bluuebunny/crossref_metadata_2025 huggingface dataset:
Random mutagenesis reveals novel host-pathogen interactions in host defense against attaching and effacing pathogens
Authors: Wan Fengyi
Published: January 2023

Understanding the host-pathogen interactions in the gut is crucial for prevention and treatment of attaching and effacing (A/E) pathogen infections. Inoculation of mouse A/E pathogen, Citrobacter rodentium, in interleukin-22 (IL-22) null mice dramatically increases mortality and morbidity rate, highlighting the crucial role of IL-22 in orchestrating antibacterial immunity against A/E pathogens. However, the host-pathogen interactions under IL-22 compromised conditions remain poorly understood. By inoculating a C. rodentium mutant library (generated by transposon random mutagenesis) in Il22−/− mice, we identified an attenuated strain (designated as Mut1), which completely abolishes wild-type C. rodentium infection-induced severe morbidity and mortality in Il22−/− mice. Compared to wild-type C. rodentium, the Mut1 mutant has no effects on the growth rate, attachment to colonic epithelial cells, and colonization. However, the Mut1 mutation dramatically impedes the inflammatory cytokine/chemokine expression and immune cell infiltration in the infected colon in Il22−/− mice. These elucidated novel host-pathogen interactions in Il22−/− mice could provide novel strategies to control A/E pathogen infections under IL-22-compromised conditions associated with systemic lupus erythematosus, chronic HIV infection, organ transplantation, and other diseases.
Blood transcriptional profiling of childhood diarrheal diseases identifies gene signatures of Shigella and rotavirus infections
Authors: DeBerg Hannah A , Zaidi Mussaret B , Khaenam Prasong , Gersuk Vivian , Linsley Peter S , Estrada-Garcia Teresa
Published: January 2023

Globally, diarrheal diseases are a leading cause of death in children under five and disproportionately affect children in developing countries. To identify changes in gene expression associated with diarrheal disease, we performed RNA sequencing on whole blood of 192 children less than 10 years of age with acute diarrhea at the Hospital General O’Horan, and 47 age-matched healthy controls from Yucatan, Mexico. We found strong transcriptional signatures associated with rotavirus and Shigella infections relative to other diarrheal infections. The other organisms in our study (Salmonella, E. coli, norovirus, and adenovirus) did not produce transcriptional changes that could be detected in whole blood. Weighted gene correlation network analysis was used to generate modules of genes with similar expression patterns that could be linked to biological functions. In the case of Shigella and rotavirus infections, differentially expressed genes with increased expression were grouped in modules associated with immune system functions. In Shigella infections, modules enriched for genes in adaptive immunity, especially in lymphocyte activation, showed decreased expression while modules enriched for innate immunity, including genes involved in complement activation, an inflammatory response and a response to bacteria, had increased expression. In rotavirus, but not other viral infections, the expression of a module containing genes involved in a type 1 interferon response was increased. Our data suggest that immune gene expression in peripheral blood may distinguish diarrheal diseases caused by Shigella and rotavirus infections from diseases caused by other organisms.
Exploring the antigen-specific memory CD4 T cell response to Chlamydia female reproductive tract reinfection
Authors: Li Lin-Xi
Published: January 2023

Chlamydia is now the most commonly reported infectious disease in the United States and more than 3 million new cases occur annually. Despite numerous attempts made by researchers to develop a vaccine strategy over the past two decades, to date, no Chlamydia vaccine is available. Among all immunization strategies tested in mouse models of Chlamydia infection, none generates protective immunity any better than a naturally resolved prior Chlamydia female reproductive tract (FRT) infection. Research in our laboratory aims to understand how immunological memory shaped by a natural episode of Chlamydia primary infection confer such potent protective immunity. Using Chlamydia-specific MHC Class II tetramers, our preliminary studies show that Chlamydia secondary infection induced massive clonal expansion of antigen-specific CD4 T cells in the FRT seven days post reinfection. In contrast, a robust recall response is not observed in local draining lymph nodes or spleen at any time point post challenge. Surprisingly, Chlamydia-specific memory CD4 T cells in the FRT do not express a common marker for tissue-resident T cells, CD69. We are now focusing our efforts on understanding the activation, effector function, and maintenance of Chlamydia-specific memory CD4 T cells in the FRT during Chlamydia reinfection.
Optimal replication and suppression of inflammation by virulent Francisella tularensis is achieved through reprogramming of host glycolysis.
Authors: Wyatt Elliott V , Diaz Karina , Griffin Amanda , Rasmussen Jed , Crane Deborah , Jones Bradley , Bosio Catharine M
Published: January 2023

