bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fusion partners matter: Factor VIII light chain binding enables CD16-mediated recombinant Factor VIII-Fc fusion protein natural killer cell activation
Authors: Ou Jiayi , Lagasse H A Daniel , Sauna Zuben , Golding Basil 
Published: August 2023

Abstract
Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with multiple Fc gamma receptors (FcγR) resulting in the activation or inhibition of a wide variety of FcγR expressing immune cells. We demonstrated that rFVIIIFc activates natural killer (NK) cells via Fc mediated interactions with FcγRIIIA or CD16. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, we assessed cellular activation by measuring inflammatory cytokine secretion (IFNγ by ELISA) or cellular degranulation (flow cytometry-based surface LAMP1/CD107a). In some cases, we introduced specific blocking molecules (anti-FVIII, anti-Fc, or anti-CD16) into our assays to act as inhibitors of NK cell activation. We identified the FVIII light chain as playing a key role in the activation of CD16+NK cells by rFVIIIFc. Using FVIII domain-specific inhibitors, we demonstrate that blocking C1 or C2 domain-mediated membrane binding potently inhibits rFVIIIFc-mediated CD16+NK cell activation, while targeting FVIII heavy chain domains does not. Our results suggest FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface and allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. This working model may help explain our previous results where we observed recombinant Factor IX-Fc fusion protein and rFVIIIFc exhibited different CD16-signaling properties despite having identical IgG1 Fc domains.
Psychological stress triggers the accumulation of myeloid-derived suppressor cells in the liver through adrenergic receptor signaling
Authors: Ninkov Marina , Rudak Patrick Thomas , Wang Nicole Ilayda , Rashu Rasheduzzaman , Inoue Wataru , Haeryfar S M Mansour 
Published: August 2023

Abstract
Chronic physiological stress affects tightly regulated crosstalk between the nervous and the immune system. However, the exact mechanism(s) of stress-induced immunomodulation is far from clear. Immature myeloid cells such as myeloid-derived suppressor cells (MDSCs) impair immune responses, which may in turn increase susceptibility to infections and cancer or their complications. In a mouse model of chronic psychological stress through physical restraint, we found mononuclear-MDSC (Mo-MDSC) cells, defined as CD11b+Ly6ChighLy6Ghigh cells, to accumulate in the liver. These cells expressed high levels of Mo-MDSC markers such as CD14, TLR4, IL-4Rα as well as markers indicative of anti-apoptotic and immunosuppressive functions (HIF-1α, G-CSFR, Bcl2, Arg2, iNOS). In addition, FACS sorted MDSCs suppressed production of IFN-ɣ by T cells in vitro. Increased levels of proinflammatory mediators were detectable in serum samples of stressed mice, with circulating levels of IL-6 and G-CSF being the most pronounced mediators. The observed intrahepatic accumulation of Mo-MDSC-like cells was mediated by IL-6 and G-CSF and could be reversed by treatment with corresponding receptor-blocking monoclonal antibodies. In addition, this phenomenon was driven by norepinephrine (NE), the main neurotransmitter released from sympathetic neurons, and could be recapitulated by in vivo administration of NE to non-stressed mice. Our work reveals a novel mechanism of immunosuppression that is governed by adrenergic and inflammatory cues and alters the cellular landscape of the liver with implications for immune surveillance against microbes and cancer.
This work was funded by NSERC
Restraint of IFN-γ expression through a distal silencer CNS–28 for tissue homeostasis
Authors: Cui Kairong , Chen Zuojia , Cao Yaqiang , Liu Shuai , Hu Gangqing , Fang Difeng , Wei Danping , Liu Chengyu , Zhu Jinfang (Jeff) , Wu Chuan , Zhao Keji 
Published: August 2023

