bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulating the ER and Golgi stress response in T cells with hydrogen sulfide signaling to enhance the anti-tumor immune response
Authors: Oberholtzer Nathaniel , Chakraborty Paramita , Mills Stephanie Ann , Choi Seungho , Mehrotra Shikhar 
Published: August 2023

Abstract
Adoptive cell therapy (ACT) continues to emerge as a novel therapeutic strategy for treating cancer. New approaches to improve ACT protocols are needed to enhance in vivo persistence of adoptively transferred tumor antigen-specific T cells and overcome tumor-induced immunosuppression and cellular stress. Here, we show that hydrogen sulfide (H2S) can be used to promote the antitumor immune response using an H2S donor compound and by enhancing endogenous production of H2S with overexpression of Cbs, a key H2S-producing enzyme. Expansion of tumor antigen-specific T cells with exogenous H2S promoted a central memory phenotype with enhanced antioxidant capacity. H2S-treated T cells and T cells overexpressing Cbs also displayed enhanced reactivity to tumor antigen, with an increase in production of cytolytic cytokines and increased overall protein translation. In in vivo models of melanoma and lymphoma, antitumor T cells conditioned ex vivo with exogenous H2S demonstrated superior tumor control upon ACT. Proteomics analysis revealed an increase in protective thiol modifications on ER-associated proteins involved in regulating ER stress in T cells treated with H2S. Further, treatment of antitumor T cells with H2S resulted in resistance to Golgi and ER stress. Golgi stress and overall Golgi mass were identified as important determinants of T cell function, with T cells possessing high Golgi mass exerting more robust tumor control when adoptively transferred. Overall, these data show that antitumor T cells exposed to H2S possess an enhanced antitumor phenotype that allows them to exert greater tumor control in ACT. Further, these studies identify the status of the Golgi in T cells as an important determinant of their antitumor capacity.
R01 CA236379
mTOR signals regulate T cell exhaustion and T cell responses during PD-1 targeted immunotherapy in chronic viral infection
Authors: Ando Satomi , Araki Koichi 
Published: August 2023

Abstract
Antigen-specific CD8 T cells gradually have impaired functions in chronic infection and cancers, which is termed T cell exhaustion. Since T cell exhaustion is associated with programmed death 1 (PD-1), the blockade of PD-1 pathway reinvigorates exhausted CD8 T cells, resulting in better control of infection and cancers.
Stem-like CD8 T cells are important for maintenance of antigen-specific CD8 T cells during chronic infection and cancers because they have self-renewal ability and provide effector-like CD8 T cells which possess transiently cytotoxicity. PD-1 targeted therapies enhance functional T cells response by promoting differentiation of stem-like CD8 T cells toward effector-like CD8 T cells.
mTOR is a serine/threonine kinase that regulates cellular functions and proliferation. However, the role of mTOR during T cell exhaustion and PD-1 targeted therapies remains elusive.
Here, we found that mTOR inhibition enhanced accumulation of stem-like CD8 T cells at the early phase of infection, leading to improved efficacy of PD-1 blockade. In contrast, after the establishment of chronic LCMV infection, mTOR inhibition suppressed the transition to effector-like CD8 T cells from stem-like CD8 T cells. Mechanistically, a cell intrinsic mTOR signal was critical for differentiation of stem-like CD8 T cells into effector-like CD8 T cells during chronic infection as well as PD-1 targeted therapy. Thus, simultaneous treatments of PD-1 blockade with mTOR inhibition failed to induce effector-like CD8 T cells, resulting in reduced efficacy of PD-1 targeted therapy. Our data demonstrate that mTOR signals regulate T cell exhaustion and PD-1 targeted therapy and have important implications for combination cancer therapies with PD-1 blockade.
Supported by grants from NIH (R01 AI139675)
A Novel Multicolor Enzyme-linked Immunosorbent Spot Assay for the Functional Assessment of CAR T Cell Products
Authors: Iraguha Thierry , Omili Destiny , Gebru Etse , Hardy Nancy M , Luetkens Tim , Rapoport Aaron P , Atanackovic Djordje 
Published: August 2023

Abstract

Introduction
Chimeric antigen receptor (CAR) T cell therapies are powerful anti-tumor immunotherapies and CD19-targeted CAR T cells have revolutionized the treatment of B cell lymphomas. Unfortunately, recurrent disease after CAR T cell therapy remains a clinical problem with up to 50% early relapses after CD19-targeted CAR T cells. Therefore, there is a critical and unmet need to identify biomarkers associated with durable responses to guide the optimization of CAR T cell therapies.