Francisella tularensis (Ft) is a gram negative, facultative intracellular bacterium that is the causative agent of pneumonic tularemia. Inhalation of as a few as 15 organisms can result in lethal infection. The virulence of Ft is attributed to the bacterium’s ability to both evade and suppress the activation of macrophages. However, the mechanisms and bacterial components required for evasion and suppression by Ft are unclear. Activation of macrophages and induction of antimicrobial responses requires a metabolic shift from oxidative phosphorylation to aerobic glycolysis. Thus, we postulated that Ft manipulates host metabolism as a mechanism of virulence. We established that virulent Ft suppresses induction of aerobic glycolysis among infected macrophages. Moreover, utilizing purified capsule and defined capsule mutants, we elucidated a novel role for Ft capsule as a component contributing to this suppression. The requirement for inhibition of host cell glycolysis in Ft pathogenesis was confirmed via direct inhibition of glycolysis using 2-deoxyglucose. Addition of 2-DG to macrophages infected with capsule mutants partially restored the early intracellular survival and replication of these bacteria, and impaired the secretion of pro-inflammatory cytokines that is typical of infection with these mutants. Together, our data demonstrate that manipulation of host metabolism is an important component of Ft pathogenesis and uncovers a previously unappreciated function of bacterial capsule in the virulence of Ft.
Gpr43, dietary fiber and composition of gut microbiota in colonic inflammation and carcinogenesis
Authors: Singh Nagendra , Sivaprakasam Sathish
Published: January 2023

Being a primary driver of gut microbiota metabolism, diet has overwhelming affect on promoting or suppressing growth of specific members of gut microbiota. Abundance of certain gut mcirobiota is also shaped by host genetics. Numerous human and animals studies show that dietary fiber consistently increase only one group of gut bacteria, which are Bifidobacterium species (spp), the primary fermenters of dietary fiber. It is unknown whether genetic make up of host influences dietary fiber mediated promotion of Bifidobacterium spp in vivo and its impact on colonic inflammation and carcinogenesis. Gpr43 is a receptor for short chain fatty acids, acetate, propionate and butyrate. Our results demonstrate that dietary fiber mediated promotion of Bifidobacterium spp and suppression of Prevotellaceae is dependent on Gpr43. Similarly, dietary fiber mediated suppression of colonic inflammation and carcinogenesis is Gpr43-dependent. Dietary fiber mediated increase in Bifidobacterium spp is relevant to suppression of colonic inflammation and carcinogenesis because administration of Bifidobacterium spp decreased susceptibility of Gpr43−/− animal to colonic inflammation and carcinogenesis. Consistent with these findings, we found that Gpr43 is down regulated and number of Bififdobacterium is decreased whereas that of Prevotellaceae is increased in human colon cancers. These findings suggest that interplay between dietary fiber and Gpr43 play a key role in promoting healthy composition of gut microbiota leading to suppression of colonic inflammation and carcinogenesis.
Notch Signaling Negatively Regulates Pathogenic IL-17 Production from Th17 cells during γ-Herpesvirus Induced Pneumonitis and Pulmonary Fibrosis Following Bone Marrow Transplantation
Authors: Gurczynski Stephen J , Zhou Xiaofeng , Maillard Ivan , Moore Bethany B
Published: January 2023

