bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective knockdown of A2AR in CD8+ T cells using CD8-targeting nanoliposomes
Authors: Newton Hannah , Arnold Michael J. , Chimote Ameet A. , Conforti Laura 
Published: January 2023

Abstract
Adenosine accumulates in the tumor microenvironment and contributes to the inhibition of T cell function via the A2A receptor (A2AR). Blockade of the A2AR has been shown to rescue T cell function and decrease tumor burden. However, A2AR is present in a variety of cells and thus a limitation of non-selective A2AR pharmacological blockers is the development of side effects. Our goal is to selectively knockdown the A2AR in cytotoxic CD8+ T cells through lipid nanoparticles (NPs) decorated with anti-CD8 antibody and containing A2AR siRNAs. The A2AR knockdown by A2AR siRNAs was verified in human peripheral blood T cells (PBT). Nucleofection of PBTs with A2AR or scramble siRNAs downregulated mRNA and protein by 61% and 14–42%, respectively (as determined by RT-qPCR and flow cytometry). A functional assay for IL-2 production (a cytokine produced by activated PBTs and suppressed by the A2AR pathway) in PBTs treated with siRNAs and activated in the presence of CGS21680 (an A2AR agonist) was used to verify the A2AR knockdown. Treatment of PBTs with A2AR siRNAs prevents the CGS21680-induced inhibition of IL-2 production by 27%. Additionally, NPs were fabricated using biotinylated lipids as previously described (Hajdu et al., Biomaterials 2013) and labeled with anti-CD8 antibody. Their selectivity and internalization were determined. Flow cytometry revealed that CD8-labeled NPs are specific to CD4− PBTs and suggested that the NPs are subsequently internalized. Thus, these data indicate the feasibility of fabrication and potential efficacy of CD8-specific A2AR siRNAs-loaded NPs that should restore the functionality selectively in CD8+ tumor infiltrating lymphocytes—a potential targeted cancer therapy with limited side effects.
Obesity-induced changes in baseline immune responses to pre-clinical breast cancer
Authors: Gibson Justin Tyler , Nagareddy Prabhakara , Norian Lyse 
Published: January 2023

Abstract
Obesity is known to increase morbidity and mortality in breast cancer patients; however, the immunological contributions to such changes remain incompletely understood. Our long-term goal is to investigate the broad impact of obesity on anti-tumor immunity, with a particular focus on understanding the mechanisms underlying obesity-induced immune alterations and how these alterations may influence the use of immunotherapeutics in breast cancer patients. For our studies, we utilized a diet-induced model of obesity (DIO) in which mice were fed either a high-fat or low-fat diet to generate DIO or age-matched lean controls, respectively, then challenged with the syngeneic E0771 mammary carcinoma cell line. Tumor outgrowth was quantified by caliper measurements, bioluminescent imaging (BLI) via firefly luciferase-expressing E0771 (E0771-fLUC) cells, and endpoint tumor weights. Immune populations were evaluated via multi-parameter flow cytometry. BLI revealed that obesity increases primary tumor outgrowth and significantly alters intra-tumoral immune populations. Changes include decreases in the frequency of CD4+ and CD8+ T cells, increases in granulocytic myeloid-derived suppressor cells (G-MDSCs) and dendritic cells (DCs), and a trending decrease in total CD45+ tumor infiltrating leukocytes. These alterations were specific to the tumor environment, as they were not present in the spleens of tumor-bearing mice. Collectively these data implicate obesity as a causal agent in impairing anti-tumor immune responses to murine breast cancer. Furthermore, they suggest several cellular candidates for mechanistic investigations into obesity-induced dysfunction in breast cancer immunity.
Changes in Chronic Inflammatory Cells by Bariatric Weight Loss Surgery
Authors: Sanchez-Pino Maria D. , Kim Yonghyan , Liu Jiao , Wyczechowska Dorota Danuta , Richardson William , Wooldridge James , Mynatt Randall , Berthoud Hans-Rudolf , Ochoa Augusto C. 
Published: January 2023

