bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of functional natural killer T cell subsets from human bone marrow derived adult hematopoietic stem progenitor cells (P4456)
Authors: Sun Wenji , Wang Yi , East James , Kimball Amy , Webb Tonya 
Published: January 2023

Abstract
Natural killer T (NKT) cells constitute an important subset of T cells that can both directly and indirectly mediate anti-tumor immunity. However, cancer patients have a reduction in both NKT cell number and function, and these deficits limit the potential clinical application of NKT cells for cancer therapy. To overcome the problem of limited NKT cell numbers, we have investigated whether NKT cells can be generated in vitro from bone marrow-derived adult hematopoietic stem-progenitor cells (HSPC).Our data demonstrate that co-culture of human bone marrow HSPC with OP9 stromal cells transduced with Notch ligand delta-like 1 (OP9-DL1), generates a CD3+ T cell population. These CD3+ precursors can be further differentiated into CD8- NKT cells by secondary culture with CD1d-Ig based artificial antigen presenting cells (aAPC), expressing anti-CD28mAb and loaded with α-GalCer. Importantly, these in vitro-generated NKT cells are functional as demonstrated by their ability to proliferate and secrete IFN-γ following stimulation. These findings demonstrated for the first time that functional human NKT cells can be effectively generated from HSPC in the absence of genetic manipulation or viral transduction. These studies will serve to help understand and design NKT cell-based approaches to enhance current immunotherapeutic strategies for cancer.
Tanshinones increase natural killer cell maturation via activating p38 phosphorylation (P4464)
Authors: Choi Inpyo , Kim Won Sam , Yoon Suk Ran 
Published: January 2023

Abstract
The maturation of natural killer cells is regulated by various growth and nuclear transcription factors. Tanshinones, such as cryptotanshinone (CTS) and tanshinone IIA (TS), are the major lipophilic constituents extracted from the rhizome of Salvia miltiorrhiza Bunge. Here, we demonstrated that tanshinones are positive regulators for NK cell maturation. The tanshinones augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells. Tanshinones also increased IFN-gamma production in NK cells in the presence of IL-15. They also induced the expression of several transcription factors, including Id2, GATA3, T-bet, and Ets-1. Additionally, tanshinones increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, the p38 inhibitor SB203580 blocked the developmental effects of the tanshinones and suppressed Id2, T-bet, and Ets-1 expression during NK cell differentiation. These results suggest that tanshinones significantly increased IL-15-induced NK cell differentiation via enhancing the p38 phosphorylation and the expression of transcription factors.
Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages (P4474)
Authors: Lee Chan Mi , Hu Jim 
Published: January 2023

Abstract
Bone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories. In this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 x 10^5 cells or 5 x 10^6 cells per 10cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b+F4/80+ macrophages (~98%) with majority (~80%) being Ly-6C-Ly-6G- and c-Fms+, while those plated at higher density produced less CD11b+F4/80+ cells and a considerably higher proportion of CD11c+ (~60%) and Ly-6C-Ly-6G+ (~80%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation. Overall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.
Cross-talk between HDAC4 and STAT6 in the transcriptional regulation of Arginase-1 during dendritic cell differentiation in mouse (P4486)
Authors: Zhou Jie , Yang Quan , Zhong Limei , Shi Maohua , Zhu Mingjiang , Qin Aiping , Yin Huiyong 
Published: January 2023