Abstract
Interferon-g (IFN-g) is a key cytokine in response to viral or intracellular bacterial infection in mammals. While a number of enhancers are described to promote IFN-g responses, no silencers for the Ifng gene have been identified. By examining H3K4me1 histone modification in naïve CD4+ T cells within Ifng locus, we identified an unrecognized silencer (CNS–28) that is responsible for restraining Ifng expression. Mechanistic study further demonstrates that CNS–28 maintains Ifng silence by diminishing enhancer-promoter interactions within Ifng locus in a T-bet independent manner. Functionally, CNS–28 restrains Ifng transcription in Th1, Tc1, and NK cells during both innate and adaptive immune responses. Moreover, CNS–28 deficiency resulted in repressed type 2 responses due to elevated IFN-g expression, shifting Th1 and Th2 paradigm. Thus, CNS–28 activity ensures immune cell quiescence by cooperating with other regulatory cis elements within the Ifng gene locus to minimize autoimmunity.
This research was supported by the Intramural Research Programs of National Heart, Lung and Blood Institute, National Cancer Institute, and National Institute of Allergy and Infectious Diseases of National Institutes of Health.
Modeling the regulation of the Il12b locus by NF-κB dimers
Authors: Oh Yeuran , Md Toufiqur Rahman Shah , Aqdas Mohammad , Sung Myong-Hee 
Published: August 2023

Abstract
The transcription factor NF-κB is composed of a family, which regulates the expression of various genes in immune and inflammatory responses. The NF-κB family consists of five members: RelA/p65, RelB, c-Rel, p50/p105, and p52/p100, which control the transcription of target genes by binding to regulatory κB sites in homo-or heterodimer forms. Unlike the widely known RelA, the mechanisms related to the regulation of target genes by c-Rel is less understood. Interleukin 12b(Il12b), which encodes the p40 subunit of IL12 and IL23, is one example of gene that depend on c-Rel for proper activation in macrophages. The differences in intrinsic binding properties of c-Rel- and RelA-containing dimers at various regulatory sites around Il12b can be a key to understand the mechanism of c-Rel-containing dimers’ role in Il12b regulation. Using c-Rel and Il12b reporter mice with fluorescence live-cell imaging, we observe the single-cell dynamics of nuclear c-Rel and Il12b gene expression in bone marrow-derived macrophages induced by NF-κB activating ligands. We analyze the live-cell imaging data and extract quantitative features of nuclear c-Rel and Il12b expression from the time-series data. For additional mechanistic insights of the Il12b gene regulation by NF-κB dimers, we propose a mathematical model of dynamic gene regulation which includes actions of RelA- and c-Rel-containing dimers at κB sites proximal and distal to the Il12b locus. Our combined experimental and mathematical modeling approach will reveal quantitative insights that may provide a more comprehensive understanding of regulatory mechanisms underlying Il12b gene expression employed by NF-κB dimers.
Analysis of STAT Protein Phosphorylation in T Cells, NK Cells, and B Cells Using High Sensitivity Flow Cytometry
Authors: Pugsley Haley Renee , Sutton Alexandra G 
Published: August 2023

Abstract
In the immune system, cytokine-induced protein phosphorylation is a key cell signaling event that modulates diverse functions, including proliferation, differentiation, apoptosis, and migration. The degree of protein phosphorylation is determined by both the cytokine concentration and the number of cytokine-specific receptors on the cell surface, which are differentially expressed across immune cell populations. Signal transducer and activator of transcription (STAT) proteins are transcription factors that translocate to the nucleus upon activation by tyrosine phosphorylation and alter gene expression. Cytokines such as interleukins (IL) initiate the signaling cascade that activates STAT proteins. In addition, phosphorylated STAT3 and STAT6 have been associated with prognosis in cancer and other malignancies.
Flow cytometry is a powerful method to study the orchestrated response of immune cell subsets to stimulation by combining immunophenotyping markers with fluorescent antibodies specific to phosphorylated signaling molecules. However, because many phosphorylation events occur on weakly expressed proteins and with low stoichiometry, flow cytometers must be highly sensitive to track the subtle changes in signaling magnitude. Flow cytometers with sensitive detection, such as the Amnis® systems, can be used to study phosphorylation events that are key to understanding the processes required for vaccine and immunotherapy development. In this study, we used the Amnis® CellStream® Flow Cytometer to monitor the phosphorylation of STAT3 and STAT6 in CD4+ T cells, NK cells, and B cells after stimulation with IL-4 and IL-6.
Mesenchymal Stem Cells (MSCs) Treatment Alleviates Smoke Inhalation and Burn-Induced Acute Respiratory Distress Syndrome (ARDS) by Inhibiting HMGB1-TLR4 Signal
Authors: Yang Zhangsheng , Thorpe Catherine R , Cancio Tomas S , Willis Robert P , Snook John K , Necsoiu Corina , Meyer Andrew D , Cap Andrew P , Cancio Leopoldo C , Batchinsky Andriy I , Salinas Jose , Young Matthew D 
Published: August 2023