Methods
We developed a novel multicolor enzyme-linked immunosorbent spot (ELISpot) assay for the functional assessment of CAR T Cell products on a single-cell level combining the numerical assessment of CAR T cell products with their functional characterization.

Results
We first used a standard single-cell interferon (IFN)γ ELISpot assay to measure CD19-targeted CAR T cell responses to CD19 protein-coated beads or to beads coated with an irrelevant target. Beads coated with anti-CD3/anti-CD28 antibodies were used as a positive control. We then developed a multicolor ELISpot assay simultaneously measuring the secretion of combinations of different cytokines on a single-CAR T cell level. We identified IFNγ/TNFα/Granzyme B as the most relevant combination of cytokines and we used our novel multicolor ELISpot assay to functionally and numerically characterize two clinical-grade CAR T cell products.

Conclusions
We have developed a novel multicolor, single-cell ELISpot for the quantitative and functional assessment of CAR T cell products. The clinical value of our novel assay will be assessed in clinical studies correlating the pre-infusion assessment of CAR T cell products with the patients’ outcome.

Supported by the Kehlert Foundation (to D. A) by the Maryland Department of Health’s Cigarette Restitution Fund Program and by the National Cancer Institute - Cancer Support Grant (CCSG) P30CA134274.
Reinvigorated exhausted T cells exhibit distinct features within 24 hours after PD-1 therapy
Authors: Leng Xuebing , Nivelo Luis , Buchness Lance , Ristin Svetlana , Rafie Christine , Villarino Alejandro , Stelekati Erietta 
Published: August 2023

Abstract
CD8 T cells are potent cytolytic immune cells. However, persistent antigens during chronic infections and cancers drive differentiation of exhausted T cells (TEX). TEXdo not generate optimal immune protection. Immunotherapy such as anti-PD-L1 provides a partial TEX reinvigoration. Some TEX regain effector functions, enter cell cycle and start proliferation by PD-1 therapy. However, the underlying molecular and epigenetic mechanisms of TEX reinvigoration are not well understood. Moreover, a key unknown question is why only some TEX gain reinvigoration. Here, we used proliferation as a surrogate for TEX reinvigoration. A transgenic mouse model carrying a cell cycle reporter (Fucci) was used. Fucci LCMV-specific CD8 T cells were transferred to mice that were then infected with chronic LCMV to induce exhaustion. Recipients were treated with anti-PD-L1 at d21 p.i. Fucci staining (cell cycle) and CTV dilution (cell division) allowed us to isolate reinvigorated TEX before first cell division and ask what molecular signals induce TEX reinvigoration. Thus, we found that TEX reinvigoration starts as early as 24 hours after a single anti-PD-L1. Proliferating cells upon treatment contained an increased progenitor-like population and a reduced terminally exhausted population compared to control, whereas non-proliferating cells were similar between groups. Single cell transcriptomics data further identified unique transcriptional signatures in reinvigorated TEX upon anti-PD-L1 compared to untreated TEX at 48 hours post treatment. Therefore, we identified the molecular signatures of TEX that have the potential to become reinvigorated upon immunotherapy and suggest novel molecular targets to enhance the potency of PD-1 therapy.
Sensing and alarm function of vaccine-elicited SIV-gag specific CD8 TRM in the reproductive mucosa of rhesus macaques
Authors: Joag Vineet , Bimber Benjamin , Quarnstrom Clare , Bollimpelli Venkata Satish , Schenkel Jason M , Bertogliat Mario , O’Flanagan Stephen , Soerens Andrew , Stolley Michael , Hunter Eric , Amara Rama Rao , Masopust David 
Published: August 2023

