bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cryopreserved highly purified immunologically important cell subsets as tools to approach immunological studies (65.27)
Authors: Di Pucchio Tiziana , Glover Chondra , Manak Mark , Anekella Bharathi 
Published: January 2023

Abstract
Cell types, including conventional B and T lymphocytes, regulatory T cells, natural killer and NKT cells, monocytes and dendritic cells are a great resource for a number of immunological studies. Cryopreserved cells are particularly useful in clinical studies as they allow the immunologic assays to be performed after conclusion of studies. Moreover, availability of purified functional cell subsets could help identification and dissection of immunological mechanisms occurring during protective immune responses and lead to development of new cell therapies and vaccine strategies. We show that highly purified human blood cell subsets retain their functionality and differentiation capability when frozen by efficient cryopreservation techniques. Our results demonstrate that frozen PBMC, purified monocytes and T cell subsets maintain their viability and recovery. Furthermore, frozen PBMC and T cell subsets maintain similar ability to proliferate and produce IFN-gamma in response to specific peptide antigen stimulation compared to fresh cell subsets. Notably, fresh and frozen purified monocytes are able to differentiate into phenotypical and functional dendritic cells with similar capability to stimulate allo-CD3 proliferation. Our results indicate that cryopreserved cell subsets could be used as a basic tool in cell-based assays for studying cellular phenotype and functions in studies testing the efficacy of new drugs and vaccines that are designed to modulate immune system.
A polychromatic antibody cocktail to investigate phenotypic and functional markers on human myeloid and plasmacytoid dendritic cells (65.3)
Authors: Jain Pooja , Betts Michael , Manuel Sharron , Makedonas George 
Published: January 2023

Abstract
Antigen presentation is a critical feature of adaptive immunity and essential in self versus nonself discrimination as well as in stimulating immune responses against pathogens. Dendritic cells (DCs) are considered to be the most potent antigen-presenting cells and have long been recognized as key regulators of the immune system. Because DCs are such crucial cells of the immune system, an extensive assessment of their quality in infected individuals is critical. Therefore, we have standardized a unique DC cocktail containing 12 different functional DC markers using an LSR II polychromatic flow cytometer. To this point, we have tested our newly standardized cocktail on the fresh, frozen, and cultured peripheral blood mononuclear cell (PBMCs) as well as on monocyte-derived DCs (MDDCs), a widely used surrogate system for primary blood DCs. This study emphasizes the practicality of utilizing frozen versus fresh cells, especially in rare disease states, wherein obtaining fresh material is virtually impossible. Furthermore, we focus on the feasibility of PBMCs compared to whole blood as starting material. To our knowledge this is the first comprehensive DC phenotyping protocol that uses an extensive list of functional markers (&gt;8). Collectively, these investigations possess great potential to enable investigators to perform immune cell monitoring and develop diagnostic and therapeutic strategies.
Tracking activation of caspases in T cell apoptosis (65.41)
Authors: Gillis Katherine , Clor Julie , Tyagarajan Kamala 
Published: January 2023

Abstract
Caspase activation is critical to immune cell apoptosis, with different caspases activated in different pathways of cell death. Thus, Caspase 8 is implicated in CD95-mediated apoptosis, Caspase 9 is crucial to the intrinsic pathway of apoptosis and Caspase 3,7 activation, is observed in multiple pathways of cell death. Caspases also play a role in immune cell inflammation and proliferation. Simplified methods that allow identification of immune cells and specific caspase activity can provide important mechanistic insights in immune cell apoptosis. We present here results from a simplified strategy to assess caspase activity in CD4 and CD8 T cells using T cell specific cocktails and fluorescently labeled caspase inhibitors. Using this approach, caspase activation with multiple compounds such as anisomycin and staurosporine were studied. Our results indicate that anisomycin causes early activation of both Caspase 9 and Caspase 8 followed by slower activation of Caspase 3,7 activity in both CD4 and CD8 T cells. Differential dose response curves were obtained for Caspase levels in CD4 and CD8 T cells on treatment with staurosporine. In some cases, donor specific differences were observed in the timing and relative activation of the different caspases. Simultaneous assessment of caspase activites in different immune sub-populations allows for a deeper understanding of mechanistic pathways of apoptosis, which is important in drug development and understanding of diseases.
A free NCRR resource for finding and using human disease models (65.46)
Authors: Mungall Chris , Haendel M. , Chatr-aryamontri A. , Oughtred R. , Rust J. 
Published: January 2023

