bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mitochondrial Ca2+ transport-dependent intercellular crosstalk between CD8 T and natural killer cells prevents tumor development and escape
Authors: Shanker Anil , Uzhachenko Roman , Buferne Michel , Boyer Claude , Goodwin J Shawn , Hofmeister William , Leserman Lee , Schmitt-Verhulst Anne-Marie , Ivanova Alla 
Published: January 2023

Abstract
Unraveling the complexity of lymphocyte interactions is key to understanding immune network and improving cancer immunotherapy. By designing various adoptive cell transfer protocols in solid tumors established in RAG−/−, RAG−/−gc−/− and RAG−/−GzmB-Tom mice and tracing lymphocytes specific to a self-tumor antigen P1A encoded by an X-linked cancer-germline gene Trap1a, we explored the functional dynamics between T and NK cells. We found that activated CD8+T cells augmented NK cell effector function in a pre-tumor T cell protocol that prevented the development of antigen-escape tumor variants. Using a silica nanofiber 3D matrix to control cellular mobility, confocal imaging showed intercellular contacts of activated CD8+T cells (CD69highCD25high) with multiple naïve NK cells, while naïve CD8+T cells formed single or no contact with NK cells. This interaction led to crossregulation of mitochondrial Ca2+ (mitoCa2+) oscillations in both cells whereby activated CD8+T cells induced differentiation of NK cell effector phenotype and NK cells polarized CD8+T cells towards a central memory phenotype. Intracellularly, CD8+T cells increased JAK1, JAK3, TYK2, STAT2 and STAT6 phosphorylation and oxidative signaling in NK cells. In turn, NK cells restrained IL-2 signaling in CD8+T cells by dampening activation-induced STAT5-dependent expression of IL-2-receptor-a chain. These effects were abrogated following the blockade of mitoCa2+ uptake. Mice deficient in mitoCa2+ handling-regulatory gene Fus1 showed increased incidence of a range of spontaneous sarcomas, lymphomas and leukemia. Data suggest that mitoCa2+ transport-guided intercellular crosstalk between CD8+T and NK cells is critical to prevent tumor development and escape.
Polymorphic UHRF1BP1 drives superior anti-tumor immunity in ovarian cancer
Authors: Payne Kyle K. , Svoronos Nikolaos , Chaurio Ricardo A. , Sanz Jairo Perez , Anadon Carmen , Calmette Joseph , Biswas Subir , Costich Tara Lee , Mine Jessica , Conejo-Garcia Jose R. 
Published: January 2023

Abstract
Despite the emergence of immunotherapy for the treatment of cancer, many of the fundamental mechanisms which characterize tumors that are amenable to immunotherapy and/or drive superior endogenous anti-tumor immune responses likely remain uncharacterized. We have identified a single-nucleotide polymorphism, rs13205210, in the gene encoding UHRF1BP1 (UBP). This polymorphism is associated with a dramatic survival benefit in ovarian cancer patients. The function of the protein encoded by this gene remains elusive, however we demonstrate UBP-ablated ovarian tumor cells display global modulation of methylated cytosine, suggesting it has a role as an epigenetic integrator. Interestingly, this polymorphism is also associated with systemic lupus erythematosus, an immune-driven pathology. Accordingly, we demonstrate that human ovarian tumors with polymorphic UBP display increased frequency of activated CD8+ T cells, as well as a type I IFN signature. In vivo, inducible autochthonous murine ovarian tumors driven by oncogenic Kras and ablation of p53, in which UBP was conditionally deleted, demonstrated a significantly enhanced overall survival with a concomitant type I IFN and CXCR3-chemokine signature, as well as an enhanced T cell infiltrate compared to controls. RNA-seq analyses of UBP-deficient ovarian tumors revealed an elevation of inflammatory cytokines and the activation of canonical inflammatory pathways. Furthermore, ectopic expression of polymorphic human UBP in ovarian tumor cells drove elevated NF-kB signaling under inflammatory conditions. Overall our work suggests that UBP functions as a regulator of inflammation, which is unleashed in the polymorphic variant leading to enhanced anti-tumor immunity.
Gene Editing in the NSG mouse strain and its genetic derivatives
Authors: Buaas F William , Wiles Michael , Low Benjamin E. , Kutny Peter M. , Grass David S. 
Published: January 2023