Abstract
Resident memory T cells (TRM) constitute a recently identified lymphocyte lineage that occupies non-lymphoid tissues (NLT) without recirculating. In murine models, upon antigenic rechallenge, TRM trigger antiviral responses in neighboring innate and adaptive immune cells, and recruit immune effectors from circulation. Collectively this is referred to as a ‘sensing and alarm’ function. However, the full range of TRM functions has not been assessed, and non-human primate/human TRM function remains almost entirely uncharacterized. Here we established a prime-boost vaccine modality in rhesus macaques to generate abundant SIV-gag-specific CD8 TRM in the female reproductive tract (FRT) and 14 other NLTs. Upon vaginal challenge with CM9 peptide from the SIV gag protein, CD8 TRM reactivation induced expression of CD69 and granzyme B in SIV-gag specific CD8 T cells throughout the rhesus FRT, in situ proliferation (Ki67 expression), and rapid antiviral and IFN response gene expression in essentially all hematopoietic and non-hematopoietic cells. Upregulation of effector genes in CD8 T cells, CD4 T cells, NK cells, and ILCs peaked at 24h and persisted at 48h. CITE-Seq analysis revealed that mucosal CD4 T cells expressed antiviral genes including HIV restriction factors and had reduced expression of the HIV-co-receptor CCR5. Increased numbers of vaginal T cells, memory B cells, and plasma cells at 24h coincided with increased expression of chemokines and VCAM-1 on endothelial and stromal cells and a concomitant reduction in circulating T and B cells. These data demonstrate that CD8 TRM trigger local activation and the mobilization of innate, cellular, and humoral immune responses to the site of antigen exposure in non-human primates.
PP4 deficiency contributes to DEP exacerbated epithelial barrier dysfunction and airway inflammation in asthmatic patients
Authors: Yang Feng-Ming 
Published: August 2023

Abstract
Diesel exhaust particles (DEP) emitted from motor vehicle or heavy-duty industries is thought to be the major source of air pollutants that lead to the rapid increase and worsening of allergic disease, such as asthma. In asthma, exposure to DEP has been linked to aberrant epithelial barrier integrity, which leads to the promotion of allergen delivery into the subepithelia and induced alarmins (IL-33, IL-25 and thymic stromal lymphopoietin (TSLP)) release to promote Th2-mediated inflammation. However, it is still not known the detail mechanism of airway epithelial barrier in response to DEP. In this study, we fund that PP4 expression was decreased in DEP-challenged human bronchial epithelial cells and air-pollution animal model. Moreover, silencing PP4 decreased E-cadherin and barrier integrity through and activated Src kinase and increased expression of Slug. Sequentially, transactivating the EGFR phosphorylation led to increased alarmin synthesis. Overexpression of PP4 wild type, but not phosphatase mutant, prevented DEP-induced loss of airway epithelial E-cadherin expression and decreased alarmin release, suggested that phosphatase activity is critical to maintain epithelial integrity and alarmin production. Primary human bronchial epithelial cells (HBECs) from mild and severe asthmatic patients showed a significantly low TJ integrity in ALI cultures in response to DEP, compared with HBECs from healthy subjects. Our data demonstrate that exposure to DEP causes a significant reduction in expression of PP4 leading to decreased epithelial barrier function might accelerate the allergen-induced airway inflammatory response in asthma.
Supported by grants from (MOST 110-2320-B-038-065-MY3) from the Ministry of Science and Technology of Taiwan.
Murine chorioamnionitis causes a dampened innate immune response that persists into infancy
Authors: Knobbe Jessica , Bermick Jennifer 
Published: August 2023

Abstract
Chorioamnionitis is infection or inflammation of the chorion, the amnion, and the placenta. Chorioamnionitis is associated with 40–70% of preterm births and increases the risk of many immune-mediated neonatal sequelae. Chorioamnionitis also increases the risk of developing wheezing and asthma later in life. With this in mind, we sought to characterize the innate immune dysfunction associated with chorioamnionitis. We hypothesized that neonatal and infant mice exposed to chorioamnionitis would display dampened innate immune responses to bacterial pathogens. To test this, genetically homogenous C57Bl/6 dams were injected IP with LPS or PBS on E18.5. Pups were born naturally and euthanized at P4–6 (neonate) or P12–14 (infant). Splenic F4/80+ cells were isolated, then unstimulated or stimulated with heat-killed Staph epidermidis, Staph aureus, or Escherichia coli for 24 hours. IL-1β, IL-6, IL-10, IL-12, KC, MCP-1, MIP-1α, and TNF-α were quantified in cell supernatants. We observed that E. coli-stimulated F4/80+ cells from chorioamnionitis-exposed infant mice displayed lower concentrations of IL-6 (p&lt;0.0001) and MCP-1 (p=.0057) compared to controls. S. epidermidis-stimulated F4/80+ cells from chorioamnionitis-exposed infant mice also displayed lower concentrations of MCP-1 (p=.0024) compared to controls. This suggests that murine chorioamnionitis impacts the developing immune system, resulting in a dampened innate immune response to bacterial pathogens that persists into infancy.
Supported by grants from the NIH (T32GM139776, R01AI141673).
Multi-parametric quantification of monocytes using live-cell analysis
Authors: McBain Kirsty A , Trigg Jasmine , Lovell Gillian , Bevan Nicola , Dale Tim 
Published: August 2023