Abstract
LAMHDI.org, a free web-based resource, helps researchers identify model systems to investigate disease mechanisms and therapies by bringing together information about diseases and model organisms. The LAMHDI portal allows search of disease models across species (non-human primates, zebrafish, mice, rats, flies, and yeast, with others in the pipeline) using gene orthology and pathway membership as key linkages. New work includes matching specific phenotypes and common pathways, a zebrafish atlas, and a graphical search based on spatial models of the brain. LAMHDI’s collaboration with BioGRID permits the exploration of networks linked to immune response, such as the NFkappaB and Interferon gamma signaling pathways. Interactions involving homologous members of these pathways provide a valuable resource for exploration across animal models. The goal is to facilitate the identification of models for disease research, make better use of existing model organisms and data about them, and provide the ability to discover new relationships between disease, phenotypes and genes that will further our understanding of disease. Companion efforts will speed the discovery and validation of novel drug candidates in areas as diverse as infectious disease, neuroscience, cardiovascular and metabolic disorders, autoimmunity, and cancer.
Single-cell multiplex RT-qPCR analysis of genes related to migration of murine long-term hematopoietic stem cells during development (65.48)
Authors: García-Ojeda Marcos , Hall Dominique , Lu Alison , De Sena Joseph , Al-Kuhlani Mufadhal , Ciriza Jesús 
Published: January 2023

Abstract
Hematopoietic stem cells (HSC) follow a defined pattern of migration in development. By E14.5, HSC are found in the fetal liver (FL) and colonize the fetal bone marrow (FBM) by E17.5, where they remain during adult life. The mechanism governing HSC migration from the FL to the FBM is not clearly understood. We performed single-cell multiplex RT-qPCR and FACS analysis of 8 genes involved in HSC migration. We show that the expression of the chemokine receptor Cxcr4 varies across microenvironments and developmental times, while N-cadherin and the calcium receptor Casr show only gene expression in FBM at E17.5. Moreover, VE-cadherin maintains high expression only during fetal development, while the integrin subunit α5 increases its variability after E14.5. The integrin subunits α4 and αL as well as the selectin ligand Psgl1 did not show differences in their expression in single HSCs irrespective of the microenvironment. Our data demonstrate that the expression pattern of phenotypically identical, single HSCs fluctuates as a function of developmental and anatomical microenvironment, an observation that could be overlooked using whole population gene expression analysis. This is the first gene expression comparison of migration-related molecules in fetal tissues across developmental times, enhancing the understanding of HSC migration during development. These findings may have potential relevance in improving BM engraftment of clinically transplanted HSC.
Optimization of loading conditions for a violet dye for use in cell proliferation studies (65.9)
Authors: Rohrer Jurg , Elia Jeanne , Rabenstein Jacob 
Published: January 2023

Abstract
Fluorescein diacetate (FDA) and its derivatives, such as carboxyfluorescein succinimidyl ester (CFSE) are nonfluorescent molecules that diffuse into cells and are hydrolyzed by intracellular non-specific esterases to become fluorescent by-products. These fluorescent dye molecules accumulate only in live cells with intact cell membranes and active esterases, while dead cells remain non fluorescent. The precise kinetics of membrane transport and intracellular hydrolysis of FDA and its analogs are related to cellular functions. FDA dyes have been shown to have multiple uses which include, in vitro or in vivo cell tracking experiments and cell proliferation. BD Horizon™ Violet Proliferation Dye (VPD450) dye contains both an esterase-cleavable and an amine-reactive succinimidyl ester group. This dye can be used in multicolor flow cytometry applications with GFP or FITC-labeled cells. Optimization of cell and dye concentrations are important considerations in experimental design with all FDA-based proliferation dyes. A comparison study was performed to determine the effects VPD450 dye loading on cell proliferation, apoptosis, and cell signaling.
Targeting PD-1 and Tim-3 pathways to reverse tumor antigen-specific CD4+ T cell dysfunction in patients with advanced melanoma (66.1)
Authors: Fourcade Julien , Sun Zhaojun , Guillaume Philippe , Luescher Immanuel , Sander Cindy , Kirkwood John , Kuchroo Vijay , Zarour Hassane 
Published: January 2023

