bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and strategic targeting of key anti-viral defense mechanisms in Natural Killer cells
Authors: Sayitoglu Ece Canan , Ozkazanc Didem , Sarac Aydan , Parlar Ayhan , Alici Evren , Erman Batu , Duru Adil Doganay , Sutlu Tolga
Published: January 2023

While genetic modification of lymphocytes proves to be an efficient approach in immunotherapy, commonly used lentiviral vectors are haunted by the activity of intrinsic restriction factors in immune cells. Natural Killer (NK) cells are members of the innate immune system that possess strikingly high resistance to viral infections, presenting a challenge to virus-based gene delivery methods. In this study, high-throughput analytical approaches were used for comprehensive characterization of NK cell anti-viral defense mechanisms resulting in the observed resistance of NK cells to lentiviral infections. Using Genome-scale CRISPR Knock-Out (GeCKO) Libraries and RNA sequencing, we show that the RLR and TLR pathways are major players influencing the dynamics of lentiviral gene delivery in NK-92 cells. More specifically, even though viral vectors can efficiently enter the cells, activation of anti-viral signaling pathways lead to intracellular elimination of the virus. Additionally, viral vector exposure increases MAPK activity in NK cells, specifically p38 and JNK phosphorylation, similar to the observations in HIV infections. To further confirm the involvement of RLR and TLR pathways, we performed targeted knock-out of 20 candidate genes, using CRISPR/Cas9 technology. Most importantly, our results demonstrate that dsRNA-induced signaling through RIG-I and TRIM25 in NK-92 cells is the key pathway restricting lentiviral gene delivery and small molecule inhibitors can help modulate key pathways. Our findings provide important insights for developing genetically modified NK cell-based immunotherapy approaches and also potentiate new targets to combat viral infections.
DUSP4 regulates STING- and RIG-I-mediated signalling in response to virus infection
Authors: Zhang Yongliang , Jiao Huipeng , James Sharmy J.
Published: January 2023

MAPK phosphatase (MKPs), also known as dual-specificity phosphatases (DUSPs), are cysteine-based protein tyrosine phosphatases that dephosphorylate phosphotyrosine, phosphothreonine, and phosphoserine residues in their substrates. They are originally identified as the major negative regulators of MAP kinases (MAPKs). Our recent study on DUSP10/MKP5 demonstrated that this molecule plays an important role in immune response to virus infection via dephosphorylating IRF3. DUSP4/MKP2 is another DUSP/MKP family member whose function in immune response to virus infection is unclear. This study aims to elucidate the regulatory function of DUSP4 in STING- and RIG-I-mediated signaling and in innate immune response to virus infection. We found that this protein is constitutively expressed in immune cells such as macrophages and its expression is increased in response to the activation of RIG-I and STING as well as virus infection including influenza and HSV. Studies using cells and mice deficient in DUSP4/MKP2 demonstrate that this molecule inhibits STING- and RIG-I-mediated signaling, thereby inhibiting the expression of proinflammatory cytokines including IL-6 and TNFa as well as type I interferon such as IFNb. Furthermore, DUSP4 deficient mice developed less severe diseases upon influenza and HSV infection, but more severe disease upon malaria parasite infection. Our studies demonstrate a novel function of DUSP4 in immune response to microbial infection and may be further explored for therapeutic purposes.
PAMP-induced monoubiquitination of PRR-associated kinase BIK1 by an E3 ligase LUCKY positively regulates plant innate immunity
Authors: Ma Xiyu , Peng Junmin , Shan Libo , He Ping
Published: January 2023

Innate immune responses are initiated by recognition of pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) in both animals and plants. In animal, perception of bacteria flagellin by TLR5 transduces signal through MyD88-IRAK1-IRAK4 to ubiquitin E3 ligase TRAF6. TRAF6 coordinates with UBC13 to produce K63-linked polyubiquitination chains serving as scaffold to mediate downstream signaling. In plant, flagellin is recognized by receptor-like kinase FLS2 and co-receptor BAK1. Upon flagellin perception, BAK1 directly phosphorylates an IRAK-like cytoplasmic kinase BIK1, leading to BIK1 dissociation from the FLS2-BAK1 complex to relay signal. How BIK1 activation is regulated and how it transduces signal to downstream remain largely elusive. We report here that flagellin perception triggers rapid monoubiquitination of BIK1 in planta. Time course and mutational analysis suggested that flagellin-induced BIK1 phosphorylation precedes its monoubiquitination. Using a yeast-two-hybrid screen we identified a RING type E3 ligase LUCKY that interacts with BIK1 and monoubiquitinates multiple residues of BIK1. Mass spectrometry analysis of in vitro-ubiquitinated BIK1 revealed nine lysine residues and the BIK19KR mutant with all nine lysine-to-arginine substitutions blocked flagellin-triggered BIK1 ubiquitination. Transgenic complementation assays indicated that BIK19KR was unable to execute normal BIK1 functions. Moreover, we found that monoubiquitination of BIK1 controls ligand-induced BIK1 dissociation from receptor FLS2. Our study revealed that the ligand-induced monoubiquitination of a key PRR complex component positively regulates innate immunity.
Amifostine activates Nrf2 by binding to Keap1, suppressing lung inflammation
Authors: Joo Myungsoo
Published: January 2023