Abstract
L-arginine and L-arginine-metabolizing enzymes play important roles in the immune system. Arg1 is one of the key enzymes in L-arginine metabolism and plays critical roles in the biological function of myeloid cells, including macrophage polarization and suppressive function of myeloid-derived suppressor cells. In this study we provide evidence for a novel role of Arg1 in the regulation of myeloid dendritic cells (DCs) differentiation. Expression of Arg1 and thus the L-arginine usage was dramatically induced during the course of mouse DC differentiation. Modulation of Arg1 expression in mouse bone marrow affects DC differentiation. Mechanistic studies showed that the induction of Arg1 in differentiating DCs was caused by an increased recruitment of HDAC4 to Arg1 promoter, which led to decreased acetylation of both Histone 3 and STAT6 proteins and subsequent transcriptional activation of Arg1. Further ChIP analysis identified a novel STAT6 binding site on Arg1 promoter that was essential for HDAC4-mediated Arg1 regulation. In addition, over-expression of Arg1 abrogated the suppressive effect of HDAC inhibitor on DC differentiation. In summary, this study reveals that Arg1 is a novel regulator of DC differentiation and that cross-talk between HDAC4 and STAT6 is the pathway that regulates Arg1 transcription during DC differentiation.
Metabolic control of dendritic cell development and function (P4490)
Authors: Wang Yanyan , Huang Gonghua , Zeng Hu , Chi Hongbo 
Published: January 2023

Abstract
Cell metabolism refers to the intracellular chemical reactions that convert nutrients and endogenous molecules into energy and biomass. Emerging evidence highlights an intimate interaction between metabolism and immunity. Whereas most of the current studies of immunometabolism have focused on T cells in adaptive immunity, we are just beginning to appreciate that activation of innate immune cells is also metabolically demanding. The evolutionarily conserved mechanistic target of rapamycin (mTOR) pathway integrates various environmental signals to regulate fundamental physiological functions, such as cell growth and proliferation, autophagy, and nutrient sensing and uptake. Although much less investigated, emerging evidence also suggests a role of mTOR signaling in the development and function of dendritic cells (DCs). Here we found that the differentiation and activation of DCs is associated with dynamic regulation of metabolic programs and the mTOR activity. Combining genetic and pharmacological modulation of mTOR activity, we found that mTOR is a crucial regulator of DC development and function. Mechanistically, mTOR signaling impinges upon distinct cell metabolic programs that in turn connect with cell cycle and apoptosis machineries. Our results demonstrate an important role of mTOR in linking cell metabolism and DC development and function.
How functional are human immune cells developed in mice? (P4376)
Authors: Lange Franziska , Scholbach Johanna , Rodewohl Anja , Koeberle Margarethe 
Published: January 2023

Abstract
After transplantation with human umbilical cord blood cells NOD-SCID IL2Rγ(null) rodents develop an human immune system which is characterized by the presence of B cells, T cells, monocytes, granulocytes and dendritic cells. The extent of the several subpopulations differs during the time after transplantation. A clear development of the immune system can be seen from immature immune system which contains mainly B cells, a few T cells in peripheral blood, during a transitional state with an additional existence of monocytes, granulocytes and denritic cells in bone marrow and spleen of the mice to a state which reminds of Graft-versus-Host-Disease. This development can be observed during a period of 10 to 20 weeks after transplantation. The B cells are able to produce high amounts of IgM in the early state and few IgG. The antigen presenting cells in the transitional state are able to express activation molecules like HLA-DR and CD86 after the injection of LPS as an immune modifying stimulus. Moreover the cells are able to produce human TNFα, IL-10, IL-1b, IFNγ and high amounts of IL-6 after stimulation with LPS in every state. Release of murine TNFα, IL10, IL-6, MCP and IL-12 was detected as well. The presence of human and murine PTX3 was also measureable after in vivo stimulation with LPS. Depending on the time point after transplantation the human immune cells are able to produce human cytokines and to express several activation markers after immune modifying stimulus.
Role of TGF-β in the creation of an immunosuppressive microenvironment during leukemia (P2025)
Authors: Carli Cedric , Caron Louis-Philippe , Jean Catherine , Dahmani Amina , Giroux Martin , Delisle Jean-Sebastien 
Published: January 2023