Abstract
The coexistence of spontaneous tumor antigen-specific immune T cell responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction and exhaustion. We have previously observed in patients with advanced melanoma that NY-ESO-1-specific CD8+ T cells upregulate PD-1 and a fraction of PD-1+ NY-ESO-1-specific CD8+ T cells upregulates Tim-3. We have found that Tim-3+PD-1+ NY-ESO-1-specific CD8+ T cells represent highly dysfunctional T cells. Importantly, Tim-3 and PD-1 pathway blockades synergized to enhance immune functions of NY-ESO-1-specific CD8+ T cells upon prolonged antigen stimulation. Whether the upregulation of PD-1 and Tim-3 is associated with tumor antigen-specific CD4+ T cell dysfunction remains unknown. Here, our findings in advanced melanoma patients show that in contrast to virus-specific and total CD4+ T cells, PD-1 and Tim-3 expression is upregulated by NY-ESO-1-specific CD4+ T cells. Interestingly, PD-1 and Tim-3 pathway blockades synergized to restore the numbers of cytokine-producing and proliferating NY-ESO-1-specific CD4+ T cells. Collectively, our findings support the use of Tim-3 and PD-1 pathway blockades to reverse tumor-induced CD8+ and CD4+ T cell exhaustion in patients with advanced melanoma. Our findings have significant implication in terms of novel immunotherapeutic strategies aiming at promoting T cell-mediated tumor regression in patients with advanced cancers.
Carcinoembryonic antigen-related cell adhesion moclecule-1 regulates myeloid-cell-dependent tumor angiogenesis by inhibition of G-CSFR-Bv8 pathway (66.19)
Authors: Lu Rongze , Pan Hao , Shively John 
Published: January 2023

Abstract
Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) promote tumor angiogenesis and are refractory to VEGF antibodies treatment. Bv8 expressed in MDSCs has recently been shown to modulate MDSCs mobilization and tumor angiogenesis. Carcinoembryonic antigen-related cell adhesion moclecule-1(CEACAM1) is highly expressed on Gr1+ myeloid cells, however the role of CEACAM1 in myeloid cells mediated tumor angiogenesis has not been investigated. We have demonstrated that tumor growth and angiogenesis was significantly enhanced in Ceacam1-/- mice, which was independent on T and B cells . Meanwhile MDSCs infiltration in tumor, spleen and blood was increased. Tumors coinjected with MDSCs from Ceacam1-/- mice exhibited increased angiogenesis compared to coinjection with MDSCs from control mice. Depletion of Gr1+ cells inhibited tumor growth and angiogenesis in control and Ceacam1-/- mice. MDSCs from Ceacam1-/- mice expressed higher Bv8 in vitro and in vivo. Further we have shown that CEACAM1 recruited Src-homology-phosphatase-1 (SHP-1) to attenuate granulocyte colony-stimulating factor receptor (G-CSFR)-Bv8 pathway. Reconsititution of CEACAM1 into Ceacam1-/- bone marrow cells restored tumor growth and angiogenesis to normal level, and mutations of immunoreceptor tyrosine-based inhibitory motifs ITIMs abolished this restoration. Therefore, we identified CEACAM1, as an inhibitory regulator of G-CSFR-Bv8 pathway, negatively regulates myeloid-cell-dependent tumor angiogenesis.
Mannose receptor regulates macrophage polarization in the tumor microenvironment (66.31)
Authors: Dangaj Denarda , Joshi Shree , Zhao Aizhi , Sandaltzopoulos Raphael , Scholler Nathalie 
Published: January 2023