Abstract
Monocytes play critical roles in innate immunity by migrating to inflamed tissue, where they clear micro-organisms and apoptotic cells, repair injured tissues, and recruit other immune cells. These highly plastic cells can change their functional phenotypes in response to a variety of cellular signals, including the Akt signaling pathway. Here we provide robust in vitro assays for the kinetic evaluation of monocytes and exemplify how these further our understanding of their biological roles.
The Incucyte® Live-Cell Analysis System was used to acquire phase and fluorescence images of monocytic cell lines, which were automatically analyzed using integrated software. To assess immune cell activation, RAW264.7 monocytes were treated with lipopolysaccharide (LPS) and phenotypic morphological changes were quantified using label-free analysis. These data showed a concentration-dependent increase in activated cells as they become larger and flatter, with a maximal response of 56% for 100 ng/mL LPS at 33 hours. To monitor Akt activity, we used cells expressing the Incucyte® Kinase Akt Green/Red biosensor, a genetically encoded fluorescent reporter whose subcellular localization is dependent on Akt activation. Cytokine analysis was performed using the iQue® Advanced Flow Cytometer and a custom Qbeads® PlexScreen Kit. LPS induced a rapid concentration-dependent activation of Akt (EC50 1.31 ng/mL) and stimulated release of inflammatory proteins TNFα and CCL3 (MIP-1α), consistent with LPS-inducing activation via the release of pro-inflammatory agents.
These data exemplify that live-cell analysis, alongside advanced analytical methods, is a powerful approach enabling multi-parametric quantification of immune cells.
Intracellular complement protein C3 promotes cell survival via Factor B (FB)
Authors: Sahu Sanjaya Kumar 
Published: August 2023

Abstract
Intracellular complement proteins such as C3 and FB play protective role during stress-induced cell death. C3−/−and Cfb−/− mice had worse Pseudomonas aeruginosa (P.a.)-induced acute lung injury at 24 h post infection compared to wildtype (WT) mice. However, the mechanism by which C3 exerts these functions is unclear.
We performed CRISPR-induced deletion of C3 and CFB in BEAS-2B cells, a human tracheobronchial airway epithelial cell line and assessed key findings in primary human airway cells. We also tested our observations in bone-marrow derived macrophages (BMDM) from Cfb−/− versus WT mice. Measures of acute lung injury were quantified in C3−/−, Cfb−/− and WT mice infected with PA57, a clinical P.a. strain.
We observed that C3 and FB-deficient airway cells have increased death overtime compared to their parent clones in the setting of both oxidative stress and P.a. infection in vitro. An increased autophagic stress response, along with increased apoptosis, was observed in both C3 and FB deficiency. On further evaluation, both C3 and FB deficiency resulted in significantly lower AKT-mTOR signaling in P.a. infection. FB deficiency resulted in lower phospho-P70 S6 kinase levels, and higher LC3-II levels in P.a. infection. A similar phenotype was observed in infected BMDMs from Cfb−/− compared to WT mice. FB-deficient BEAS-2B cells and Cfb−/− mouse BMDMs had higher intracellular bacterial burden compared to FB-sufficient controls, suggesting that a lack of intracellular FB altered intracellular processing of P.a. The protective effect of C3 was present even when its secretion was inhibited but was abolished in FB deficiency.
We conclude that intracellular complement protein C3 plays an essential role in cell survival via FB.
Neutrophil Inhibitory Receptors Expression Is Distinct During Gram-Positive and -Negative Pneumonia and Affects Neutrophil Function
Authors: Pihl Riley M F , Martins Kevyn , Traber Katrina E 
Published: August 2023

Abstract

Rational
Despite effective antimicrobial therapy, morbidity and mortality from pneumonia remains high. Honing our understanding of immune dysfunction in pneumonia is now crucial for developing targeted therapeutics. Our objective is to understand how different lethal bacterial pneumonias affect neutrophil phenotype and function over time and identify regulators of neutrophil function.

Methods
Mice were intratracheally instilled with E. coli or S. pneumoniae (SP3) for 6, 24 or 48h and neutrophils were isolated from either bronchioloalveolar lavage fluid (BALF) or peripheral blood. Neutrophil surface marker expression (SME) was analyzed via 25-color panel run on a Cytek Aurora spectral flow cytometer (SFC). Data was analyzed in FlowJo and gated for live, single cell, CD45+Ly6G+ neutrophils, opt-SNE was run for data visualization, and Phenograph was run for unbiased clustering. For bacterial clearance assays, bone marrow neutrophils were isolated via Percoll, then incubated with blocking antibodies against inhibitor receptors (IRs) (PD-L1, SIRPα, VISTA, or CD200R), soluble ligands, or media only. Neutrophils were then cocultured with either SP3 or E. coli. Media from bacteria-neutrophil cultures was then plated on agar and the developing colonies were counted.