Abstract
Obesity is characterized by chronic low grade systemic inflammation, where myeloid cells play a critical role. Myeloid-derived suppressor cells (MDSC; Monocytic [M-MDSC] and Granulocytic [PMN-MDSC] subsets) have been identified in cancer and other chronic inflammatory diseases. A few studies have shown a significant accumulation of MDSC in blood and different tissues in diet-induced obese mice and we also observed a significant accumulation of MDSC in mice made obese by a high fat-high sucrose diet compared with lean animals. The mechanisms linking obesity and MDSC activation or the effects of weight loss are unknown. We have compared the frequency of MDSC in blood and omental adipose tissue in obese patients (BMI≥40) with non-obese controls (BMI≤25), and evaluated the effect of bariatric surgery over time (3, 6 and 12 months). Our preliminary data shows that PMN-MDSC are significantly increased in patients prior to surgery, but drastically decrease 3 months after bariatric surgery, associated with improvement of obesity-related metabolic dysfunction. These data support the hypothesis that increased caloric intake and obesity stimulate the induction of MDSC, which promote a chronic inflammatory environment, while weight loss intervention reduces MDSC, eliminating the inflammation and controlling the metabolic dysfunction condition. Furthermore, our in vitro studies demonstrated that the uptake of lipoproteins transforms normal myeloid cells into highly immunosuppressive MDSC by inducing the expression of Arginase I. Thus understanding the cellular and molecular mechanisms by which obesity leads to the induction of MDSC is essential to understand the role of those MDSC in the obesity-related pathologies.
The Force Awakens: Illuminating the role of kynurenine in cancer progression and treatment.
Authors: Ashoura Norah E. , Dekker Joseph D. , Triplett Todd , Garrison Kendra , Blazeck John , Karamitros Christos , Lamb Candice , Tanno Yuri , Ehrlich Lauren I. , Zhang Michelle , Manfredi Mark G. , Stone Everett , Georgiou George 
Published: January 2023

Abstract
Cancer is the second leading cause of death in the US and, despite progress in treatment options, there is a critical need for novel treatments that specifically target cancerous cells. Our immune system routinely identifies potential cancer cells and eliminates them without the need for clinical intervention. However, to evade immune clearance, many cancers elevate tryptophan catabolism in the tumor microenvironment (TME) by upregulating the enzymes indoleamine 2, 3-dioxygenase (IDO) or, alternatively, tryptophan 2, 3- dioxygenase (TDO). This results in greater tryptophan turnover, accumulation of IDO/TDO product, kynurenine (L-kyn), and immune suppression in the TME. Whether the resulting immunosuppression arises from tryptophan depletion or L-kyn accumulation remains highly controversial. This work aims to (1) clarify L-kyn’s effect on T-cells and (2) whether its depletion can relieve tumor burden. Exposing T cells to L-kyn in vitro results in gene expression changes consistent with regulatory T-cell generation and the suppression of naïve T-cell proliferation; establishing L-kyn as a key therapeutic target for depletion to relieve TME immune suppression. Using a pharmacologically optimized kynureninase (KynU) enzyme, we tested L-kyn depletion therapy in murine cancers. KynU administration potently inhibits tumor growth, reduces L-kyn concentration, and results in a significant increase in the infiltration and proliferation of polyfunctional T-lymphocytes. Our ongoing study of KynU’s efficacy and L-kyn’s in vitro effects will illuminate details of L-kyn’s elusive mechanism of action, resolving critical mechanisms of tumor tolerance while creating a more innovative and effective cancer treatment strategy.
Nicotinamide adenine dinucleotide (NAD) oxidation preserves T cell function under lactic acidosis characteristic of the tumor microenvironment (TME)
Authors: Beier Ulf H. , Quinn William J. , Jiao Jing , TeSlaa Tara , Stadanlick Jason , Hancock Wayne W. , Eruslanov Evgeniy B. , Rabinowitz Joshua D. , Baur Joseph A. 
Published: January 2023