Abstract
Tumor-associated macrophages (TAMs) share properties with M2 macrophages (MΦ),including high IL10 and mannose receptor (MR) expression. MR cross-linking differentiates DCs into APCs inducing T-cell anergy. However,although MR is abundantly expressed on M2 MΦ,its role on TAMs remains poorly understood. To address this question,we isolated 3 novel recombinant antibodies(scFv) against the human mannose binding domain of MR (CRD4-MR),and set up transwell cocultures allowing chemical exchanges between MΦ and ovarian cancer cells. MΦ phenotype was assessed by FACS,qPCR and bead-based FACS. We also challenged wild type,MR+/- and MR-/- mice with ovarian cancer. We first confirmed that MΦ coculture with tumor cells triggers a tumor associated phenotype and showed that 1 anti-CRD4-MR scFv could block the phenotype switch. We further demonstrated that tumor-released mesothelin (MSLN), but not GPI-anchor truncated MSLN,bound to MR-expressing MΦ,and that MSLN binding could be blocked by all 3 anti-CRD4-MR scFvs. Finally, although tumor challenged MR-/- mice did not survive significantly longer than the wild type,MR+/- mice exhibited lower tumor burden and prolonged survival. MR+/- ascites MΦ secreted significantly higher levels of TNF-a consistent with a pro-inflammatory phenotype. Altogether,tumor-released mesothelin binds to MR and blocking of CRD4-MR binding inhibits TAM phenotype and promotes MΦ with proinflammatory proprieties. Blocking MR function could have therapeutic potential.
NF-κB pathway orchestrates the tumor promoting role of monocytes/macrophages in human renal cell carcinoma (66.45)
Authors: Chittezhath Manesh , Dhillon Manprit , Teo YiLing , Chen Jinmiao , Yang Henry , Lum Josephine , Shalova Irina , Kamaraj Revathy , Singh Rajeev , Wong Alvin , Biswas Subhra 
Published: January 2023

Abstract
Growing evidences suggests that monocyte/macrophages play crucial role in cancer progression. Even though studies in transgenic mouse tumor models have demonstrated the role of myelomonocytic cells in various aspects of tumor progression, information on the role of these cells in human cancers is limited. We investigated the role of monocytes/macrophages in the progression of a human renal Cell Carcinoma (RCC). Gene expression studies have shown that RCC associated monocytes (RCC-Mo) under basal condition exhibit a proinflammatory and protumoral phenotype characterized by high expression of genes such as TNFA, IL6, VEGFA, IL8 and MMP9. Surprisingly, when these cells were exposed to inflammatory stimuli, such as Lipid A or TNF-α, they failed to show an up- regulation of proinflammatory cytokines such as TNFA, CCL3, IL6, and IL1B. This observation correlated to the tumor-induced immunosuppression observed in the patients. Signaling studies showed that the inflammatory/protumoral phenotype of TAMs under basal condition was regulated through NF-κB pathway.Further studies demonstrated defective NF-κB signaling was responsible for the “refractory state” of this RCC-Mo to further inflammatory stimuli. In vivo studies in a human RCC mouse model validated our above findings. In conclusion these results are the first to characterize the transcriptomal and functional phenotype of tumor associated monocytes/macrophages in human cancer which suggested their tumor promoting role.
Blockade of cystine transport inhibits glutathione synthesis and survival of inflammation-induced myeloid-derived suppressor cells (66.6)
Authors: Beury Daniel , Fitzgerald Phillip , Ostrand-Rosenberg Suzanne 
Published: January 2023

Abstract
Tumor-induced myeloid derived suppressor cells (MDSC) are responsible for immune suppression in tumor bearing individuals and are a major obstacle to effective immunotherapy. MDSC suppress T cell activation through several mechanisms including their production of reactive oxygen species (ROS). MDSC accumulation, suppressive activity, and ROS production are enhanced by inflammation. However, MDSC production of ROS does not negatively impact MDSC themselves. Since MDSC sequester cystine/cysteine from their local environment, and cysteine is the rate limiting reagent for glutathione (GSH), which is the predominant antioxidant, we hypothesized that inflammation-induced MDSC utilize high GSH production to attenuate ROS-mediated damage. This hypothesis is supported by our data showing that inflammation-induced MDSC upregulate the subunits of the xc- (cystine/glutamate) transporter, resulting in greater uptake of cystine and higher GSH synthesis. Since MDSC only generate cysteine via the uptake of cystine, the xc- inhibitor sulfasalazine (SASP) may decrease MDSC survival and suppressive activity. This concept is supported by our findings that MDSC treated with SASP are depleted of GSH and are less viable and that inflammation further enhances these effects. Because of their ability to reduce MDSC survival and block MDSC-mediated suppression, xc- inhibitors are potential supplements to cancer immunotherapies.
Response of human natural killer cells to monocyte-derived dendritic cells infected with dengue virus (67.10)
Authors: Lim Daniel , Ooi Eng Eong , Li Na , Yeong Lai Han , Wong Soon Boon Justin 
Published: January 2023