Abstract
The NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mouse strain is highly immunodeficient and has dramatically improved the ability to work with human tumors in an organismal context using xenograft approaches. Technical innovations have expanded the utility of this personalized tumor platform via the reconstitution of parts of the human immune system. An example of this is the NSG-SGM3 inbred mouse strain, in which transgenes expressing human IL3, GMCSF and SCF have been stably integrated into the NSG background, and can effectively support the stable engraftment of human myeloid lineages and the regulatory T cell populations. Direct modification of the NSG genome in mouse embryos was a key to attain this NSG “derivative” strain rapidly and avoid extensive breeding. Modification of NSG, which is built on the parental NOD/ShiLtJ background, a commonly utilized polygenic model of autoimmune type 1 diabetes, can optimize the engraftment of other human lineages. Modern genome modification technology must overcome strain specific characteristics. DNA repair is involved in creating mutations by resolving double strand breaks. The SCID mutation (Prkdcscid) in the NSG background compromises some DNA repair pathways. It was not clear whether nuclease assisted recombination technologies would be a robust tool to modify NSG genomes at the zygote stage. We demonstrate CRISPR-Cas9 is an effective tool for introducing genome modifications in NSG and present a specific example using the Hprt locus. We will also present our experience modifying NSG and its derivatives, demonstrating that direct modification of these genomes is technically feasible and is enabling the rapid sequential genetic tailoring in this high value immunodeficient strain.
Single-Cell Transcriptomic Analysis of Tumor-Immune Microenvironment in Pancreatic Cancer
Authors: Li Xiao , Zhao Yi , Yi Gang , Wang Jingwan , Wang Xie , Guo Shiwei , Jin Gang , Li Bin , Liu Xiao 
Published: January 2023

Abstract
Compelling evidence has suggested the pivotal role of immune cells in the development of carcinogenesis and metastasis in pancreatic cancer (PC). Interrogation on the cell diversity is essential to delineate the tumor microenvironment and the cross-talks between immune cells and tumor. In this study, we systemically profiled single-cell transcriptomes at various stages of PC and found that malignant cells between tumors expressed varied gene repertoire related to proliferation and differentiation. Interestingly, fibroblasts, pancreatic stellate cells and vascular cells also displayed heterogeneity in cell composition and gene signatures, potentially leading to varied degrees of desmoplastic reaction. Strikingly, such dynamics in desmoplastic reaction was evidently associated with the enrichment of specific subsets of immune cells, providing rich resources for detailed mechanistic studies. Our analyses highlighted the connection between infiltrated immune cells and tumor cells and provided insight into the cellular cross-talk of PC at single-cell resolution. Results from this study will also further our understanding of immune landscape in PC with implications for therapeutic development.
Selective TRAF over-expression enhances CD19-targeted 41BB CAR T function by increasing NF-κB
Authors: Li Gongbo , Boucher Justin C. , Kotani Hiroshi , Zhang Yongliang , Shrestha Bishwas , Yu Bin , Davila Marco L. 
Published: January 2023

Abstract
The prototypical second-generation CD19-targeted CAR (chimeric antigen receptor) features a costimulatory domain, either CD28 or 41BB, with both designs achieving comparable clinical outcomes against B cell malignancies. However, clinical application of CAR T cells has outpaced the understanding of their functions in vivo. We evaluated fully murine CD19-targeted CAR T cells containing CD28 or 41BB endodomain in an immune competent B-ALL (B cell acute lymphoblastic leukemia) mouse model. Mouse 41BB CAR T cells imparted inferior survival to mouse CD28 CAR T cells at a stress-test dose. Replacing mouse 41BB endodomain with human 41BB sequence significantly increased NF-κB signaling in 41BB CAR T cells and enhanced survival and persistence to match the mouse CD28 CAR T cells. TRAF binding and its subsequent NF-κB signaling are more important to 41BB than CD28 CAR T functions as evidenced by TRAF1 deficient 41BB but not CD28 CAR T cells showing inferior in vivo B cell killing and CAR T persistence to wild type CAR T cells. Also, 41BB CAR T cells with impaired TRAF binding showed remarkably decreased NF-κB signaling, viability and proliferation in vitro. Over-expressing TRAF2 or TRAF3 but not TRAF1 significantly enhanced CD19 targeted 41BB CAR T functions, such as cytotoxicity, viability and proliferation. Our study demonstrates how co-stimulatory domains impact CAR T functions and selective TRAF over-expression could be a new strategy to improve current CAR T therapy.
Non-viral and viral delivery solutions for next generation cell therapy
Authors: Chang Sean , Yu Xin , Ji Yongchang , Liang Xiquan , Andronikou Nektaria , de Mollerat du Jeu Xavier 
Published: January 2023