Abstract
ARDS is one of the leading causes of high morbidity and mortality in trauma patients. Previously our data demonstrated autologous MSCs treatment improved survival in a swine model of smoke inhalation and burn injury. However, the pathophysiological mechanisms are still largely unknown.
Twenty anesthetized female swine underwent smoke inhalation injury and 40% TBSA burns, were then randomly assigned to either mock treatment (IC, n=10), or autologous MSCs treatment (MSCs, n=10), followed by ICU care up to 72 hours except in case of early death. Three doses of MSCs collected by bone marrow aspiration and concentrated using a bedside cell concentrator device were applied at 2, 24, and 48 hours of post-injury (PI). Blood and tissue samples were collected for ELISA and IHC analyses.
In IC group, 10/10 injured pigs developed ARDS, but only 6/10 pigs in MSCs group developed ARDS. Serum analysis revealed that the HMGB1 level gradually increased after injury and reached a peak at 48h PI (5.7-fold increase vs. baseline). The SDC-1 and C3a levels also increased after injury but reached a peak at 24h (2.9-fold) and 72h PI (2.2-fold), respectively. The MSCs treatment significantly reduced the HMGB1 level in the serum, especially at 6h and 12h PI compared to the IC group but failed to inhibit the SDC-1 and C3a increases. IHC analyses showed that injury triggered a significantly higher expressions of HMGB1 and TLR4, as well as co-localization of HMGB1 and TLR4 in the lung; while MSCs treatment was able to disrupt the HMGB1-TLR4 interaction and significantly reduce their expressions.
Our data indicate that the way MSCs treatment mitigates ARDS might be by reducing HMGB1 release and inhibiting the HMGB1-TLR4 signal pathway activation in pigs after smoke and burn injury.
This work was supported by the US Army Medical Research and Development Command (USAMRDC) under Grant No. W81XWH-13-2-0005.
The nuclear receptor Nurr1 regulates the expression and activity of PPARγ in human pro-inflammatory macrophages
Authors: Solís Miguel Ángel , Santana Eduardo , Segovia Norma Cristina , Sánchez-Torres Carmen 
Published: August 2023

Abstract
Nurr1 is a member of the orphan nuclear receptor family NR4A that modulates inflammation in several cell lineages through inhibition of the NF-κB signaling pathway. Nurr1 was found to be able to interact with the peroxisome proliferator-activated receptor (PPAR)γ, as well as to be recruited to the PPARG promoter in LPS-activated microglial cells; however, the functional relationship between Nurr1 and PPARγ is not clearly established. Here, we aimed to investigate the role of Nurr1 on PPARγ activity in human macrophages. Blood monocytes were cultured with GM-CSF or M-CSF to generate pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages, respectively. GM-MDMs showed enhanced expression of both Nurr1 and PPARγ compared to M-MDMs, and they were preferentially located at the cytoplasmic (Nurr1) and nuclear (PPARγ) compartments. PPARγ activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. Conversely, Nurr1 activation with the agonist C-DIM12 increased PPARγ expression. This was concomitant with enhanced expression of two PPARγ target genes, CD36 and PLIN2, while silencing Nurr1 expression led to a decrease in the amounts of CD36 transcripts. Co-treatment of GM-MDMs with C-DIM12 and rosiglitazone synergistically enhanced the transcriptional activity of PPARγ, whereas C-DIM12 alone had no effect. PPARγ and Nurr1 agonists alone exerted anti-inflammatory effects on GM-MDMs, and also showed synergistic activity in combined form. In sum, our data suggests that Nurr1 positively regulates PPARγ activity at least partially through induction of PPARγ expression, which may represent a novel mechanism of Nurr1 targeting metabolic pathways and inflammation.
Supported by grants from the Secretaría de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)
Age-Related Changes in B1a Derived IgM Repertoire Affect their Atheroprotective function
Authors: Bontha Sai Vineela , Srikakulapu Prasad , Deroissart Justine , Binder Christoph J , Rothstein Thomas , Holodick Nichol , McNamara Coleen 
Published: August 2023