Although intracellular reactive oxygen species (ROS) produced during phagocytosis function as a key arsenal for innate immunity, inordinate production of ROS is detrimental to host cells, exacerbating inflammation. Nrf2/Keap1 axis is a key countering measure against ROS. Keap1 senses ROS via its thiols, resulting in Nrf2 activation and subsequent production of enzymes that nullify ROS. Amifostine is a cytoprotective thiophosphate that scavenges intracellular free radicals by donating hydrogen ions. Therefore, we tested whether amifostine can be used to suppress inflammation that involves intracellular ROS. Our results show that amifostine down-regulated the production of intracellular ROS induced by LPS. Interestingly, amifostine activated Nrf2 and suppressed the ubiquitination of Nrf2. Amifostine bound to Keap1 at S273, suggesting that amifostine suppresses the ubiquitination of Nrf2 by forming a complex with Keap1, which results in Nrf2 activation. In an LPS-induced acute lung injury mouse model, amifostine suppressed neutrophilic lung inflammation with decreased production of prototypic pro-inflammatory cytokines. Amifostine ameliorated lung damage and protected mice from LPS-induced sepsis. Together, our findings suggest that along with directly scavenging ROS, amifostine contributes to suppressing inflammation by activating Nrf2.
Defensamide, a new synthetic compound, could regulate epidermal innate immunity by stimulating cathelicidin antimicrobial peptide production
Authors: Mann Shivtaj , Park Kyungho
Published: January 2023

Sphingosine-1- phosphate (S1P), a signaling sphingolipid, has multiple biological functions, including stimulation of cathelicidin antimicrobial peptide (CAMP), an innate immune element. Recently, we described production of CAMP in human epidermal keratinocytes via a novel S1P→NF-κB-dependent mechanism. The catalytic activity of Sphingosine kinase (SPHK)-1 is widely described in literature to be essential in the production of S1P. In this study, we investigate whether defensamide, a new synthetically derived compound, can modulate the innate epidermal immune response by potentiating SPHK1 activity and inducing CAMP production. In cultured keratinocytes, treatment with defensamide (100uM) increased CAMP mRNA and protein expression, whereas blockade of SPHK1 by treatment of siRNA significantly attenuated defensamide-induced CAMP expression. Exogenous defensamide increased SPHK1 activity and cellular S1P levels but it did not alter the activity of S1P lyase, the key enzyme responsible for S1P degradation. Defensamide treatment also activated NF-κB which was assessed by nuclear translocation of NF-κB. Conversely, pretreatment with a specific inhibitor of SPHK1 attenuated defensamide-induced nuclear translocation of NF-κB. Accordingly, blockade of NF-κB activation with a specific inhibitor also attenuated defensamide-mediated increase in CAMP expression. Taken together, our results show the following: 1) defensamide induces CAMP production, and 2) induction of CAMP is likely via defensamide → SPHK1 activation → increased cellular S1P levels → NF-κB activation. These results highlight a potential role of defensamide in modulating innate epidermal antimicrobial defense by upregulating CAMP production.
Identification of Trim29 as a Key checkpoint inhibitor of natural killer cell functions
Authors: Dou Yaling , Xing Junji , Li Xianchang , Zhang Zhiqiang
Published: January 2023