The Notch signaling pathway is an important mediator of T-cell cytokine responses in a variety of immunological maladies such as GVHD, rheumatoid arthritis, and systemic lupus erythematosus. Using a genetically altered mouse model wherein CD4+ and CD8+ T-cells were ablated for all Notch signaling through expression of a dominant negative version of the Notch transcriptional regulator Mastermind Like Ligand (DNMAML) we have identified Notch as a regulator of cytokines during Murine Gamma Herpesvirus 68 (MHV-68) infection following bone marrow transplantation (BMT). Wild type syngeneic BMT mice infected with MHV-68 develop severe pneumonitis and pulmonary fibrosis by 21 dpi driven in part by a switch from TH1 to TH17 mediated immunity. Expression of the Notch ligands DLL1, DLL4, Jagged1, and Jagged2 were decreased in the lungs following BMT and CD4+ T-cells isolated from the lungs and spleens of infected WT BMT mice displayed defects in Notch signaling resulting in decreased expression of Notch target genes. T-cell Notch deficient DNMAML BMT mice developed increased fibrosis 21 dpi in comparison to wild type BMT mice, and displayed elevated levels of IL-17 and IL-6 in the lungs as well as an expansion of TH17 cells and a decrease in TH1 cells. Further, both in vitro culture of BMT lung leukocytes with recombinant DLL4 and adoptive transfer of WT CD11C+ APCs after BMT restored T-cell Notch signaling, decreasing production of IL-17 and rescuing fibrotic pathology. Thus, alterations in T-cell Notch signaling during γ-herpesvirus challenge following BMT correlated with increased expression of IL-17 from TH17 cells shifting the immunologic balance from a protective TH1 response towards a pathogenic TH17 response.
New cross-strain protective influenza vaccine using self-attenuated influenza virus
Authors: Wu Lihong , Li Junwei , Arévalo Maria T , Chen Yanping , Yan Yongyong , Wang Haiyan , Zeng Mingtao
Published: January 2023

Live attenuated vaccines (LAVs) are derived from disease-causing pathogens and are effective way for prevention of infectious disease. Classical LAVs are produced experimentally by repeated passaging of a virus in cultured cells, but this method is not always reliable and safe because in some cases, there is a reversion to wild-type virulence. Several methods have been employed to improve them, but the efficacy and safety are still a challenge. In this study, we have developed a self-attenuated RNA virus expressing a functional and mammalian species-specific artificial microRNA. Using influenza virus as a model, we designed an artificial miR-93 cassette for insertion into NS gene segment of influenza viral genome, which produces a specific amiRNA for inhibiting NP gene expression that would result in a virus which is attenuated in mammalian cells. The resulting engineered self-attenuated influenza virus, PR8-amiR-93NP, produced mature and functional artificial microRNA against NP in mammalian cells, but could be propagated at a high yield with the traditional influenza virus production method in embryonated chicken eggs. Furthermore, PR8-amiR-93NP was attenuated by 100 fold in mice compared with its wild-type counterpart. In animal experiments, intranasal vaccination with PR8-amiR-93NP induces antigen-specific antibody responses and protective immunity against heterologous influenza virus strains. In conclusion, our results demonstrated that this self-attenuation approach provides a safe and effective platform for generation of live attenuated influenza vaccine, as a next generation nasal spray influenza vaccine.
A practical approach to high complexity multiplexed chromogenic immunohistochemistry.
Authors: Blenman Kim RM
Published: January 2023

Histology is a pathologist’s first step to diagnosing and monitoring diseases but we still rely heavily on methods created in the mid-1800s. A major gap in histology is the ability to assess multiple cellular targets within the tumor microenvironment simultaneously and quantitatively. Immunohistochemistry (IHC) is an essential tool for clinical diagnostic and translational research laboratories. It is typically used to qualitatively assess morphology and cell types present within tissue. IHC involves staining a tissue section with a purified primary antibody followed by a peroxidase- or phosphatase-conjugated secondary antibody and developing the stain with a chromogenic substrate such as 3,3′-diaminobenzidine tetrahydrochloride (DAB). Currently, for clinical diagnostic purposes the majority of IHC staining is a two-color stain, which includes DAB chromogen and hematoxylin dye. Many diseases such as autoimmunity and cancer require assessment of multiple cell types simultaneously in order to fully unravel the complexity surrounding the disease, metastasis, treatment, and management. We describe a high complexity 7-color IHC chromogen-based staining method to assess in situ tumor microenvironments and show how the staining method can be utilized for quantitative analysis. In comparison with current standard clinical laboratory techniques, this method could reduce the amount of biospecimens used, reduce time for processing, and increase the amount of clinically meaningful data extracted from a single biospecimen. The method can be used broadly to assess any disease and any tissue type with an intact tissue microenvironment.
Optogenetic stimulation of brain cholinergic networks suppresses inflammation
Authors: Pavlov Valentin A , Lehner Kurt R. , Silverman Harold A. , Tsaava Tea , Chavan Sangeeta S. , Tracey Kevin J.
Published: January 2023