Abstract
Cancers are adept at employing metabolic events to evade host immunity. The TME is often glucose depleted and enriched with lactic acid, weakening effector T cell (Teff) function and interferon (IFN)-γ production. By contrast, immunosuppressive T-regulatory (Treg) cells are well adapted to low glucose and elevated lactate and enriched in the TME. Overcoming immunosuppressive metabolic conditions in the TME may be an important step towards improving anti-tumor immunotherapy. Here we show that NAD oxidation can protect Teffs from the immunosuppressive effects of lactic acid. We found that Teff proliferation and IFN-γ production can be controlled through NAD redox metabolism and NAD-dependent enzymes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Metabolomic and bioenergetics studies showed that reducing NAD led to accumulation of pre-GAPDH glycolytic metabolites and impaired glycolytic flux similar to GAPDH inhibition, which was reversed by NAD oxidation. Human and murine Teffs exposed to lactate, oligomycin, rotenone, or antimycin lost GAPDH function despite sufficient glucose, limiting glycolysis and IFN-γ mRNA translation, while the Treg transcription factor Foxp3 was stabilized. Oxidizing NAD (pyruvate, mitochondrial uncoupling, β-lapachone, α-ketobutyrate) rescued Teff glycolysis and restored their function despite ambient lactate, while Foxp3 turnover increased in a poly-(ADP-ribose) polymerase dependent manner, using NAD as substrate. Our results identify a novel therapeutic approach to overcoming metabolic barriers to anti-tumor immunity, and provide new mechanistic insights into the role of NAD redox metabolism in controlling T cell function in the TME.
Cellular and molecular mechanisms regulating the development of tertiary lymphoid structures in tumor
Authors: Rodriguez Anthony B. , Peske J David , Woods Amber N. , Melssen Marit Martina , Cyranowski Salwador , Parriott Geoffrey Raymond , Engelhard Victor H. 
Published: January 2023

Abstract
Tertiary lymphoid structures (TLS) have been described in association with several tumor types, and they are usually associated with enhanced patient survival. However, mechanisms regulating their formation remain unknown. We found that intra- and peri-tumoral TLS develop in intraperitoneal (IP) but not subcutaneously (SC) implanted tumors. These TLS are characterized by a robust accumulation of B cells, and an associated reticular network of podoplanin+ fibroblasts expressing high levels of CXCL12, CXCL13, and the B-cell survival factors BAFF and APRIL. These results suggest that IP tumor-associated fibroblasts are polarized into organizer cells that orchestrate TLS formation. We’ve previously demonstrated that CD8 T effectors induce the development of lymph node-like vasculature in IP tumors. To determine whether these cells also regulate TLS development, we evaluated IP tumors grown in Rag1−/− mice and Rag1−/− mice repleted with bulk CD8 T cells. CD8 T cells upregulated the number of podoplanin+ fibroblasts and CXCL12 expression. However, CXCL13, BAFF and APRIL expression was not dependent on CD8 T cells. Thus, the representation of podoplanin+ fibroblasts and their expression of CXCL12 is regulated by adaptive immunity, while their expression of CXCL13, BAFF and APRIL is mediated by an innate element within the IP tumor microenvironment. We also found that increasing the load of the model tumor antigen ovalbumin led to an increased formation of peri-tumoral TLS with a classical organization, on top of the non-classical intratumoral structures evident at a low antigen load. These results identify novel mechanisms regulating different elements involved in organizing tumor-associated TLS.
Atrophied thymus can serve as a tumor reservoir for harboring melanoma cells
Authors: Sizova Olga , Kuriatnikov Denis , Su Dong-Ming 
Published: January 2023

Abstract
Tumor metastatic relapse is responsible for main cancer associated mortality and potentially arises from undetectable minimal number of tumor cells, which are able to resist radio-chemotherapy at a dormant state hiding in certain organs (termed: tumor reservoirs). A central T cell immune organ, the thymus, has been suggested as this kind of pre-metastatic tumor reservoir for B-lymphoma cells, which are a risk for eventual relapse. It remains unknown whether the thymus is able to harbor non-lymphoid solid tumor cells, why chemotherapy cannot thoroughly eliminate cancer cells in the thymus, and what the state of thymic occult cancer cells is during chemotherapy. With melanoma inoculated and genotoxic doxorubicin treated mouse model, we determined that the thymus, particularly the atrophied thymus, was able to harbor blood stream-circulating melanoma cells. We found that chemotherapy-resulted DNA-damage response triggered p53 activation in non-malignant thymic cells, which in turn resulted in thymocyte death and thymic epithelial cell senescence to develop an inflammatory thymic microenvironment. This inflammatory condition induced thymic occult minimal tumor cells to acquire heterogenic chemo-resistant dormancy. Therefore, the thymus, which becomes a pre-metastatic reservoir for non-lymphoid solid tumor cells under chemotherapy, should be a novel target in antitumor therapy for considering and preventing from tumor metastatic relapse.
IgD Ligation Promotes B Cell Proliferation in a Model of the Tumor Microenvironment.
Authors: Riggs James Edward , Londregan Jennifer E. , Goldman Naomi , Somerville John 
Published: January 2023