Abstract
Perivascular adipose tissue (PVAT) harbors immune cells and locally regulates atherosclerosis. The B1a cells present in PVAT secrete IgM in young mice. This IgM has been shown to confer atheroprotection by binding to ox-LDL and other oxidation specific epitopes (OSEs). The impact of aging on B1a-B cells in PVAT, the PVAT B-1a cell IgM repertoire and how aged B-1a cells affect atherosclerosis has not been explored. We sequenced the V-D-J gene segments of IgM in aged B-1a cells from hyperlipemic mice with atherosclerosis. Results demonstrated greater N-additions as well as changes in the usage of the VDJ gene regions in PVAT B1a cells when compared to the B1a cells residing in peritoneal cavity: the primary niche of B1a cells where they self-renew. Further, our previous results have shown similar changes in IgM repertoire in secondary antibody producing sites like spleen and bone marrow. ELISA demonstrated that these changes in IgM repertoire during aging reflect in the systemic IgM pool. The ratio of atheroprotective IgM (IgM to OSEs) to total IgM was significantly decreased with age when 10-, 50- and 100-week-old ApoE−/− mice plasma IgM levels were compared. Further, when B1a cells from young and aged CD45.2 Apoe−/− mice were adoptively transferred to PCSK9 injected CD45.1 STEM mice, those that received B1a from young mice had lower atherosclerosis compared to those that received B1a cells from aged mice. With these results we conclude that with age B1a derived IgM repertoire substantially changes to be less atheroprotective.
“Supported by grants from NIH (RO1 HL107490 and RO1 HL136098 (CM); Ro1AI154539 (NH)); and AHA (award number 18CDA34110392 (PS)).”
The prognostic implications of macrophage polarization in advanced and therapy-resistant prostate cancer
Authors: Martinez Shannalee R , Muriel Nicole Torres , Barrientos Risso Carla M , Wall Nathan R , Dutil Julie , Diaz Osterman Carlos J 
Published: August 2023

Abstract
Tumor-associated macrophages (TAMs) are components of the spatial microenvironment of many solid neoplasms. In prostate cancer (PCa), TAMs are linked to a poor prognosis. PCa is characterized by enhanced tumor lipid production and TAM lipid accumulation. However, the interplay between lipid-producing tumor cells and TAM lipid uptake has yet to be determined. Human prostate tumor tissue microarrays were evaluated for TAM (CD68+) and M2/M1 (CD206+/CD86+) levels correlated with tumor aggressiveness by multiplex immunohistochemistry. Standard PCa cells 22rv1, PC3 and DU145 were used to develop chemoresistant sublines which were validated using dose-response viability assays and immunoblotting for resistance-associated markers and lipid-producing enzymes. Cancer cell-derived extracellular vesicles (EVs) were isolated from cell culture supernatants by size exclusion chromatography and were incubated with U937-derived macrophages, followed by flow cytometric evaluation of M1 and M2 markers. Proteomic evaluation of human prostate tumors indicates that CD68+ TAMs correlate with aggressive, metastatic PCa. Furthermore, a higher M2-to-M1 ratio also correlates with metastatic disease compared to normal or localized tumors. Chemoresistant PCa cells exhibited an enhanced lipogenic phenotype compared to naive cells and the over-production of lipids was linked to EV-mediated reprogramming of macrophages toward an M2-like phenotype. These findings indicate that alternative activation of macrophages is linked to aggressive, therapy resistant PCa. Moreover, our data suggest that tumor lipid metabolism may influence tumor-associated macrophage phenotype and function through EV-mediated intercellular signaling.
Puerto Rico Science Research and Technology Trust
Essential role of IL-17A in activating Treg cells in autoimmune uveitis
Authors: Chong Wai Po , Zhong Yajie , Zhang Xuan , Deng Yujie , Mak Kingston King-Lun 
Published: August 2023