Natural killer (NK) cells play an important role in immune surveillance and protective immunity, mainly through rapid cytokine release and cytolytic activities. But how such responses are regulated remains poorly defined. Here, we demonstrated that E3 ubiquitin ligase Trim29 is a crucial regulator of NK cell functions. NK cells lacking Trim29 show increased production of IFN-γ. NK cell-derived IFN-γ has previously been demonstrated to be protective against murine cytomegalovirus (MCMV) infection, and we show here that Trim29−/− mice as well as Trim29lox/loxNKp46-iCre mice exhibit decreased hepatic viral load upon challenge with MCMV, indicating that Trim29 acts within NK cells to regulate antiviral responses. In addition, we discovered that Trim29 is up-regulated in activated NK cells during MCMV infection in response to signals from the proinflammatory cytokines IL-12 and IL-18. Mechanistically, Trim29 suppresses NK cell IFN-γ expression by ubiquitinating the TGF-β activated kinase 1 binding protein 2 (TAB2). These results identify TRIM29 as a previously unknown regulator that restricts NK cell functions.
Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death
Authors: Kambara Hiroto , Liu Fei , Zhang Xiaoyu , Liu Peng , Bajrami Besnik , Teng Yan , Zhao Li , Zhou Shiyi , Yu Hongbo , Zhou Weidong , Silberstein Leslie E. , Cheng Tao , Han Mingzhe , Xu Yuanfu , Luo Hongbo R.
Published: January 2023

Gasdermin D (GSDMD) is considered a pro-inflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, establishing GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived N-terminal fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for anti-bacterial and anti-inflammatory therapies.
LincRNA-EPS and lincRNA-Cox2 regulate expression of KC, IL-6 and CCL5 in macrophages and mice treated with TLR2 and TLR4 agonists or during K. pneumoniae infection, but are dispensable for endotoxin tolerance
Authors: Agliano Federica , Karlinsey Keaton , Carpenter Susan , Medvedev Andrei E.
Published: January 2023

Long non-coding RNAs (lncRNAs) are critical regulators of immune responses, but their role in sepsis is largely unknown. During sepsis, TLR sensing of microbes triggers the “cytokines storm”, disseminated intravascular coagulation and organ damage. Patients surviving systemic inflammatory response syndrome develop profound immunosuppression that resembles TLR tolerance and often succumb to secondary infections. To study the sepsis-associated “cytokine storm”, we used in vivo exposure of mice to LPS (endotoxemia) and Pam3Cys, while endotoxin tolerization of macrophages in vitro was employed to examine features of sepsis-associated immunosuppression. This study showed that LPS-treated macrophages decreased expression of lincRNA-EPS while upregulating lincRNA-Cox2 levels. Using knockout mice, we demonstrated that lincRNA-EPS acts to restrain KC, IL6 and CCL5 expression in the serum, spleen, lungs and liver of LPS or Pam3Cys-treated mice. Infection of lincRNA-EPS−/− macrophages with Klebsiella pneumoniae led to increased expression of IL-6 and CCL5 compared to wild-type cells. K. pneumoniae-infected wild-type and lincRNA-EPS−/− macrophages exhibited similar levels of TNF-α and bacterial 16S RNA. Compared to wild-type animals, pulmonary K. pneumoniae infection of lincRNA-EPS−/− mice resulted in higher levels of serum and lung IL-6 but decreased lung TNF-α. Finally, we showed that lincRNA-EPS and lincRNA-Cox2 are not required for LPS tolerance induction in macrophages. Thus, lincRNA-EPS and lincRNA-Cox2 shape macrophage-elicited cytokine induction upon exposure to microbial agonists and regulate host responses to K. pneumoniae.
Phosphatase PTEN-mediated blockade of IL-2 production is essential for Th17 cell differentiation
Authors: Kim Hyeong Su , Sohn Hyogon , Lee Gap Ryol
Published: January 2023

T helper 17 (Th17) cells are one of CD4+ T cell subsets that mediate inflammation and autoimmunity. Unlike other subsets, interleukin 2 (IL-2) inhibits differentiation of Th17 cells but the mechanism that regulates Th17 differentiation is poorly understood. Here, we show that phosphatase and tensin homologue (PTEN) plays key role in Th17 cell differentiation by regulating IL-2 expression. We generated Th17-specific Pten knock-out mice (Pten fl/fl Il17a CRE) to investigate intrinsic role of PTEN. Th17-specific Pten deletion inhibited Th17 differentiation by inducing excessive expression of IL-2 in vitro. Over-expressed IL-2 recognized by IL-2 receptor and induced phosphorylation of STAT5, eventually triggered Foxp3 expression in Th17 cells. Next, we used experimental autoimmune encephalomyelitis (EAE) as in vivo model, Th17-specific deficiency of Pten ameliorated symptoms of EAE. Additionally, we used SF1670 which is PTEN-specific inhibitor for therapeutic approach. Treatment of SF1670 to EAE induced mice showed ameliorated symptoms of EAE, therefore PTEN may be a potential therapeutic target for Th17-mediated diseases.
Orally administered poly-γ-glutamic acid enhances mucosal immune responses through production of intestinal chemokines and activation of mucosal dendritic cells
Authors: Yang Jihyun , Kim Eunjin , Kim Jaemoo , Poo Haryoung
Published: January 2023

