carotid artery or retrograde external carotid injections) reduced the tumor burden in peripheral organs intracardiac injection, but turned out being too invasive procedures for this brittle mouse strain. Stereotaxic intrastriatal injection in NSG mice resulted in tumor growth at the implantation site within brain, but also in skull bone and dura. Therefore, the NSG background appeared unsuitable for our purpose. We then evaluated intracardiac injections of tumor cells in a Foxn1nu nude mouse strain on a C57BL/6 background, which developed predominantly brain tumors and no tumors in major peripheral organs (lung, liver, kidney). Cross-breeding with C57BL/6 transgenic HLA-A2 mice was initiated and yielded a novel HLA-A2 transgenic nude mouse strain.
Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for CAR T cell immunotherapy.
Authors: Lomueller Jason Jakob , Sato Shuji , Popova Lana , Chu Isabel M , Tucker Meghan A , Barberena Roberto , Innocenti Gregory M , Cudic Mare , Ham James D , Cheung Wan Cheung , Polakiewicz Roberto D , Finn Olivera J 
Published: January 2023

Abstract
Hypoglycosylated MUC1 is a tumor-associated protein that is expressed on over 80% of all human cancers including adenocarcinomas of the colon, pancreas, breast, lung, prostate, and ovary, and multiple myeloma. In our recent clinical trial, individuals at-risk for colon adenocarcinoma received a prophylactic MUC1 cancer vaccine. Many individuals responded producing high titers of anti-MUC1 IgG antibodies with no detectable toxicity. This trial provided a rare source of human antibodies elicited and affinity-matured in a healthy human host to abnormal MUC1. Using our recently developed proteomics method, we isolated and identified 13 anti-MUC1 antibodies representing 7 different clonotypes. These antibodies bind to several different epitopes on the MUC1 vaccine peptide with a range of affinities (15.7μM to 130pM). They also stain MUC1 on human cancer cell lines and colon, breast, lung, and pancreas adenocarcinoma tissue sections while showing no reactivity against a large panel of normal tissues that express MUC1. We constructed lentiviral vectors encoding chimeric antigen receptors (CARs) using scFv’s of several of the antibodies as antigen binding regions and a variety of co-signaling domain architectures. Several of the scFv’s were able to retarget human primary T cells to become activated and produce cytokines in a MUC1-dependent manner and to lyse a variety of MUC1+ human tumor cell lines. Preclinical testing in mouse tumor xenograftgrafts is underway. Being of fully human origin and showing a high-degree of tumor specificity and efficacy in preclinical experiments, these antibodies will be tested in future clinical trials for potential approval for therapy of cancer patients.