bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbiota-derived butyrate negatively regulates RORγt+ ILC3 in Peyer’s patch of the terminal ileum
Authors: Jang Yong-Suk , Kim Sae-Hae , Kim Ju , Kim Yu Na
Published: January 2023

In the intestinal immune system, anatomical and physiological distinctions in the gut contribute to its regional specialization by microbiota and immunomodulatory agents from the diet. Given that the ileal Peyer’s patch (PP) belongs anatomically to the small intestine, while it is physiologically exposed to an environment similar to the large intestine with respect to microbes and microbial metabolites, its characteristics may differ from those in PPs in the jejunum. As the terminal ileal PP is a key mucosal organ where host defense and oral immune tolerance in the gut develop, identification of the specific factors regionally specialized in the tissue is essential to understand mucosal homeostasis. Among the various cell types present in PPs, group 3 innate lymphoid cells (ILC3s) are closely associated with the regulation of commensal bacteria through the suppression of commensal bacteria-specific CD4+ T cells, although the regulation of ILCs in ileal PPs is poorly defined. In this study, we found that butyrate plays a role as a regional specific factor involved in the repression of ILC3s in PPs of the terminal ileum. This butyrate-mediated negative regulation of ILC3s alleviates the tolerogenic mucosal microenvironment by suppressing regulatory T cells in PPs. Collectively, we conclude that the inhibition of ILC3s by microbiota-derived butyrate can confer the functional ability to induce antigen-specific immunity, and that this network contributes to homeostatic regulation of the mucosal immune inductive site.
Discovery of a novel subset of lung resident macrophages that regulate pathogen clearance and immune homeostasis after respiratory infection
Authors: Cicek Basak B , Khanna Kamal Mohan
Published: January 2023

The importance of tissue resident macrophages (TRMϕ) for pathogen clearance and homeostasis is beginning to emerge. However, our understanding of the multifaceted roles these macrophages play in mucosal tissues such as the lung remains incomplete. Currently the major population of TRMϕs in the lung is known to be alveolar macrophages (AMϕ) that express CD11c and CD169. Here we report a novel subset of CD169+ lung resident macrophages that are phenotypically and developmentally distinct from the alveolar or interstitial macrophages. These novel TRMϕ were CD169+CD11C−CX3CR1+Ly6C−CSF−1R+. A remarkable quality of these TRMϕs was their geographical location, which was important for their function. They were exclusively localized around the airways of the lung close to the airway epithelial cells. These airway-associated macrophages (AAMϕ) were radio-resistant, self-renewing and failed to recirculate. To determine the function of lung TRMϕs, we used genetic mouse models and bone marrow chimeric mice to exclusively deplete AMϕ or AAMϕ following influenza infection. We show that AAMϕ cell numbers dramatically increased during infection due to local proliferation and differentiation of infiltrating monocytes. Depletion of CD169+ Mϕ in mice during influenza infection resulted in elevated morbidity. In the absence of CD169+ Mϕ, we observed dramatic changes in the recruitment of innate immune cells such as neutrophils and monocytes in the lungs following infection. Overall, our study relates structure to function at a fundamental level and provides essential insights into a novel subset of AAMϕ in the lung that along with alveolar Mϕ are crucial in mediating protection and maintaining immune homeostasis during influenza infection.
The impact of obesity onset on intraepithelial lymphocyte homeostasis at different ages.
Authors: Jameson Julie M , Cheung Kitty , Limon Natalie , Yee Christine , Costanzo Anne , Park Christa S
Published: January 2023

According to the Centers for Disease Control and Prevention, one in six children and adolescents in the United States are obese. The obesity epidemic is caused by chronic excess energy intake and low energy expenditure. This energy imbalance creates excess adipose tissue that produces systemic inflammation in the body. Obesity is detrimental to barrier tissues that protect the body from foreign pathogens and environmental stressors. This metabolic disease disrupts normal homeostasis within the epithelium, which then results in complications that include more severe inflammatory bowel disease. We have identified that as obesity progresses, T cell numbers are reduced within the epithelial layer of the intestine and proliferation of these immune cells in the tissue becomes dysfunctional. Interestingly enough, we show that these same T cells are partially restored in number with a low-fat diet. We examined the relationship between obesity onset and the ability of intraepithelial lymphocytes to seed and proliferate in the intestine to maintain homeostasis in the barrier tissue. Here we demonstrate that obesity is particularly devastating in young mice that are still establishing lymphocyte populations in their epithelia. In addition, the long-term effects of chronic obesity from childhood to adulthood shows a dramatic reduction in the number of intraepithelial lymphocytes in the intestine. This study helps shed light on how epithelial barrier maintenance differs in early versus late onset of obesity and will direct research to explore alternative approaches to restore intestinal intraepithelial T cells and promote optimal barrier integrity in obese patients.
Continuous, antigen independent re-seeding of lung resident memory CD8+ T-cells after Influenza A virus infection.
Authors: Slutter Bram , Budimir Natalija , Varga Steven M , Harty John T
Published: January 2023