Abstract
Potent immune suppression within tumor microenvironments (TMEs) is a hallmark of cancer. We model the TME by culture of C57BL/6J peritoneal cavity (PerC) cells. PerC cells include a significant fraction of immune-suppressive macrophages that, depending on the form of activation, temper T and B cell proliferation. PerC B cell responses to both BCR (F[ab′]2 anti-IgM) and TLR4 (LPS) ligation are suppressed. Here, we show that IgD receptor ligation (anti-IgD) induces modest B cell proliferation in this suppressive environment. Unlike other forms of B cell stimulation, the anti-IgD response is optimal at high PerC cell concentrations. Both polyclonal anti-IgD and monoclonal anti-IgDa(AMS 9.1) reagents trigger B cell proliferation. Strain comparisons revealed better responses with PerC cells from BALB/c (“Th2-like”) mice. Interestingly, while IL-4 enhanced spleen B cell proliferation, this cytokine suppressed the PerC cell response to IgD ligation. We are testing other cytokines to optimize this response and are assessing the impact of IgD ligation on T cell survival in the PerC cell/TME model. These studies may inform strategies to enhance lymphocyte activation within TMEs.
Surface and cytoplasmic PD-L1 regulate distinct cell-intrinsic signaling and functional outcomes
Authors: Gupta Harshita B. , Lao Shunhua , Padron Alvaro , Zhou Mei , Liu Jinyou , Clark Curtis A. , Drerup Justin Michael , Garcia Myrna , Sareddy Gangadhara , Hurez Vincent , Li Rong , Vadlamudi Ratna , Curiel Tyler J. 
Published: January 2023

Abstract
We reported that PD-L1 regulates tumor cell-intrinsic mTOR, autophagy and tumor initiating cell (TIC) generation. We found significant cytoplasmic PD-L1 in various mouse and human tumor cell lines by Western, flow cytometry and confocal. To assess subcellular localized PD-L1 effects, we CRISPR knocked out PD-L1 (KO) from mouse B16 melanoma cells and re-expressed PD-L1 only on surface (surfKI; Display vector) or cytoplasm (cytoKI; PcDNA vector). KO cells proliferated slowly vs ctrl in vitro, partially restored by surfKI and cytoKI. KO suppressed mTORC1 (p70S6K), which was reversed by cytoKI but not surfKI. Rapamycin (mTORC1 inhibitor) reduced surfKI and KO proliferation equally, but both &gt; cytoKI (57% vs 88%, p&lt;0.0001). AZD8055 (mTORC1/2 inhibitor) effects were similar. In WT mice surfKI grew faster (p &lt;0.0001) than KO and cytoKI. SurfKI grew similar to total KI when injected into immunocompromised NSG mice, whereas cytoKIgrew slower than either (p&lt;0.01). CytoKI and surfKI partially restored TIC numbers in vitro. We similarly generated PD-L1KO, cytoKI and surfKI cells in human ES2 ovarian cancer cells and found similar growth, rapamycin and AZD8055 effects thus far. Thus, surface and cytoplasmic PD-L1 contribute distinctly to tumor-intrinsic signals and pathogenesis. Current works tests PD-1/PD-L1 cooperation and PD-L1 cytoplasmic tail effects. Understanding specific cell-intrinsic PD-L1 signals affords novel biology insights and suggests new treatments and drug discovery.
Utilization of High-Frequency Irreversible Electroporation (H-FIRE) to modulate the tumor microenvironment and promote systemic immune system activation in breast cancer
Authors: Allen Irving C. , Ringel-Scaia Veronica , Coutermarsh-Ott Sheryl , Brock Rebecca , Lorenzo Melvin , White Natalie , Oestreich Kenneth J. , Verbridge Scott , Davalos Rafael 
Published: January 2023