Abstract
The successes of chimeric antigen receptor (CAR) T cells in treating blood cancers have highlighted the cell therapy era. However, the difficulty of delivering molecules into immune cells has been an obstacle to more rapid advancement. Here we present an innovative large-scale Lentivirus (LV) production system as a solution to lower the cost and time of viral production. On the other hand, the next generation cell therapy will rely heavily on gene editing, especially in a safer non-viral integration manner. We have demonstrated that our novel non-viral all-in-one electroporation method provides high efficiency of gene knock-in in primary T cells.
The new LV production system was developed for the clinical grade production of lentiviral vectors (LVVs) on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. The system is able to deliver greater than 1 × 108 (TU/mL) functional titer with un-concentrated LVVs.
For CRISPR/Cas9 gene editing in primary T cells, we were able to reach more than 90% knockout efficiency for most genes we tested, including T cell receptor (TCR), with Cas9 RNP electroporation using Neon Transfection System. More importantly, gene knock-in efficiency can be reached to greater than 30% with all-in-one electroporation, which delivers Cas9 RNP and donor DNA in one reaction using our newly developed electroporation buffer. Moreover, TCR knockout in addition to a knock-in at another locus can also be done in a single electroporation using our all-in-one method, which is ideal for developing next generation CAR and TCR-T cells.
Generating suppression-resistant natural killer cells as an enhanced immunotherapeutic for neuroblastoma
Authors: Burga Rachel A. , Williams Elizabeth , Yvon Eric , Fernandes Rohan , Cruz C. Russell Y. , Bollard Catherine M. 
Published: January 2023

Abstract
Refractory neuroblastoma (NB) has a poor prognosis and there is a need to explore novel therapies. Natural killer (NK) cells serve as a promising immunotherapy for NB, however they are impaired by the immunosuppressive TGFb-rich NB tumor microenvironment. Critically, TGFβ impairs NK cell proliferation and cytolytic activity. To overcome these detrimental effects, we genetically modified NK cells to express a variant TGFβ receptor – composed of a truncated extracellular TGFbRII domain fused to a synthetic Notch-like receptor coupled to RELA – which may be able to associate with exogenous TGFβ and convert downstream signals to enhance NK cell function. NK cells were isolated from umbilical cord blood (CB; universal donor source) and expanded ex vivo. The modified TGFβ receptor was cloned into an SFG vector backbone. Expanded CB-derived NK cells were transduced with the modified TGFβ receptor (42.5±5.5%). Transduction did not negatively affect the endogenous expression of the activating receptors NKG2D, NKp44, or NKp30 (p&gt;0.05) or NK cell proliferative capacity (p&gt;0.05). Non-transduced (NT) NK cells were cytotoxic against K562 target cells in a dose-dependent manner (45.2±4.69% killing at 40:1), however NK cell cytolytic activity was impaired following exposure to TGFβ (p&lt;0.05). In contrast, transduced-NK cells exhibited dose-dependent cytotoxicity (40.2±4.42% killing at 40:1), which was unaffected following exposure to TGFβ (p&gt;0.05). Administration of transduced-NK cells in a xenograft model of human NB delayed tumor progression by &gt;14 days and significantly prolonged survival compared to control NK cells (p=0.04). In summary, this novel immunotherapeutic may translate as a potent treatment modality for patients with NB.
Effector responses of cytotoxic CD8+ T cells generated by utilizing an FcR-targeting vaccine strategy in an intracellular bacterial infection model.
Authors: Bitsaktsis Constantine , McCauley James 
Published: January 2023