Resident memory CD8+ T-cells (Trm) are a subset of memory T-cells, defined by their permanent localization in non-lymphoid tissues and are associated with potent protection against local infections. Lung Trm can mediate rapid control of respiratory pathogens like influenza A Virus (IAV), but in contrast to other non-lymphoid tissues like the skin, the number of lung Trm is not maintained long term. Here we observed through intranasal CFSE labeling that CD69+/CD103+ memory CD8 T-cell reside in the lung parenchyma longer then their CD69−/CD103− counterparts, but the average residence time of a Trm cell in the lung parenchyma is less than a week. We show that, in contrast to skin, Trm in the lung are prone to apoptosis and require continuous recruitment of memory CD8 T-cells from the circulation to maintain their numbers. Of note, the lung microenvironment drives the continuous conversion of circulating memory CD8+ T-cells into Trm, but does not require the presence of antigen in the lung. Moreover, intrinsic changes to the circulating memory CD8 T-cell population over time decrease their capacity to adopt a Trm phenotype. Thus our data could provide an explanation as to why lung Trm are not maintained long term.
Validating impedance-based methodology for in vitro dynamic analysis of reagents and effector cells efficacy in cancer immunotherapy protocols
Authors: Cerignoli Fabio , Guenther Garret , Xi Biao , Zhu Jenny , Zhao Leyna , Radecke Chris , Abassi Yama
Published: January 2023

In vitro characterization of the efficacy of reagents for cancer immunotherapy is a necessary step before moving to more expensive animal models and clinical studies. However, current in vitro assays are difficult to implement in high throughput environment and are mainly based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we present the adaptation of an impedance-based platform to monitoring cytotoxic activity of immune cells activated trough different means. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent cell lines and primary tumor cells with different expression levels of the EpCAM antigen, which is currently under evaluation as a target for cancer immunotherapy. Using PBMCs and NK92 as effector cells and combinations of EpCAM/CD3 BiTE and anti-EpCAM antibodies, we demonstrated the suitability of the impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. We also validated the same approach with suspension cells immobilized on the surface of the electrodes. Overall, our results demonstrate the value of such approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays. Furthermore, the availability of 384-wells format and minimal sample handling place the technology in an ideal spot for applications in personalized medicine, like therapeutic protocol validation directly on patient samples.
Flow Cytometry Multiplexing as a High Throughput Screening Method to Analyze Metabolic Activity for Pharmaceutical Drugs
Authors: Low Quentin , Anderson April , Calderon Veronica , Clark Scott , DeMarco Carolyn , Finnessy Carmen , Kihn Kathy , O’Grady Michael , Wickett Marcy , Yan Michelle
Published: January 2023

High throughput screening (HTS) is an extremely effective method that allows researchers to identify putative active compounds for therapeutics. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry is a fast and sensitive platform aiding in the study of protein-protein interactions, metabolic activities, as well as DNA content in a single or multi-parametric assay format. For this study, we screened several compound libraries and compared the results using a plate reader with flow cytometer multiplexing.

Peripheral blood mononuclear (PBMCs) and Jurkat cells were used as cell models. Cells were cultured under standard conditions and either at normoxic (≤5%) or hyperoxic (≥19%) oxygen levels for varied lengths of time; 24, 48, or 72 hrs. Membrane integrity and metabolic activity were measured as an output for evaluating cellular viability. To further assess cellular activity, post-screening analysis was implemented to establish a drug dose-response and EC50s of “hits” from compound library.

Screening analysis identified target drugs that were taken on to further tests using different mechanisms of action to assess compound toxicity. Results indicate that compound “hits” and potency differed in the screen depending on cell type, the mechanistic readout, length of compound exposure, and mode of readout used to perform the HTS experiments.
Early diagnostics of septicemia using microRNA flow cytometry
Authors: Stein Erica Vanessa , He Hua-Jun , Cole Kenneth D , Wang Lili
Published: January 2023

From 2000 to 2008 the incidence of sepsis in hospitals increased by 83%, but we still lack effective diagnostic methods. Recent advances in probe chemistry have enabled scientists to better detect RNA using flow cytometry. This technique could potentially be used for acute and efficient detection of sepsis. However, it still requires substantial validation before it reaches full potential in the clinic.

We are in the process of creating a robust microRNA flow cytometry assay that can be used in the clinic for early detection of sepsis. In order to do this, first a reference control and diagnostic microRNA biomarkers, specific for detection of septicemia, must be identified and validated. Next, a microRNA flow cytometry assay will be developed and validated with the use of the reference control and diagnostic microRNAs both intracellularly and in serum. Lastly, the developed flow cytometry assay, including controls and diagnostic biomarkers will be verified against blood samples of clinical patients both positive and negative for sepsis.

An effective sepsis diagnostic will save lives, aid in drug discovery, and decrease health care costs. This project provides a unique opportunity to create a robust flow cytometry assay to measure expression differences in microRNAs to detect septicemia in the clinic. We are hopeful that our continued efforts to validate and standardize previously published microRNA detection assays and expand the possible new targets will lead to quicker and more robust methods to survey for sepsis in clinics.
A Framework for Meta-Analysis of Cytometry Data
Authors: Jujjavarapu Chethan , Hughey Jacob , Gherardini Federico , Bruggner Robert , Nolan Garry , Bhattacharya Sanchita , Butte Atul
Published: January 2023

Flow cytometry has been used to analyze cell populations with a certain condition, but because of the limitations of a single flow cytometry experiment, results only present a small portion of cell populations. Meta-analysis has been used to compare and contrast multiple datasets in hopes of discovering new insights. Scientists can then identify more cell populations linked to the condition. Therefore, we are developing a method to standardize and analyze multiple flow cytometry datasets.
89Zr positron emission tomography detects migration of donor cells to the ileum in a mouse bone marrow transplantation model.
Authors: Sato Noriko , Asiedu Kingsley O. , Choyke Peter L.
Published: January 2023