The oral administration of poly-gamma glutamic acid (γ-PGA), a safe and edible biomaterial naturally secreted by Bacillus subtilis sups. Chungkookjang, has been reported to enhance the anti-tumor immunity. After the oral uptake of γ-PGA, the underlying mechanism to affect the mucosal and systemic immune responses has not been displayed yet. Thus we checked the mucosal immune response induced by γ-PGA, such as the production of intestinal chemokines and the activation of mucosal dendritic cells. Upon oral administration of γ-PGA in mice, the presence of γ-PGA was observed in subepithelial dome region of peyer’s patch (PP). Orally administered γ-PGA enhanced levels of various chemokines in intestines, activation of DCs in PPs and accumulation of CD8− DC subsets in mesenteric lymph nodes. Oral administration of γ-PGA mixed with ovalbumin (OVA) as a model antigen increased activation of OVA-specific T cells and levels of both intestinal IgA and sera IgG compared with mice received OVA alone. Moreover, the γ-PGA-OVA group robustly suppressed tumor growth and increased survival in a murine cancer model seeded with the B16mOVA cells, which are B16 expressing membrane-bound OVA, compared to the OVA group. Taken together, these results suggest that γ-PGA induce the mucosal immune responses by enhancing the production of intestinal chemokines and the activation of mucosal dendritic cells, which lead to the increase of the anti-tumor immunity.
Lymphotoxin receptor beta expression by neutrophils is a critical regulator of colitis pathogenesis
Authors: Giles Daniel A. , Zahner Sonja Petra , van der Gracht Esmé , Morris Venetia , Tumanov Alexei , Mikulski Zbigniew , Kronenberg Mitchell
Published: January 2023

Inflammatory bowel disease, while multifactorial, is largely characterized by an exacerbated intestinal immune activation. However, the critical mechanisms regulating immune activation remain to be fully defined. We have previously reported that a member of the TNF superfamily, TNFSF14/LIGHT, is required for prevention of severe disease in a mouse model of dextran sodium sulfate (DSS) induced colitis. Additionally, antibody-mediated blocking of lymphotoxin beta receptor (LTβR), which recognizes LIGHT, also led to exacerbated colitis pathogenesis. Thus, we aimed to determine the cell type(s) and mechanism critical to LTβR mediated exacerbation of DSS driven colitis. Employing the use of LTβRflox/flox mice and appropriate Cre strains, we were able to evaluate the role of LTβR signaling during DSS-induced colitis in a variety of immune and intestinal cell types. For all experiments, body weight, colon length and intestinal histopathology were assessed. These studies revealed that neutrophils were the critical LTβR expressing cell type and that specific deletion of neutrophil LTβR expression, via MRP8 Cre, resulted in exacerbated DSS-induced colitis similar to that in LIGHT−/− mice. RNASeq analysis of neutrophils revealed alterations in cellular metabolism and mitochondrial function, further confirmed through ex vivo analysis of LTβR deficient neutrophils, which had increased mitochondria. In sum, our results demonstrate that neutrophil LTβR activation, likely through LIGHT signaling, plays a critical role in the immune activation associated with DSS-induced colitis.
Targeting fatty acid amide hydrolase (FAAH) in diffuse large B-cell lymphoma (DLBCL).
Authors: Woods Christian Tyler Rutan , Sun Jeff , Henderson Jared , Mohan Chandra
Published: January 2023

Background

DLBCL is an aggressive disease and the most common type of non-Hodgkin lymphoma among adults. Endocannabinoids have been shown to produce anti-proliferative and pro-apoptotic actions in a broad spectrum of tumors. FAAH is the primary enzyme responsible for regulating levels of endocannabinoids. Here we explore the role of FAAH inhibition in DLBCL.

Methods

Western blot analysis was used to probe for FAAH expression in the human DLBCL cell lines, U2392 and Ly-7. The apoptotic effect of an FAAH inhibitor (URB 597) was tested. Next, DLBCL was co-treated with URB 597 and an endocannabinoid (AEA) to simulate endocannabinoid accumulation in cancer tissues. Cellular apoptosis rates were measured by Annexin V and Caspase 3/7 staining and flow cytometry.

Results

High levels of FAAH were observed in both DLBCL cell lines. AEA alone induced significant apoptosis over vehicle control in a dose dependent manner. The greatest change was observed at 30 μm AEA treatment, which resulted in a 9.8 fold increase in apoptosis rate (p<0.001). Together with URB597, AEA increased DLBCL death dramatically in a supra-additive manner. The most significant change was observed at co-treatment levels of 10 μM URB597 and 30 μM AEA, which resulted in a 16.8 fold increase (p<0.001) in apoptosis rate compared to vehicle control.