Abstract
F. tularensis is a highly virulent (Category A) gram-negative intracellular mucosal pathogen. It is believed that while humoral immunity delays the progression of infection, cellular immunity is critical for protection. Consequently, by identifying a vaccine strategy that would generate a pathogen-specific, cell-mediated, cytotoxic immune response will be key for protection against intracellular bacterial pathogens such as F. tularensis, which has no FDA approved vaccine available. For this purpose, we have successfully developed an unadjuvanted vaccine delivery system, which targets Ag to FcγR on antigen presenting cells (APC). However, there is a significant lack of information regarding the role of FcR in the generation of mucosal immunity and more specifically the protective role played by cytotoxic CD8+ T cells. For this reason, we have utilized our F. tularensis FcR-targeting vaccine platform to analyze the generation of F. tularensis-specific, memory CD8+ T lymphocytes and their effector, cytotoxic functions during lethal respiratory challenge with the bacterium. In our studies, we have concluded that our FcR-targeting strategy generates antigen-specific effector memory CD8+ T cells that have significantly improved cytotoxic capabilities against macrophages infected by the bacterium and are capable to transfer protection in naïve recipients.
Immune Competition of HIV Epitopes in Draining Lymph Nodes
Authors: Lu Zhongyan , Hu Xintao , Valentin Antonio , Rosati Margherita , Mullins James I. , Pavlakis George N. , Felber Barbara K. 
Published: January 2023

Abstract

Background
Using HIV-derived DNAs encoding only conserved elements (CE) within Gag or Env, we were able to alter the immune hierarchy redirecting to otherwise subdominant viral targets. This hierarchy was preserved after boosting with DNA expressing full-length (FL) Gag or Env proteins. A vaccination strategy including simultaneous delivery of CE and FL molecules in separate anatomical sites was compared to CE prime/co-delivery CE+FL booster vaccination to examine the magnitude and breadth of epitope recognition to variable and conserved regions.

Methods
The DNA vaccine components were administered by IM route followed by in vivo electroporation in macaques. Antigen-specific cellular immune responses in blood were monitored by flow cytometry.

Results
The T cell responses targeting CE were similar in magnitude, function and breadth in both regimens. Separating anatomical sites of vaccination CE resulted in increased levels of cytotoxic T cell responses targeting variable regions. Thus, separating the immunogens allows development of T cell responses with overall increased breadth including conserved and variable epitopes.

Conclusion
The CE prime/CE+FL DNA boost vaccine induces immune responses targeting the CE epitopes. The vaccination strategy that separates CE and FL DNA plasmids to different anatomical sites, thereby targeting different draining lymph nodes, induces responses to both CE and variable regions. This vaccine regimen has the advantage of avoiding interference of the different vaccine components (CE, FL) during the priming of the adaptive immune response, redirecting immune dominance, and eliciting robust cytotoxic T cell responses targeting conserved and variable regions in the viral proteome.
Novel strategy to enhance antigen presentation and CD8 T cell immunity for curative efficacy of intranasal therapeutic HPV peptide vaccination in preclinical vaginal HPV tumor model
Authors: Galvan Gloria , Sastry Jagannadha , Dorta-Estremera Stephanie , Nookala Sita , Yanamandra Ananta 
Published: January 2023

Abstract
Immune checkpoint modulation to enhance antitumor immunity while minimizing immune suppressive responses within tumors has proven to be a highly successful cancer treatment strategy, but, often with toxic side effects and financial constraints. Using preclinical models of human papillomavirus (HPV) cancers, we tested vaccination employing adjuvants with diverse modes of action to promote antigen presentation and T cell immunity. We employed the combination of α-galactosylceramide (α-GalCer), a potent inducer of NKT cells and CpG-containing oligo-deoxynucleotide (CpG-ODN) that function through the TLR9 pathway. Since most HPV tumors occur at mucosal tissues, we used the mucosal intranasal route for vaccination with E6 and E7 peptides representing tumor-specific antigens to treat vaginal HPV+ TC-1 tumors in mice. Previously, we observed that vaccination using α-GalCer adjuvant alone reduced tumor growth but, complete and durable tumor regression required supplementing the vaccine with anti-4-1BB checkpoint modulating antibody that has clinical safety concerns. We now observed that intranasal HPV peptide vaccine incorporating α-GalCer and CpG-ODN adjuvants, rendered tumor-free survival advantage. Correlates of tumor protection included doubling the number of CD8 T cells exhibiting enhanced cytotoxic potential within the tumor and 84% increase in the ratio of CD8 T cells to regulatory T cells relative to vaccination with either adjuvant alone. Intranasal vaccination delivering α-GalCer plus CpG-ODN allows for the activation of dendritic cells through both classical and alternative licensing pathways and permits the secretion of a diverse set of chemokines to attract enhanced populations of naïve CD8+ T cells.
Detecting neoepitope-specific intra-tumoral T cell responses in a glioblastoma patient treated with personal neoantigen vaccine
Authors: Anandappa Annabelle J. , Keskin Derin B. , Hu Zhuting , Sun Jing , Tirosh Itay , Mathewson Nathan D. , Shukla Sachet A. , Li Shuqiang , Olive Oriol , Neuberg Donna , Fritsch Edward F. , Livak Kenneth J. , Hacohen Nir , Ott Patrick A. , Reardon David A. , Wu Catherine J. 
Published: January 2023