In hematopoietic stem cell (HSC) transplantation, the efficacy of bone marrow homing and engraftment of HSCs directly correlates with the success of treatment. At the same time, reduction of graft-versus-host-disease (GVHD) incidence would be critical. Development of a non-invasive, real time monitoring system for the donor cells would be important for optimizing the HSC transplantation conditions. Recently, we have developed a 89Zr-oxine complex cell labeling technique that enables visualization of cell trafficking at a high sensitivity using positron emission tomography (PET)/computed tomography (CT) imaging. We employed this technique to monitor bone marrow transplantations in mice. PET/CT imaging visualized that 89Zr-labeled donor bone marrow cells rapidly migrated to the bone marrow of the recipient, as well as to the spleen and liver. More than 20% of the donor cells homed to the bone marrow within 4 hours, irrespective of prior bone marrow ablation. While monitoring the donor cell homing, we have found that a small fraction of cells migrating to the intestine. This was observed only when the recipient mice had undergone a conditioning by a whole-body irradiation and the cells targeted the distal part of the small intestine. The most of the cells expressed CD45 and CD103. These cells could be the cause of GVHD in case of allogeneic transplantation. 89Zr PET/CT would be useful to monitor donor cell trafficking in HSC transplantations.
Novel nanofibrous DNA-histone structures mediate reproducible, high-throughput analyses of neutrophil extracellular traps and their effects in vitro
Authors: Louttit Cameron , Weerappuli Priyan , Kojima Taisuke , Maeda Midori , Takayama Shuichi , Moon James J.
Published: January 2023

Termed the “double-edged swords of innate immunity” by Kaplan and Radic, neutrophil extracellular traps (NETs) are vital contributors to innate defenses but also participate in the pathogeneses of manifold diseases including systemic lupus erythematosus, rheumatoid arthritis, thrombosis, and cancer. Despite their vast clinical impact, NETs have proven difficult to study in high quantity and isolation; indeed, over 1.9E9 neutrophils are required for full NET coverage of a 96-well plate. Our group has developed a novel NET-mimicking structure for adaptable and reproducible high-throughput analyses of NETs in vitro. Rooted in a base composition of DNA and histones interacted by slow diffusion across an aqueous two phase system, these structures exhibit the same 10–100 nm diameter fibers as their physiological counterparts and can incorporate additional NET-associated proteins and peptides. This platform therefore enables unprecedented high-throughput probes of the roles of individual NET components in health and disease. In a pilot study exploiting this opportunity, our group expanded upon the previously-reported protection of NETs from nuclease-mediated degradation by the peptide LL-37. The comparison between LL-37-containing structures and control structures without the peptide, a data point unavailable endogenously, revealed a 4.5-fold increase in structural half-life conferred by LL-37 after exposure to DNase I. Similar ongoing experiments are exploring the lupus-linked activation of plasmacytoid dendritic cells by NETs. In this way, the reported platform provides new investigative angles for NET studies while also conferring a reproducibility and statistical power unattainable with cell-based methods.
A direct, bioluminescent caspase-1 assay for monitoring inflammasome activation
Authors: O’Brien Martha A. , Moehring Danielle , Munoz-Planillo Raul , Nunez Gabriel , Callaway Justin , Ting Jenny P , Scurria Mike , Ugo Tim , Bernad Laurent , Cali James , Lazar Dan
Published: January 2023

Caspase-1, an essential component of the inflammasome, is activated in response to inflammatory stimuli, resulting in 1) the processing and release of IL-1b and IL-18, and 2) pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent added directly to cultured cells. The assay can also measure caspase-1 activity released into the medium. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG-132, in the reagent and by use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. Using this assay system, caspase-1 activity has been monitored in THP-1 cells, J774A.1 mouse macrophages, mouse bone marrow-derived macrophages (BMDMs), and human primary monocytes treated with known inflammasome inducers. Caspase-1 activity was not detected in BMDMs derived from Casp 1−/−mice. Measuring caspase-1 activity released into medium enables multiplexing of the sample to monitor additional cell parameters. Multiplexing to monitor caspase-1 activity, cell death, and IL-1b release revealed that unprimed, undifferentiated THP-1 cells stimulated with nigericin or a-hemolysin activate caspase-1 and undergo pyroptosis, but do not release any IL-1b. This suggests that K+ efflux alone can engage the inflammasome and activate caspase-1 in THP-1 cells, providing an interesting system to assess pyroptosis in the absence of IL-1b. This convenient caspase-1 assay will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.
Real-time imaging of non-adherent cells maintained in contact with adherent cell layers
Authors: Yeh Victor , Chen Cindy , Hung Paul
Published: January 2023