Abstract
Tumors develop a milieu that fosters their development and counteract the total efficacy of immune system through different mechanisms. TGF-β, a potent growth suppressor and immunosuppressive cytokine has the capacity to inhibit the anti-tumor immune response in several models. However, all the mechanisms underlying the effects of TGF-β are not well known. Specifically, the role of TGF-β in shaping the cancer microenvironment is ill-defined. Our objective is to characterize the role of TGF-β in shaping the neoplastic microenvironment using a leukemia model. Using the TGF-β producing EL4 cell line, we characterized the immune cell infiltration and the levels of several cytokines in the context of systemic TGF-β neutralization. When compared to tumor extracted from untreated animals, leukemic masses that had evolved in the context of TGF-β inhibition had increased levels of inflammatory cytokines and a tendency to accumulate more activated T cells and surprisingly more myeloid cells, unveiling a potential role for these cells in leukemia specific immunity. More precisely, secretion of cytokines involved in migration and activation of leucocytes, namely IL-2, GM-CSF, KC and MIP-1α was significantly induced in treated animals. Our data suggest that TGF-β blockade alters the leukemic microenvironment. Further studies will confirm the impact of TGF-β on the leukemic milieu including immune cells, stromal cells and angiogenesis and how this cytokine impedes anti-cancer immunity.
Myeloid-derived suppressor cells are associated with disease progression and decreased overall survival in advanced-stage melanoma patients (P2027)
Authors: Jordan Kimberly , Amaria Rodabe , Ramirez Oscar , Callihan Eryn , Borakove Michelle , Manthey Elizabeth , Borges Virginia , McCarter Martin 
Published: January 2023

Abstract
Myeloid-derived suppressor cells are increased in the peripheral blood of advanced-stage cancer patients and prevent effective antitumor immune responses. Although these cells have been well characterized in mouse studies, few studies have shown a correlation of these immunosuppressive cells with functional and clinical outcomes in melanoma patients. We characterized the frequency and suppressive function of two subsets of myeloid-derived suppressor cells in the peripheral blood of 34 patients with Stage IV melanoma. The frequency of CD14+ MDSCs (Lin- CD11b+ HLA-DR- CD14+) and CD33+ MDSCs (Lin- CD11b+ HLA-DR- CD14- CD33+) were increased in the peripheral blood of Stage IV melanoma patients relative to healthy donors. These frequencies correlated with increased expression of PD-1 on peripheral T cells and an increased frequency of regulatory T cells. CD33+ MDSCs isolated from the peripheral blood of Stage IV melanoma patients, but not healthy donors, suppressed T cell activation and the frequency of these cells correlated with disease progression and decreased overall survival. Our study provides evidence that the frequency of CD33+ MDSCs negatively correlates with clinical outcomes in advanced-stage melanoma patients. These data indicate that suppressive MDSCs should be considered as targets for future immunotherapies.
ABCG1 as a novel link between cholesterol homeostasis and tumor immunity (P2031)
Authors: Sag Duygu , Cekic Caglar , Wu Runpei , Linden Joel , Hedrick Catherine 
Published: January 2023

Abstract
ATP-binding Cassette Transporter G1 (ABCG1) promotes cholesterol efflux from cells for intracellular cholesterol homeostasis. ABCG1 has been shown to regulate macrophage and lymphocyte immune responses. In this study, we demonstrate a role of ABCG1 as a novel modulator of tumor immunity. MB49 bladder carcinoma or B16 melanoma cells were injected into Abcg1-/- or B6 mice subcutaneously. Both groups of mice were fed with either high-fat diet or chow diet starting a week before injection of tumor cells. Abcg1-/- mice on high-fat diet had dramatically reduced (80 %) tumor growth compared to B6 mice. Abcg1-/- mice on high-fat diet also showed diminished tumor metastasis and prolonged survival. Furthermore, we showed that reduced tumor size in Abcg1-/- mice on high-fat diet was associated with an increase in the frequency of NK cells and T cells and decrease in the frequency of macrophages and Tregs in tumor. Selective inhibition of ABCG1 in myeloid cells, but not in T cells, reduced tumor growth in vivo, demonstrating that impaired tumor growth in the absence of ABCG1 is mediated through myeloid cell-intrinsic factors. Moreover, ABCG1 deficiency caused an increase in apoptosis of macrophages and a shift of macrophages to an M1 phenotype in the tumor under high-fat diet conditions. Overall, our study shows that absence of ABCG1 inhibits tumor growth through regulation of macrophage function within the tumor and provides a link between diet, cholesterol homeostasis, and cancer.
TGF-β dependent suppression of effector T cell function within the tumor microenvironment (P2033)
Authors: Budhu Sadna , Schaer David , Li Yongbiao , Houghton Alan , Silverstein Samuel , Merghoub Taha , Wolchok Jedd 
Published: January 2023