Abstract
Breast cancer is the most common malignancy among women, where almost 1.7 million new patients worldwide are diagnosed annually. Despite promising treatments for breast cancer, metastatic disease and recurrence remain significant challenges. There is no highly effective cure currently available once metastatic disease has developed. Thus, new therapeutic paradigms and treatment strategies are direly needed. High Frequency Irreversible Electroporation (H-FIRE) is a particularly novel and emerging therapeutic approach for tumor ablation. This technique uses a series of high-frequency, bipolar electric pulses applied through electrodes inserted directly into the targeted tumor to induce cancer cell death. Our overarching hypothesis predicts that local treatment of the breast tumor with H-FIRE will significantly stimulate the innate and adaptive immune system, resulting in improved survival outcomes. To test this hypothesis, we utilized a mouse 4T1 mammary tumor model. Here, we show that H-FIRE treatment of the primary tumor results in near complete ablation and a shift in the local tumor microenvironment from immunosuppressive to pro-inflammatory. This shift results in the recruitment of increased numbers of antigen presenting cells and T lymphocytes. Local H-FIRE treatment also significantly reduces 4T1 metastases in animals with an intact immune system, indicating increased engagement of a systemic anti-tumor immune response and improved activation of the adaptive immune system. We anticipate that this novel tumor ablation technology will improve conventional treatment strategies and complement emerging immunomodulatory approaches targeting primary tumors, metastatic lesions, and preventing recurrence.
A novel and easy-to-use approach for assessment of chimeric antigen receptor (CAR) modified immune cell cytotoxicity
Authors: Xiong Wei , Weber Nikki , Liu Dongfang 
Published: January 2023

Abstract
Cancer immunotherapies have shown promising results for treating various cancers. One such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), thus combining tumor antigen specificity with immune cell activation in a single receptor. Currently, assessment of this CAR-modified immune cell cytotoxicity is limited by conventional tools (e.g., standard Chromium-51 release assay), which are time-consuming, costly, and labor-intensive. In this study, we developed a novel, easy-to-use, and non-radioactive approach for assessment of CAR-modified immune cell cytotoxicity on the basis of luciferase bioluminescent signal. After a 4- hour incubation of CAR-modified immune cells in a 96-Well optical-bottom microplate, which was pre-seeded with targeted cell stablely expressed eGFP -firefly luciferase fusion gene (GFP-FFluc), the chemical bioluminescent signal of GFP-FFluc was quantified by a fluorescent microplate reader. The positive correlation between FFLuc signal and number of target cells was observed. The dose-dependent specific lysis is comparable with Chromium-51 release assays. The cytotoxicity activity measured by this approach can be further quantified under a common inverted fluorescence microscope to evaluate the morphology and dynamics of CAR-modified immune cells. In conclusion, this newly developed approach provides the scientific community with a powerful tool to study the CAR-modified immune cell cytotoxicity.
Targeting HLA-E for prostate cancer immunotherapy
Authors: Frueh Klaus , Verweij Marieke , Hansen Scott , Mansouri Mandana , Nair Sujit , Malouli Daniel , Tewari Ashutosh K. , Uebelhoer Luke , Ventura Abigail , Selseth Andrea , Axthelm Michael , Bhardwaj Nina , Picker Louis J. 
Published: January 2023

Abstract
Genetically modified strain 68-1 rhesus cytomegalovirus (RhCMV)-derived vaccine vectors uniquely elicit CD8+ T cells recognizing peptides presented by non-polymorphic MHC-E instead of conventional MHC-I molecules. Since MHC-E (HLA-E in humans) is a ligand for inhibitory receptors on NK cells, the upregulation of MHC-E is a known immune evasion strategy for both cancers and intracellular pathogens. Indeed, using tissue arrays we observed that early stage prostate cancer tissues express high levels of HLA-E, as shown by immunohistochemistry, whereas healthy prostate samples were negative. To determine whether MHC-E-restricted T cells recognizing prostate antigens could be used to target prostate cancer we inserted rhesus prostatic acid phosphatase (rhPAP) into RhCMV. Upon inoculation of rhesus macaques (RM) we observed a T cell response comparable to heterologous antigens suggesting that CMV-based vectors excel at eliciting T cell responses to tumor antigens, including self-antigens. Furthermore, all CD8+ T cells recognized rhPAP either in the context of MHC-II or MHC-E, but not MHC-I. Importantly, MHC-E-restricted CD8+ T cells recognized PAP and MHC-E-expressing K562 cells suggesting that endogenous PAP-derived peptides can be loaded onto MHC-E. We further demonstrate that PAP and HLA-E-positive human prostate adenocarcinoma cells stimulated PAP-specific rhesus CD8+ T cells restricted by MHC-E. We conclude that prostate cancer cells load highly conserved HLA-E molecules with prostate antigen-derived peptides that can be targeted by HLA-E-restricted CD8+ T cells thus exploiting a previously unexplored immunological vulnerability.