Cell-cell interactions are crucial to the survival and function of cells. Imaging these interactions in real-time is a challenging task with non-adherent cells due to their mobility in suspension, not to mention their communication with other heterotypic cells. Here we present a novel method to trap non-adherent cells in precise locations with a layer of adherent cells in order to study and image their interactions while in contact over time. This platform uses a well plate format to integrate 8 individual microfluidic units. Each unit has a 3 mm × 3 mm × 40 μm (L × W × H) chamber for cell culture, and an array of one hundred 72 × 72 × 15 μm (L × W × H) trap areas within each chamber to immobilize non-adherent cells underneath. The chamber in each unit can be addressed with programmable and on-demand perturbation of up to 2 different reagents to perform live cell imaging assays without disrupting the cell cultures. We have successfully cultured MCF-7 cells using this design, with Jurkat cells subsequently loaded and trapped above the layer of MCF-7. Upon reagent introduction, time-lapse images confirmed that the Jurkat cells remained in place. In conclusion, the microfluidic design allows investigation of cell-cell interactions in a wide range of applications requiring the confinement of non-adherent cells in contact with a different adherent cell layer. The on-demand perturbation feature further increases the capability to control the microenvironment exposed to the cell culture.
Increasing the palette of fluorophores for intravital 2P-imaging of the vascular and immune system in mice
Authors: Saunders Brian T , Li Wenjun , Williams Jesse W. , Kim Ki-Wook , Randolph Gwendalyn J. , Zinselmeyer Bernd H.
Published: January 2023

Disruption in the movement of blood or lymph leads to potentially fatal diseases, most commonly atherosclerosis in the blood and lymphedema/lymphangitis in the lymph. There have been an enormous amount of studies done on blood vasculature in the past few decades, but our understanding of lymphatics in health and disease remains poor. Lymphatics interact systemically and locally with the blood vasculature and we have recently proven that dendritic cells are an important regulator in lymphatic function. Some of these processes can only be proven in a living animal as the interactions and impact of immune cells on vascular flow dynamics and vice versa cannot been mimicked in vitro. Our most recent intravital imaging studies allow the visualization of resident macrophage dynamics within a plaque. Due to the fast movement of cells in the vasculature and the sensitivity of cells to phototoxicity it was challenging to generate a palette of dyes which are low in cytotoxicity able to label the structures and cell populations of interest. It is possible by using fluorophores (Fluorescent proteins and conventional dyes) efficient enough to be excited with low laser power and an emission sufficient enough for being detected. Recently, a femtosecond pulsed Titanium Sapphire (Ti:sap) laser with averaged power ≥ 1W above 1200 nm became available. We have coupled a laser of this type (InSight DS+) together with an conventional Ti:sap-laser (Mai Tai DeepSee) to an customized ultra fast and sensitive scanning Microscope. Using these settings we can image up to five fluorophores in intravital settings. This approach enables us to visualize lymphatics and blood vessels together with myeloid cells and lymphocytes in vivo.
IDO1-targeting intervention by proteasome inhibition in autoimmune diabetes
Authors: Orabona Ciriana , Albini Elisa , Mondanelli Giada , Volpi Claudia , Pallotta Maria Teresa , Puccetti Paolo , Grohmann Ursula
Published: January 2023

Indoleamine 2,3-dioxygenase 1 (IDO1) is a metabolic enzyme involved in the catabolism of tryptophan. Its metabolic activity has become pivotal in the regulation of the immune responses. The functional expression of IDO1 enzyme confers a tolerogenic phenotype to different subsets of dendritic cells (DCs). Our previous data suggested a defective IDO1 expression in a prediabetic phase of nonobese diabetic (NOD) mice, a prototypic model of human type 1 diabetes. Based on the recent finding that IDO1 may be subjected to regulatory proteolysis, which is triggered by IL-6 and mediated by the immunoproteasome, this pathway was explored in splenic plasmacytoid dendritic cells (pDCs) of NOD mice. The in vitro conditioning of murine NOD pDCs with the proteasome inhibitor Bortezomib, increased the functional expression of IDO1 enzyme, conferring those cells an immunoregulatory phenotype that is strongly inhibited for presenting in vivo the diabetogenic antigen IGRP, an effect accompanied by increased IGRP-specific regulatory T cells in pancreatic lymph nodes. The inhibition of IDO1 by 1-methyl tryptophan (MT) completely abolished the immunoregulatory effect of Bortezomib in NOD pDCs, indicating the involvement of the enzyme. Moreover, the pharmacologic treatment of NOD female mice with Bortezomib during prediabetic phase (8–10 weeks old) prevented the onset of hyperglycemia, reducing the diabetes incidence. Overall, our data suggest that the control of proteasomal degradation in pDCs may represent an innovative strategy for inducing/potentiating IDO1-mediated immunoregulation in autoimmune diabetes.
Reinstalling immune regulatory tryptophan catabolism in juvenile diabetes via interleukin 6 receptor blockade
Authors: Mondanelli Giada , Orabona Ciriana , Pallotta Maria Teresa , Albini Elisa , Volpi Claudia , Grohmann Ursula
Published: January 2023

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunoregulatory enzyme that catalyses the degradation of the essential amino acid tryptophan (Trp) along the kynurenines pathway. Significant changes in systemic Trp catabolism have been reported in many diseases, including cancer and autoimmunity. In female nonobese (NOD) mice, IDO1 expression and hence immune tolerance to pancreatic b-cell autoantigens are defective in conventional dendritic cells stimulated with IFN-γ, the main IDO1 inducer. Although the evidences in NOD mice suggest that IDO1 function is impaired, the existence of the IDO1 defect in human T1D (type 1 diabetes) has not been proven yet. Here we monitored the IDO1 expression and activity in peripheral blood mononuclear cells (PBMCs) of children with T1D as compare to age-matched control subject, in response to IFN- γ. Results from kynurenines assay and Western blot analysis demonstrate that the majority of patients with T1D is characterized by defective Trp catabolism. Moreover, our data indicated that this defect is mainly imputable to a SOCS3-mediated, dysregulated IL-6 signaling that would favor IDO1 proteasomal degradation in inflammatory environments, i.e. dominated by IFN-γ. To confirm this, we measured IDO1 expression and activity in PBMCs co-incubated with IFN-γ and Tocilizumab (TCZ), a licensed IL-6 receptor blocker. Results showed that TCZ is able to restore normal levels of IDO1 catalytic activity in response to IFN-γ in approximately 30% of the examined T1D population. Besides further confirming the heterogeneity of the disease, our data indicate the existence of a subset of individuals with T1D who may gain clinical benefit in restoring immunoregulatory mechanisms by treatment with tocilizumab.
Dendritic cell-CD8+ T cell communication is essential for Kinsenoside, a potential immunosuppression drug for autoimmune hepatitis
Authors: Liu Tingting , Xiang Ming , Tan Wanyue , Liu Junjun , Li Hua , Liu Xiulan , Cao Hui , Zhu Hucheng , Tuo Yali , Zhang Yonghui
Published: January 2023