Abstract
Natural Killer (NK) cells are involved in innate responses triggered early during viral infections. However, the factors that regulate the response of primary human NK cells to primary human cells infected with dengue virus (DV) remain poorly characterized. In this study, we demonstrate that naïve NK cells co-cultured with DV-infected monocyte-derived dendritic cells (DCs) produce IFN-γ, upregulate cell surface CD107a, and lyse DV-infected DCs. Type I interferons and TNF-α produced by the DV-infected DCs were crucial for this IFN-γ response, since blocking IFNAR combined with neutralization of TNF-α completely abolished the IFN-γ response in these co-cultures. Cytokines previously reported to play important roles in NK cell activation, e.g. IL-12, IL-15, IL-18, IL-21, and IL-27, were not produced in significant amounts by DV-infected DCs. Transwell experiments suggested that membrane-bound ligands were also important for NK cell recognition of DV-infected DCs. However, we failed to detect the upregulation of ligands for NKG2D or DNAM-1 on DV-infected DCs. Ligands for the natural cytotoxicity receptors were determined not to contribute to the IFN-γ response. In summary, our results have uncovered a previously unappreciated role for the synergy between type I interferons and TNF-α in triggering the anti-dengue response of naïve human NK cells.
Cross reactive TCR modulate the antiviral efficacy of protective HLA class I alleles (67.17)
Authors: Chen Huabiao , Ndhlovu Zaza , Porter Lindsay , Trocha Alicja , Fang Justin , Pereyra Florencia , Yu Xu , Allen Todd , Walker Bruce 
Published: January 2023

Abstract
HLA class I alleles B27 and B57 are associated with protection against HIV-1 disease progression. However, the majority of persons expressing these alleles experience progressive infection, and factors modulating the HLA protective effect remain unclear. Here, we performed a detailed analysis of HIV-1-specific CD8 T cell responses in both progressors and controllers expressing HLA B27 or B57 in terms of TCR recruitment, polyfunctionality, proliferation, avidity, differentiation phenotypes, and functional ability to inhibit viral replication. The data show that HLA B27- or B57-restricted CD8 T cells targeting the same epitopes could be clearly differentiated between controllers and progressors based on potency and cross-reactivity of recognition of HIV-1 and viral variants. This in turn was associated with distinct TCR usage. Superior control of replication of HIV-1 and viral variants was observed by cross-reactive TCR-equipped CD8 T cells while highly antiviral efficacy was associated with polyfunctional CD8 T cells. These data indicate that the efficacy of protective alleles is associated with their ability to cross recognize viral variants, and this in turn is related to specific TCR clonotypes that are generated in natural infection. Clonally diverse CD8 T cell responses to a viral epitope increase the likelihood that multiple divergent strains of viruses may be recognized by cross-reactive CD8 T cells, and have potentially important implications for immunogen design.
Role of B cell memory immunity in secondary heterotypic dengue virus infection in mice (67.2)
Authors: Zompi Simona , Santich Brian , Williams Katherine , Beatty P. Robert , Harris Eva 
Published: January 2023

Abstract
While severe dengue disease has been associated with heterotypic secondary (2°) DENV infection, most 2° infections result in asymptomatic or mild disease. The role of the memory B cell response in mediating cross-protection against a 2° heterotypic infection is not well understood. DENV infection of AG129 (interferon-a/b and -g receptor-deficient) mice reproduces features of human disease. Prior infection with DENV1 protects mice against a lethal 2° infection with DENV2 strain D2S10. DENV1-immune mice treated with cyclophosphamide (CP) and infected with D2S10 died on day 7.5-8.5, demonstrating a role for the cellular immune response in protection. In contrast, naïve CP-treated mice experiencing a 1° infection with D2S10, died on day 4, suggesting that pre-formed Ab play a role in protection. Adoptive transfer of immune memory B cells into naïve mice prior to 2° infection delayed mortality but was not sufficient to protect against a lethal infection. Mice depleted of T cells developed sign of disease but recovered after 2° infection. We are further investigating the respective role of B cells by treating mice with anti-CD20 and by comparing the avidity of post-1°, pre-2° infection serum (LPC response) with that of the supernatant obtained after polyclonal B cell stimulation (B cell memory response). Understanding the role of immune memory in protection will be useful for dengue vaccine development and evaluation.