Abstract
Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation had a similar effect, reducing the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs.
FOXO3 regulates tumor-associated DC tolerogenicity by binding and sequestering NF-kB. (P2037)
Authors: Watkins Stephanie , Hurwitz Arthur 
Published: January 2023

Abstract
Tumor-associated dendritic cells (TADC) are a major contributor of immune tolerance in the tumor microenvironment. We previously reported that the transcription factor FOXO3 is a critical mediator of TADC tolerogenicity and that silencing Foxo3 expression promoted anti-tumor immunity. In the current study, we sought to identify the mechanism by which FOXO3 regulates TADC function. It was previously reported that FOXO3 to inhibits NF-kB activity in T cells through the transcriptional regulation of IkB proteins. In contrast, our data demonstrate that unlike in non-DC populations, FOXO3 was principally detected in the cytoplasm of TADC. Further analysis revealed that despite extensive phosphorylation, cytoplasmic FOXO3 was stable. These findings suggested that FOXO3 may not solely function as a transcriptional regulator. Instead, our findings show for the first time that FOXO3 binds both the RelA and p50 NF-kB subunits and sequesters them to the cytoplasm. The formation of this protein complex prevents nuclear translocation and downstream pro-inflammatory signaling by NF-kB. In the absence of FOXO3, NF-kB re-localizes to the nucleus and turns on expression of pro-inflammatory genes. We further demonstrate that NF-kB enhances cytoplasmic FOXO3 stability. These new insights into the regulation of DC tolerogenicity provide new potential targets for the enhancement of immune-based therapies for cancer, where FOXO3 may reduce pro-inflammatory activities of TADC.
Soluble CD80 restores T cell activation and overcomes tumor cell Programmed Death Ligand-1-mediated suppression (P2041)
Authors: Haile Samuel , Dalal Sonia , Clements Virginia , Tamada Koji , Ostrand-Rosenberg Suzanne 
Published: January 2023

Abstract
Many tumor cells escape anti-tumor immunity through their expression of Programmed Death Ligand 1 (PDL1 or B7-H1), which interacts with T cell-expressed PD1 and results in T cell apoptosis. We previously reported that transfection of human tumor cells with a membrane-bound form of the costimulatory molecule CD80 prevented PD1 binding and restored T cell activation. We now report that membrane-bound CD80 similarly reduces PDL1-PD1-mediated suppression by mouse tumor cells, and that a fusion protein consisting of the extracellular domains of CD80 fused to an Fc domain of IgG1 overcomes PDL1-mediated suppression by human tumor cells. T cell activation experiments assessing costimulation indicate that the soluble CD80 fusion protein mediates its effects by binding to PDL1 and inhibiting PDL1-PD1 interactions. Comparison of the CD80 fusion protein to antibodies specific for PD1 or PDL1 demonstrate that soluble CD80 treatment is more effective in restoring T cell activation than treatment with mAb to either PD1 or PDL1. These studies identify soluble CD80 as an alternative and potentially more efficacious therapeutic agent for overcoming PDL1-induced immune suppression and facilitating tumor-specific immunity.
Deregulation of the Fas-mediated apoptosis pathway confers MDSCs ability to evade apoptosis to persist in tumor-bearing hosts (P2055)
Authors: Paschall Amy , Hu Xiaolin , Bardhan Kankana , Yang Dafeng , Waller Jennifer , Park Mary , Nayak-Kapoor Asha , Samuel Thomas , Abrams Scott , Liu Kebin 
Published: January 2023