Autoimmune hepatitis (AIH) is a chronic auto-immune inflammation against liver tissue. Here, using T cell-deficient mice, acute AIH mice model and a novel AIH mice model induced by dendritic cells loaded with murine hepatocellular carcinoma cells (DC/Hepa1–6) vaccination, we reveal that Kinsenoside (KD), a major component of Anoectochilus roxburghii, has an powerful therapeutic effect on AIH by limiting the metabolism of immune cells such as DCs and CD8+ T cells, which results in the immunosuppression against autoimmune liver injury. The immunosuppressive and anti-inflammatory effect of KD is triggered by increased the expression of co-inhibitory molecules PD-L1/PD-1 to inhibit DC cross-priming of CD8+ T cell responses but not modulate the hepatotoxicity of CD8+ T cells directly. Furthermore, KD reduces immune cells metabolism and executes immunosuppressive and anti-inflammatory action by binding to the potential target VEGFR2 and blocking the cross-talk of metabolism related PI3K-AKT and inflammation related JAK2-STAT3 signaling pathways.
Programmed cell death-ligand 1 ameliorates psoriatic inflammation by inhibiting TCR-induced IL-17A production of T cells overexpressing PD-1
Authors: Kim Jong Hoon , Choi Young Joon , Lee Byung Ha , Song Mi-Young , Ban Chae Yeon , Kim Jihye , Park Junsik , Kim Song-Ee , Kim Tae-Gyun , Park Su-Hyung , Kim Hyoung-Pyo , Sung Young-Chul , Kim Soo-Chan , Shin Eui-Cheol
Published: January 2023

Psoriasis is one of the most common chronic inflammatory diseases in the skin. Recently, IL-17-producing T cells have been shown to play a critical role in psoriatic inflammation. Programmed cell death-1 (PD-1) is a co-inhibitory receptor expressed on T cells in various chronic inflammatory diseases; however, it remains to be elucidated whether PD-1 is overexpressed on T cells in psoriasis and whether PD-1 agonist alleviates psoriatic inflammation. We examined PD-1 expression on IL-17A-producing T cells from Imiquimod (IMQ)-treated mice and psoriasis patients. Additionally, we investigated the therapeutic effect of recombinant programmed cell death ligand-1-Fc (PD-L1-Fc) protein on IMQ-induced psoriatic inflammation. During IMQ-induced psoriatic inflammation, PD-1 is overexpressed on CD27−Vγ1− γδ T cells. Further, PD-1 expression on IL-17A+ T cells was confirmed in tissue samples from patients with psoriatic skin. In the CD27−Vγ1−γδ T cell population, Vγ4−γδ T cells with Vγ6 mRNA expression showed a high level of PD-1 expression. Further, these PD-1hiVγ4−(Vγ6+) γδ T cells were specialized for T-cell receptor (TCR)-induced IL-17A production, which was inhibited by PD-L1-Fc protein treatment. In IMQ-treated mice, PD-L1-Fc protein reduced psoriatic inflammation when given alone and enhanced the therapeutic effect of anti-p40 when given in combination. In conclusion, PD-1 is overexpressed in IL-17A-producing T cells in both IMQ-treated mice and psoriasis patients, and recombinant PD-L1-Fc protein alleviates psoriatic inflammation in IMQ-treated mice.
Constitutive expression of LAG-3 defines the exhaustion of intratumoral PD-1+ T cells in B-cell non-Hodgkin lymphoma
Authors: Yang Zhi-Zhang , Kim Hyojin , Price-Troska Tammy , Novak Anne , Ansell Stephen
Published: January 2023