Conclusions

FAAH inhibition is a promising approach to induce cell death in DLBCL tumors. Studies are in progress to test the in vivo efficacy of FAAH inhibition and the underlying pathogenic mechanisms.
Distinct innate immune responses to different murine mammary carcinoma are evident within the first 72 hours of tumor injection
Authors: Kurt Robert A. , Zhang Chenyu , schwartz Jennifer , Newsom-Stewart Catherine , Baranovic Alison , Chilel-Martin Silvia
Published: January 2023

We used three different murine mammary carcinoma, each syngeneic in Balb/c mice, to study the early anti-tumor immune response. Initial analysis showed that the number of cells at the tumor sites almost doubled between 12–72 hours going from about 3e5 cells at 12 hours to about 6e5 cells at 72 hours. Surface staining revealed that the immune response during this time period was predominated by Gr1+ and F4/80+ cells. Strikingly, similar cell numbers and cell types were recruited to saline injected sites suggesting that these cells may be recruited non-specifically. However, closer examination revealed significant differences not only between the saline and tumor injected sites, but also among different tumor sites. For instance, at 12 hours post injection there were significantly more Gr1+ cells at the EMT6 tumor site than at the 4T1 tumor site, and at 48 hours there were significantly more Gr1+ cells at the 168 tumor site than at the saline and 4T1 tumor sites. At 24 hours post injection there were significantly more F4/80+ cells at the EMT6 tumor site than at the 4T1, 168 and saline injected sites, and at 72 hours post injection there were significantly more F4/80+ cells at the EMT6 tumor site compared to the 4T1 and 168 tumor sites. Next, we collected the Gr1+ and F4/80+ cells from the sites 24 hours after tumor delivery and using qRT-PCR we found several additional differences. For instance, the Gr1+ cells from the EMT6 tumor site expressed more arginase and less CCL2 than the Gr1+ cells from the 4T1 and 168 tumor sites, and the F4/80+ cells from the EMT6 site expressed more MMP-9 and TNF-a than F4/80+ cells from the 4T1 and 168 tumor sites. Collectively, these data indicated that a unique anti-tumor immune response was evident within hours of tumor delivery.
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

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

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

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

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

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

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

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>0.05) or NK cell proliferative capacity (p>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<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>0.05). Administration of transduced-NK cells in a xenograft model of human NB delayed tumor progression by >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

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

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

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

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

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.
‘Plasma monocyte-derived extracellular vesicles are associated with neurocognitive impairment in HIV infected individuals.’
Authors: de Menezes Erika Grasiela Marques , Inglis Heather C. , D’Antoni Michelle , Shikuma Cecilia M. , Ndhlovu Lishomwa C. , Norris Philip
Published: January 2023

Monocytes contribute to the neuropathogenesis of HIV-related cognitive impairment (CI) as they enter the central nervous system and initiate a cascade of neuroinflammatory events. Extracellular vesicles (EVs) are potent vehicles of intercellular signaling and may reflect the state of cells in the brain under healthy and pathological conditions. We measured EVs in plasma from 79 HIV-infected adults enrolled in the Hawaii Aging with HIV Cohort, classified as having either normal cognition (n=16) or CI based on meeting criteria for HIV-Associated Dementia (n=25) or Mild Cognitive Motor Disorder (n=38). We tested whether EV phenotypes differed between those with and without CI. EVs expressing monocyte-associated markers (EVs/μl) were significantly higher in HIV+ donors with CI compared to those without CI: CD14 (819 vs. 118.5, p=0.017), CD4 (261 vs. 45, p=0.001), CD16 (898 vs. 215, p<0.0001), CD163 (1660 vs. 55, p=0.010), and CCR5 (202 vs. 43.5, p=0.009). In addition, increased levels of EVs expressing neural markers were found in HIV+ adults with CI compared to those without CI: MAP2 (531 vs. 103.5, p=0.013), GFAP (1810 vs. 775, p=0.036), and CD200 (1216 vs. 36.5, p=0.005). There was no significant difference between the two groups in age, CD4 count, viral load, and, EVs expressing CD3, CD41a, CD62p, CD63, CCR2 and CD11b. Levels of CD16-expressing EVs negatively correlated with the degree of CI using a composite of 8 neuropsychological (NP) tests to compute a summary NPZ-8 score (r=−0.224, p=0.046). Our findings reveal that EVs expressing monocyte activation and neural damage markers correlate with CI in HIV+ adults. These techniques reveal novel circulating biomarkers of CI, and neuronal pathogenesis markers utilizing peripheral blood.