Results and Conclusions
Using SFC, we identified pathogen- and timepoint-specific differences in SME of the IRs PD-L1, SIRPα, VISTA and CD200R. Modulating IR signaling by blocking or stimulating them impacts neutrophils’ ability to clear bacteria. In sum, our data suggests that neutrophils modulates their response to adapt to specific pathogens. IRs may play a role in regulating these dynamic neutrophil responses, making them a potential therapeutic target.

NIH grants K08 HL130582 (KET) and R01 HL158732 (KET).
Double DAMPs – NLRP3 priming of murine bone marrow derived mast cells with IL-33 leads to robust IL-1β secretion in a Gasdermin-D independent manner
Authors: Foster Cheyanne , Lee Elizabeth , Dubyak George R 
Published: August 2023

Abstract
Activation of the NLRP3 inflammasome involves hierarchical activation and assembly of upstream protein complexes which can lead to a Gasdermin-D (GSDMD) dependent form of lytic cell death coined pyroptosis. Upon cleavage by Caspase-1, GSDMD oligomerizes in the plasma membrane to form pores which act as a conduit for IL-1β release – a potent proinflammatory cytokine that is implicated in a variety of physiological and pathological responses. Prior studies have utilized macrophages as a model system for interrogating the NLRP3 inflammasome, and recent studies with mast cell (MC) extrapolate these macrophage mechanisms to MCs. However, the mechanisms underlying IL-1β secretion in MCs have yet to be interrogated. We are characterizing the detailed mechanisms underlying NLRP3 activation in MCs, with a focus on GSDM-family members in the pathways of IL-1β release. Our data using primary murine MCs indicate that IL-1β is released independently of GSDMD. This correlates with low GSDMD expression in contrast to high expression of GSDME. Thus, our current studies focus on defining roles for GSDME or other mechanisms of IL-1β release from MCs. Notably, our studies demonstrate a critical role for IL-33 priming vs. LPS in the signal 1 priming of MCs for NLRP3 signaling. Taken together we demonstrate that the MC NLRP3 inflammasome signaling and IL-1β release utilize mechanisms distinct from those in macrophages.
Supported by grants from NIH (P01-AI141350)
Time Course scRNAseq Analysis Reveals Mechanisms of Impaired Lung Recovery Post Influenza Infection during Aging: Zooming into Macrophages
Authors: Wu Yue , Tang Jinyi , Gao Xiaochen , Li Chaofan , Zhu Bibo , de Almeida Santos Gislane , Narasimhan Harish , Li Ying , Zhang Ruixuan , Fain Cori E , Ayasoufi Katayoun , Johnson Aaron J , Dong Haidong , Sun Jie 
Published: August 2023

Abstract
Aging is considered to be a major risk factor for host morbidity and mortality upon acute respiratory virus infections. To better elucidate the immune determinants of viral pathogenesis during aging, we chronological characterized lung cellular and molecular profiles following influenza virus (PR8) infection in young or aged mice till 60 days post infection. Time-course scRNAseq coupled with high dimensional flow cytometry enabled the description of changes in both immune and stromal compartment between young and aged hosts. We further zoomed into the myeloid compartment and focused on the dynamic responses of lung macrophages given their complex roles in viral pathogenesis, tissue repair and lung fibrosis. Enlarged Monocyte-derived Macrophage (MoM) population as well as decreased resident-Alveolar Macrophage (AM) population were observed in the aged mice when compared to those of the young mice. Despite the self-sustain nature of AMs during homeostasis, it has been reported that Ly6Chi monocytes can gain AM identity following severe lung damage like influenza virus infection. We found that the gain of AM identity from monocytes was abrogated in the aged hosts, which likely contributed to the age-associated impairment in lung recovery after influenzas virus infection. Additionally, trajectory analysis with scRNAseq allowed us to identify IFNα/γ signaling to be potential pathways contributing to such defect. This notion is also supported by elevated IFNγ level in BAL from the aged host during lung recovery. Our results thus have improved our understanding on the cellular and molecular mechanisms by which aged hosts exhibit severe diseases and delayed recovery after respiratory viral infection.