We have previously shown that not all PD-1+ cells are exhausted and that PD-1 is differentially expressed on two distinct T-cell subpopulations, with high expression on T follicular helper cells and dim expression on exhausted T cells. We observed that while PD-1bright T cells lacked the ability to produce cytokines and granules, PD-1dim T cells from biopsy specimens of B-cell NHL were the major source of cytokine and granule production compared to PD-1− T cells, suggesting that PD-1dim are functionally active. Therefore, other markers are needed to more clearly identify exhausted intratumoral T cells. We first phenotyped intratumoral PD-1+ (brightly- and dimly-stained) T cells and found that LAG-3 was exclusively expressed on dimly-stained PD-1+ T cells, forming a defined population of intratumoral PD-1+LAG-3+ T cells. We found that LAG-3 expression was strongly upregulated on CD4+ or CD8+ T cells by IL-12, a cytokine that has been shown to induce T-cell exhaustion. Functionally, with a substantial numbers of PD-1dim T cells being cytokine- or granule-producing cells, it is PD-1dimLAG-3−, but not PD-1dimLAG-3+, T cells that were responsible for the capacity of producing cytokines and granules. Supporting this finding, we found that blockade of both PD-1 and LAG-3 signaling by using the combination of PD-1 and LAG-3 blocking antibodies increased cytokine production by T cells significantly more than PD-1 or LAG-3 antibody alone, suggesting that LAG-3 is important in the impairment of PD-1+ T cells. Taken together, these results suggest that constitutive expression of LAG-3 was critically involved in and defines the exhaustion of PD-1+ T cells in B-cell NHL.
Tumor B7-H1 regulates cancer stem cell generation and virulence
Authors: Gupta Harshita B. , Hurez Vincent , Clark Curtis A , Sareddy Gangadhara R , Vadlamudi Ratna , Li Rong , Curiel Tyler J
Published: January 2023

Cancer stem cells (CSC) resist chemotherapy and radiation, leading to treatment failures and reduced patient survival. Thus, CSC are clinically relevant therapy targets. B7-H1 is an immune checkpoint molecule and a target for αB7-H1 immunotherapy. We generated control (native B7-H1+) and B7-H1lo B16 melanoma lines by shRNA. B16*ctrl cultures were 4.2% CSCs (CD44+CD133+CD24+) vs. 0.19% in B16*B7-H1lo. CSC from B16*B7-H1lo cultures formed fewer and smaller (<50 μm) microspheres vs. many, large (100–150 μm) microspheres from control CSC. B16*ctrl CSC were more tumorigenic in mice than B7-H1lo CSC. B7-H1 expression was 2-fold higher on CSC vs. non-CSC. αB7-H1 reduced CSC numbers in B16*B7-H1lo cultures (~4-fold) but not control cultures. PD-1 expression was 2-fold higher in B16*B7-H1lo non-CSC vs B16*ctrl non-CSC. PD-1 was 5-fold higher in B7-H1lo CSC vs. B7-H1lo non-CSC, but equal in B16*ctrl CSC vs. non-CSC, suggesting PD-1 regulation by B7-H1. αPD-1 reduced CSC only in B7-H1*ctrl cultures (18.9% to 2.4%). 0.1 ng/ml IFN-γ for 48 hours reduced total cell counts in control vs. B7-H1lo cultures (by 55% and 65%, respectively). Strikingly, IFN-γ increased B7-H1lo CSC numbers 9.5-fold vs. 5.6-fold for control CSC. In parallel work, we tested ID8agg ovarian cancer cells. CSC (CD24+CD44+) were 12.2% vs. 2.2% in ID8*ctrl vs. ID8agg*B7-H1lo culture. B7-H1 and PD-1 expression was similar to B16. RNA-seq of total ID8agg cells showed significantly lower expression of CSC associated genes c-Kit, Lin28A and CD24 in B7-H1lo cells. Classic CSC genes were too low to read. B7-H1 is a candidate CSC regulator. It affects CSC PD-1 expression and alters αB7-H1, αPD-1 and IFN-γ effects differentially in CSC vs non-CSC suggesting novel pathogenic mechanisms.
FATS deficiency in mice limits melanoma progression via converting the tumor immune microenvironment to an anti-tumor state
Authors: Zhang Rongxin , Zhang Kai , Zhang Lijuan , Wang Huafeng , Zhang Qi , Fu Shuyu , Xue Zhenyi , Li Yan , Da Yurong , Yao Zhi
Published: January 2023

C10orf90 is a newly identified common fragile sites gene named fragile-site associated tumor suppressor (FATS) according its tumor suppressive function in FATS overexpressive cancer cell lines. However, our FATS knockout mice data suggested that FATS may service as an important immune regulator.

In this study, we found that FATS deficiency in mice can significantly inhibit the tumor incidence and growth of melanoma implanted with B16 cells. In addition, FATS deficient mice obviously increased the numbers and frequency of tumor suppressor cells CD3+ T cell, γδT and NK cells, and decreased tumor promoter cells PMN-MDSC and Treg cell, in tumor tissues. Notably, DCs which play a key role in immune response initiation were dramatically increased in tumor tissues of the FATS deficiency mice. Furthermore, IL-2 which is crucial to the activation of T cells, significantly increased in serum of the FATS deficiency mice compared with the WT mice, meanwhile, other cytokines, such as IFN-γ and IL-12 were increased as well. Importantly, FATS deficiency increased the expression of IFN-γ in CD8+ T cells and CD4+ T cells and facilitated the activation of these T cells separated from the mice tumor tissues. Also, perforin and granzyme, the pivotal effector molecules in tumor cytotoxicity, raised in the CD8+ T cells from the melanoma tissues of FATS deficiency mice. Consistently, in the ex vitro study, DC separated from FATS deficiency mice with melanoma could significantly promote the proliferation and activation of T cells. These results demonstrate the inhibitory effect of FATS deficiency on the progression of melanoma via regulating tumor immune microenvironment and suggest that FATS could be a potential immune therapeutic target for malignant tumors.
Distinct mechanisms of B and T lymphocyte accumulation generate tumor-draining lymph node hypertrophy
Authors: Habenicht Lauren M , Iritani Brian M , Ruddell Alanna
Published: January 2023