Abstract
Recent advances in single cell T cell receptor (TCR) sequencing have allowed detailed analysis of TCRαβ pairs. However, efficient methods for probing the specificity of discovered TCRs remain limited. We developed a streamlined approach for cloning and expressing TCRs and screening against candidate antigens. A key innovation was the establishment of a plasmid library to encode all variable (V) regions of the TCR, from which any TCR of interest can be assembled with only custom synthesis of short CDR3 regions. A reporter cell system allows rapid detection of TCR antigen specificity and assessment of functional avidity based on cytokine production.
We applied this pipeline to study the intra-tumoral TCR repertoire of a patient with glioblastoma treated with personal neoantigen vaccine. The vaccine consisted of up to 20 synthetic long peptides containing predicted neoepitopes and poly-ICLC adjuvant. Single cell TCR sequencing of CD3+ tumor infiltrating lymphocytes (TILs) and peripheral T cells in vitro expanded against immunizing peptides showed that 4 CD4+ and 2 CD8+ T cell clonotypes in peripheral blood were identical to TILs. The cloning and expression system was used to test their specificity. We identified a TCR from a CD4+ TIL specific for ARHGAP35, a neoantigen targeted by vaccination, and capable of discriminating between mutant and wildtype peptide. This result provides the first demonstration that neoepitope-sensitized T cells can traffic from the periphery to an intracranial GBM. Overall, we demonstrated our ability to identify TCRαβ pairs, express them on demand, and probe antigen specificity. Ongoing studies are characterizing the functional avidity of neoepitope-specific TCRs and reactivity against autologous tumor.
Depo-Provera alters cervical immune responses and increases HIV infectivity ex vivo: a longitudinal study.
Authors: Tasker Carley , Davidow Amy , Roche Natalie E. , Chang Theresa Li-Yun 
Published: January 2023

Abstract
Depo-Provera (Depot medroxyprogesterone acetate, MPA) is associated with an increased risk of HIV acquisition; however, its impact on the cervicovaginal immune milieu and HIV infectivity ex vivo is not well characterized. In a longitudinal study, we investigated the impact of Depo-Provera use by healthy women on levels of cervical immune mediators, expression of immune markers for HIV preference (CCR5, integrin a4b7, CD38) on cervical CD4+ T cells, and HIV infectivity ex vivo at baseline (visit 1), one month (visit 2) and three months (visit 3) after Depo-Provera treatment. Forty-seven healthy women who met the eligibility criteria were enrolled (median age 26, IQR 22.5–30). Cervical cytobrush cells and secretions from vaginal, endocervical, and rectal swabs were collected. Flow cytometry analysis indicated a significant increase in the frequency of cervical CCR5+CD4+T cells at visit 3 compared to visit 1. There was also an increasing trend in the frequency of cervical CD4+ cells expressing integrin a4b7 in response to Depo-Provera. Levels of several cytokines including G-CSF, GM-CSF, IL-6, MIP-1b, and VEGF in vaginal and cervical secretions were significantly decreased at visits 2 and 3 compared to visit 1. At visit 3, cervical levels of immune mediators remained suppressed despite a decreased serum concentration of MPA, suggesting a prolonged effect of Depo-Provera on mucosal immune responses. Cervicovaginal secretions from women after receiving Depo-Provera (visits 2 and 3) promoted HIV infectivity ex vivo compared to those at visit 1. Further studies on the modulation of mucosal immune responses and HIV transmission by Depo-Provera will offer insights into developing better strategies for HIV prevention.