Abstract
Myeloid-derived suppressive cells (MDSCs) are potent immune suppressive cells that accumulate with tumor progression. Compelling data from mouse models and human cancer patients have shown that tumor-induced inflammation induces MDSC differentiation. However, the mechanisms underlying MDSC persistence is unknown. Here, we made use of orthotopic mammary and colon carcinoma mouse tumor models and demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis in vivo, as well as decreased sensitivity to FasL-induced apoptosis in vitro, as compared to myeloid cells with the same phenotype from tumor-free mice. Furthermore, tumor-induced MDSCs accumulated in a greater rate in Faslgld mice than in wild type mice. Analysis of gene expression profiles revealed that IRF8 is dramatically down-regulated in MDSCs from tumor-bearing mice and from human cancer patients as compared to tumor-free mice and normal human donors, respectively. Further analysis of IRF8 target gene expression indicated that Fas and Bax are down-regulated, whereas Bcl-xL is up-regulated in MDSCs from tumor-bearing mice. ABT-737 therapy increased MDSCs spontaneous apoptosis and decreased MDSC accumulation in vivo. Our data determine that MDSCs use down-regulation of IRF8 to deregulate the Fas-mediated apoptosis pathway to evade elimination by apoptosis in tumor-bearing hosts, and targeting Bcl-xL is potentially an effective approach to suppress MDSC persistence in cancer therapy.
Neuropilin-1 mediates infiltration of regulatory T cells into tumors favoring immune escape (P2063)
Authors: Buer Jan , Hutzler Marina , Helfrich Iris , Schadendorf Dirk , Hansen Wiebke 
Published: January 2023

Abstract
Infiltration of tumors by Foxp3+ regulatory T cells contributes to tumor development and progression by dampening an effective anti-tumor immune response. We show that the surface receptor Neuropilin-1, highly expressed by Foxp3+ regulatory T cells, controls the immunological anti-tumor response by directing regulatory T cells into the tumor in a Vascular Endothelial Growth Factor dependent mode of action. Deletion of Neuropilin-1 in CD4+ T cells or abrogation of Vascular Endothelial Growth Factor production by the tumor cells itself resulted in a major impairment of tumor development and progression accompanied by an increased CD8+ T cell response. Most importantly, the frequency of tumor-infiltrating Foxp3+ regulatory T cells was clearly reduced within tumors from both T cell-specific Neuropilin-1-deficient mice and wild type mice transplanted with Vascular Endothelial Growth Factor-deficient tumor cells. From this, we conclude that Neuropilin-1 represents a key mediator of Foxp3+ regulatory T cell infiltration into Vascular Endothelial Growth Factor producing tumor sites.
Immune checkpoint protein VISTA regulate autoimmunity and anti-tumor immunity (P2069)
Authors: Wang Li , LeMercier Isabelle , Lines Janet , Sergent Petra , Li Jiannan , Noelle Randolph 
Published: January 2023

Abstract
V-domain Ig Suppressor of T cell Activation (VISTA) is a newly discovered immune checkpoint ligand that suppresses T cell activation. Our study demonstrates the critical role of VISTA in controlling autoimmunity and anti-tumor immunity. Within the hematopoietic compartment, VISTA is highly expressed on myeloid antigen-presenting cells, naïve T cells and Foxp3+ Tregs. Aged VISTA ko mice developed inflammatory phenotypes. When bred onto a TCR transgenic background that is specific for myelin oligodendrocyte glycoprotein (MOG), VISTA ko mice developed severe autoimmune disease at 6-7 weeks of age. VISTA also regulates the suppressive function of Foxp3+CD4+ regulatory T cells (Tregs), as well as the induction of adaptive Tregs in the periphery. During tumor development, VISTA expression is upregulated within the tumor microenvironment. A VISTA-specific monoclonal antibody (mab)-mediated blockade enhanced tumor-specific T cell priming, as well as altered the suppressive character of the tumor microenvironment by reducing tumor-infiltration of myeloid-derived suppressor cells while enhancing the infiltration and effector function of tumor-specific effector T cells. Consequently, VISTA mab-mediated blockade as a single reagent significantly suppressed the growth of both transplantable and inducible melanoma tumors, and when combined with a cancer vaccine, effectively eradicated established tumors. VISTA thus represents a novel and promising target for cancer immunotherapy.