Tumor-draining lymph nodes (TDLNs) enlarge and remodel in many human cancer patients and murine tumor models, featuring extensive lymphocyte accumulation, lymphatic sinus growth (lymphangiogenesis), and increased lymph flow. B cells can drive these lymphatic alterations, however the cellular and molecular mechanisms that direct lymphocyte accumulation in TDLNs are unknown. This study investigated whether lymphocyte accumulation in TDLNs is due to increased entry via high endothelial venules. The popliteal lymph node of C57Bl/6 mice bearing unilateral footpad B16-F10 melanoma tumors exhibits an 8-fold increase in B cells and 3-fold increase in T cells. Comparative measurements of labeled-lymphocyte entry into TDLNs versus the contralateral non-tumor draining lymph nodes by flow cytometry only partially explained TDLN hypertrophy. Further investigation of TDLN lymphocyte exit, proliferation, and apoptosis identified distinct mechanisms regulating B and T cell trafficking through TDLNs in response to tumors, including preferential retention of B cells. Surprisingly, B and T cell accumulation in TDLNs occurred without increased lymphocyte activation, suggesting an ineffective anti-tumor immune response. These insights could provide new therapeutic targets to manipulate lymph node lymphocyte content and immune responses to cancer and other diseases.
The role of CXCR3 in the microenvironment of chronic lymphocytic leukemia
Authors: Rößner Philipp M. , Hanna Bola S. , Zenz Thorsten , Stilgenbauer Stephan , Lichter Peter , Seiffert Martina
Published: January 2023

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy that is stringently associated with a tumor-supportive microenvironment and defective anti-tumor immunity. T cells from CLL patients show features of exhaustion, including expression of PD-1, and are highly impaired in their activity.

Our previous work showed that immune checkpoint blockade using anti-PD-L1 effectively prevents disease and restores T-cell activity in the Eμ-TCL1 mouse model of CLL. Development of disease in these mice was shown to be associated with a relative loss of naïve T cells and an enrichment of effector T cells displaying an exhausted phenotype. Our work showed that T cells in the Eμ-TCL1 mice express high levels of the chemokine receptor CXCR3 accompanied with elevated serum levels of the corresponding chemokines CXCL9 and CXCL10, suggesting a role for CXCR3 in T-cell skewing in CLL. Intracellular flow cytometric analysis showed an enrichment of CXCL9+ CD45+ hematopoietic cells in leukemic spleens which were identified as Ly6C+ monocytes. Along this line, depletion of phagocytic cells by Clodronate Liposomes resulted in significantly decreased serum levels of CXCL9, a reversal of T-cell skewing and slowed down development of CLL in mice. To evaluate the role of this receptor in CLL in vivo, adoptive transfer of murine CLL cells in CXCR3 knockout mice is currently ongoing. We further perform an in-depth phenotypical analysis of T-cell subsets in blood samples of CLL patients from different prognostic subgroups to characterize T-cell aberrations along with disease progression. Taken together, our data suggest a pathomechanistic role for CXCL9, CXCL10 and their receptor CXCR3 in CLL. Their potential as novel drug targets needs to be explored in the future.
DUB-3 regulation of tumor associated inflammation and tumor growth
Authors: Lu Chih-hao , Yeh Da-Wei , Lai Chao-Yang , Liu Yi-Ling , Chuang Tsung-Hsien
Published: January 2023

The tumor microenvironment includes blood vessels, immune cells, fibroblasts, and tumor cells. Inflammation in tumor microenvironment leads to the initiation, promotion, invasion, and metastasis of cancer. Inflammation stimuli such as TNF-α, IL-1β and TLR ligands are capable of activating the NF-κB controlled inflammatory signaling in cancer cells. The activations by these stimuli are modulated by various signaling molecules including those control ubiquitination and deubiquitination in the NF-κB signaling pathway. The DUB-3, has been identified as a deubiquitinating enzyme that belongs to cytokine inducible DUBs. In this study, we investigated the function of DUB-3 in control of inflammatory responses in different cancer cells. Expression of DUB-3 in these cancer cells regulated IL-1β, LPS, and Pam3Cys induced cytokine and chemokine productions and generated favorable conditions for tumor growth. DUB-3 promoted in vivo tumorigenesis and tumor growth. H&E staining of tumor sections revealed higher leukocyte infiltration in the tumors. The expression of genes associated with inflammation were investigated, and higher expression of inflammatory cytokines and chemokines were observed in tumors. Taken together, these observations suggest that DUB-3 regulates tumor growth by modulating inflammatory associated function.
Enterobacteria-mediated the overproduction of intestinal exosomes S1P to drive Th17-mediated colon tumor growth
Authors: Deng Zhongbin , mu jingyao , Zhuang xiaoying , zhang lifeng , Haribabu Bodduluri , Miller Donald , zhang huang-ge
Published: January 2023