Abstract
The balance between Th17 cells and regulatory T (Treg) cells is critical for host homeostasis. The signature cytokine of Th17 cells, IL-17A, is a pro-inflammatory cytokine associated with various autoimmune diseases in patients and in animal models. Our previous study demonstrated that IL-17A limits pathogenicity of Th17 cells by inducing IL-24. In this study, we investigate the role of IL-17A in regulating Treg cells. In-vitro studies showed that IL-17A promotes both the differentiation and suppressive functions of Treg cells. Adoptive transfer of IL-17A-treated Treg cells is more potent to ameliorate experimental autoimmune uveitis (EAU) in mice when compared to the control Treg cells. We found that the induction of Treg cells is impaired in Il17ra−/− mice after EAU immunization and Il17ra−/− naïve T cells are less effective in their differentiation into Treg cells in EAU compared to wild-type cells. Mechanistic studies revealed that IL-17A activates NF-kB signaling and inhibits TAZ, a Foxp3 suppressor, during Treg cell differentiation. In conclusion, our data suggest that IL-17A is involved in Tregs induction and/or maintenance, and that this has a role in controlling the autoimmune response.
Natural Science Foundation of Guangdong Province (2019A1515010956, 2021A1515010569, 2022A1515012450) Fundamental Research Fund of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center.
Spatial phenotyping of cytokine signatures reflecting the immunotherapy responses in head and neck cancer
Authors: Ji HaYeun , Jhaveri Niyati , Pratapa Aditya , Ma Ning , Ben Cheikh Bassem , Monkman James , O’Byrne Ken , Hughes Brett , Kulasinghe Arutha , Braubach Oliver 
Published: August 2023

Abstract
Immune checkpoint inhibitor (ICI) therapy has drastically improved the treatment strategies for mucosal head and neck squamous cell cancer (HNSCC). To increase treatment response rates for highly targeted ICI therapies, predictive and prognostic biomarkers are being actively explored. Specifically, the cytokine expression patterns in the tumor microenvironment (TME) are now recognized as key to understanding immune responsive and resistant phenotypes. Cytokines play an essential role in the regulation of the TME, specifically in modulating the proliferation and differentiation of immune cells. Here, we have studied spatial signatures of various cytokines within the TME of metastatic/recurrent HNSCC tumors treated with Pembrolizumab/Nivolumab. We utilized the PhenoCycler-Fusion to perform whole-slide, single cell resolution spatial phenotyping of the TME of HNSCC tumors from a cohort of n=40 patients. The discovery cohort consisted of patients who had complete vs. partial vs. stable vs. progressive responses to ICI therapy. Transcriptomic profiling of more than 60 RNA targets for various subfamily of chemokines, interleukins, and immune cell lineages was achieved using Akoya’s novel high plex RNA detection technology. Our study identified distinct spatial signatures that implicate certain cytokines in either tumor progression or regression. Specifically, we have identified areas of high and low CXCL9 and CXCL10 expression in several tumor regions that reflect immune-cell landscapes associated with resistance and sensitivity to immunotherapy. Our study demonstrates the power of unbiased spatial phenotyping with whole-slide imaging to identify biomarkers associated with response to ICI therapy in HNSCC.
CD244 loss on monocytes promotes differentiation into M1 macrophages and potentiates PD-L1 blockade in melanoma
Authors: Kim Jeongsoo , Kim Tae-Jin , Chae Sehyun , Ha Hyojeong , Park Yejin , Lee Hyemin , Hwang Youngsoo , Hwang Daehee , Lee Kyung-Mi 
Published: August 2023

Abstract
Accumulating data have highlighted the role of monocytes/macrophages in immune escape by generating immunologically “cold” tumors that do not respond to immunotherapy. CD244 (SLAMF4, 2B4), a member of the signaling lymphocyte activation molecule family, is expressed on myeloid cells, but its precise role has not been elucidated. Using monocyte lineage-specific CD244-deficient mice challenged with B16F10 melanoma, we report for the first time that CD244 negatively regulates tumor immunity by inhibiting the differentiation and functional maturation of monocytes into macrophages within the tumor microenvironment. CD244-deficient macrophages more effectively activated antigen-specific T cell responses compared to WT macrophages, thus delaying tumor growth in the B16F10 melanoma model. Moreover, combinatorial intervention of anti-PD-L1 antibodies with CD244-KO BMDM markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with WT BMDM. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset, revealed DEGs of CD244− monocytes and macrophages were associated with phagocytosis, antigen presentation, and autophagy. Additionally, cell type deconvolution analysis within melanoma patients bulk RNA-seq datasets from TCGA database, revealed presence of CD244-monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Hence, we proposed that CD244 serve as a critical immunoregulatory receptor on macrophages, and CD244-deficient macrophages may represent a novel therapeutic modality, which can function synergistically with checkpoint blockade therapies.
Supported by grants from National Research Foundation of Korea (NRF-2020R1A2C2103061, NRF-2018M3A9D3079288, NRF-2016M3A9B6948342)