The tumorigenic function of exosomes has become widely recognized. However, the potential role of intestinal mucosa-derived exosome-like nanoparticles (IDENs) in the regulation of the intestinal immune system of a colon tumor–bearing host has not been studied. We hypothesize that, along with epithelial cells, intestinal bacteria and immune cells, IDENs in the intestinal mucosa play a crucial role in gut immune regulation. We find that enterobacteria-secreted particles (ET-BSPs)induce the overproduction of exosome-like nanoparticles from gut mucosal cells. These nanoparticles promote colon cancer by influencing the recruitment and proliferation of Th17 cells in the intestine. A key component of these bacterially induced nanoparticles is their ability to induce the production of lipid sphingosine-1-phosphate in mucosal cells. Moreover, the release of sphingosine-1-phosphate causes elevated levels of chemokine (C-C motif) ligand 20 (CCL20) and prostaglandin E2 (PGE2) in the exosome-like nanoparticles. CCL20 and PGE2 are required for the recruitment and proliferation, respectively, of Th17 cells, and these processes also involve the MyD88-mediated pathway. We demonstrate the biological effect of sphingosine-1-phosphate production by exosome-like nanoparticles on tumor growth in APCMin/+ and MC38 tumor-bearing mouse models. These findings provide deeper insights into how host-microbe relationships are mediated by particles secreted from both bacterial and host cells
Chronic Lymphocytic Leukemia-Derived Extracellular Vesicles Mediate NFκB Signaling and Pro-Inflammatory Cytokine Release in Monocytes
Authors: Seiffert Martina , Haderk Franziska , Cid Laura Llao , Göbel Maria , Dürig Jan , Lichter Peter
Published: January 2023

Chronic lymphocytic leukemia (CLL)is a B-cell malignancy associated with an inflammatory milieu and impairedanti-tumor immunity. This study aimed at characterizing CLL-derived extracellularvesicles (EVs) and their role in the tumor microenvironment.

EVs were isolated from blood plasma or culture supernatant of the CLL cell line MEC-1 by a serialcentrifugation protocol. Characterization of EVs by electron microscopy, Nanoparticle Tracking Analysis (NTA) and Western blotting revealed vesicles 30to 350 nm in size, which were positive for various EV marker proteins. Quantificationof EVs by NTA and ELISA indicated an enrichment of B-cell derived EVs in plasmaof CLL patients compared to healthy controls, although absolute EV counts were not different. As we observed an accumulation of small RNAs in EVs, small RNAsequencing was performed which revealed a unique microRNA signature of MEC-1 EVs, with CLL-relevant miRNAs such as miR-21, miR-155 and miR-146a being the mostabundant. Moreover, full length and 5′ end fragment forms of Y RNAs, another class of small non-coding RNAs, were enriched in MEC-1 and CLL plasma EVs. In vitro and in mice, CLL EVs were rapidly taken up by monocytes and macrophages which resulted in NFκB signaling and the release of multiple pro-inflammatory cytokines such as CCL2, CCL3, IL-6 andIL-8, proteins known to be elevated in CLL plasma. Moreover, several surface markers on monocytes were altered upon EV uptake, including an increase of the immune checkpoint molecule PD-L1 as well as downregulation of the chemokine receptorCCR2. Of interest, liposomes containing Y RNA transcripts induced a similar response in respective assays suggesting a novel role for these molecules in the tumor microenvironment of CLL.
Creating a T cell-inflamed tumor microenvironment overcomes resistance to checkpoint blockade
Authors: Tang Haidong , Wang Yang , Chlewicki Lukasz K , Zhang Yuan , Fu Yang-Xin
Published: January 2023

Recent clinical trials with checkpoint blockade antibodies have shown unprecedented durable responses in some patients with a variety of cancers. However, such therapies fail to induce responses in majority of cancer patients. How to increase the objective response rate becomes an urgent challenge. A T cell-inflamed tumor microenvironment is associated with clinical responses to immunotherapies, including checkpoint blockades. Previous studies in our lab have shown that local expression of the tumor necrosis factor superfamily member LIGHT (also known as TNFSF14) by tumor cells may promote the formation of lymphoid-like structures for direct T-cell sequestration and activation, leading to tumor regression. To better evaluate the therapeutic efficacy of LIGHT, we target LIGHT to tumor tissues through an antibody-LIGHT fusion protein. We demonstrated that targeting tumors with LIGHT activates lymphotoxin b receptor signaling, leading to the production of chemokines that recruit massive numbers of T cells. Furthermore, targeting non-T cell-inflamed tumor tissues by antibody-guided LIGHT creates a T cell-inflamed microenvironment and overcomes tumor resistance to checkpoint blockade. Our study indicates that targeting LIGHT may be an effective strategy for cancer immunotherapy, especially those who do not response to checkpoint blockade due to the lack of infiltration.
MEK inhibition by trametinib impairs the mobilization of monocytic MDSCs to facilitate anti-tumor T cell immunity
Authors: Allegrezza Michael , Svoronos Nikolaos , Perales-Puchalt Alfredo , Payne Kyle K , Nguyen Jenny , Conejo-Garcia Jose R
Published: January 2023

The MEK inhibitor trametinib is approved for clinical use in melanoma and being investigated in many other solid tumors, yet the effects of trametinib on non-tumor cells in the cancer microenvironment remain understudied. Although MEK inhibition potently suppresses T cells in vitro, cytotoxic T cell activity is unaffected in trametinib-treated mice. In a preclinical Kras-driven breast cancer model we found that the anti-tumor activity of trametinib requires CD8+ T cells and occurs even in the absence of direct inhibition of tumor cell proliferation. Instead, trametinib selectively impairs the mobilization of Ly6C+ Myeloid-Derived Suppressor Cells (MDSCs) from myeloid precursors and also abrogates the production of MDSC-chemotactic osteopontin by tumor cells. These combined effects reduce MDSC accumulation at tumor beds, enhancing T cell-mediated protection. Our results demonstrate that the combined anti-inflammatory activity of trametinib on multiple cell types, even without direct influence on tumor cell proliferation, could be responsible for its anti-tumor effects.