Brain cholinergic muscarinic acetylcholine receptor (mAChR) signaling regulates peripheral inflammation via the inflammatory reflex (Nat Rev Endocrinol, 2012, 8:743). Here we provide further insight into this regulation by utilizing optogenetic stimulation, a method allowing precise spatiotemporal neuronal activation. Laser light (473 nm) and transgenic mice expressing light activated channelrhodopsin-2 coupled to a fluorescent protein (ChR2-YFP) under the control of the choline acetyl transferase (ChAT) promoter were used. Optogenetic stimulation of the medial septum, a major source of brain cholinergic output to areas with abundant M1 mAChR localization significantly suppressed serum TNF levels, as compared to sham stimulation (P<0.0398) in ChAT-ChR2-YFP mice during endotoxemia. In contrast to ChAT-ChR2-YFP mice, using the same approach in control (non-carrier) endotoxemic mice did not alter serum TNF. In addition, intracerebroventricular administration of the selective M1 mAChR agonist benzyl quinolone carboxylic acid (5 μg/kg) significantly decreased serum TNF, as compared to vehicle administration (P<0.0180) in C57Bl/6 mice during endotoxemia. These results from ongoing studies reveal a role for forebrain cholinergic M1 mAChR-mediated signaling in controlling peripheral inflammation. These findings may be of interest for development of new approaches for selective brain modulation for the treatment of inflammatory conditions.
Sequence-specific detection of different strains LCMV in a single sample using tentacle probes
Authors: Franco Lina S , Holechek Susan A , Caplan Michael , Blattman Joseph N
Published: January 2023

Viral infections often result in quasi-species of virus strains that can have dramatic impacts on disease outcomes or experimental results. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity in order to design appropriate treatment, and can be useful for many experimental systems. We have developed a novel molecular method for simple and accurate detection of RNA virus genetic variants using Tentacle probes coupled with quantitative PCR. Tentacle probes are modified molecular beacons that are less expensive and dramatically improve false positive rates in routine qPCR. To validate this approach we have designed two tentacle probes for two strains of Lymphocytic Choriomeningitis Virus (LCMV): the parental Armstrong strain and the more virulent clone-13 strain, which differ by only 3 nucleotide substitutions. Using this approach we were able to distinguish these two strains of LCMV. Furthermore, in virus mixing experiments we were able to detect down to 5% of clone-13 diluted in Armstrong, and vice versa. Thus, we have developed a fast, cost-effective approach for differentiating between closely related viruses.
Diferuloylmethane prevents type 1 diabetes development via inhibition of IFNγ-producing T cells.
Authors: Alard Pascale , Harder James W , Fausnaught Thomas , Kosiewicz Michele M
Published: January 2023

Diferuloylmethane (cucurmin), a natural phenol chemical isolated from the plant Curcuma longa (turmeric, is thought to be anti-inflammatory, and has been shown to ameliorate autoimmune diseases, i.e., multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease. We examined the effects of diferuloylmethane on type I diabetes development and the immune response in NOD mice. Treatment of NOD dendritic cells (DC) with diferuloylmethane decreased pro-inflammatory cytokines (e.g., IL-12) in vitro, but had no effect on the anti-inflammatory cytokine, IL-10, or DC viability. Interestingly, the ability of diferuloylmethane-treated NOD DC to induce IFNγ-producing T cells in vitro was decreased by comparison to controls. In in vivo studies, NOD mice treated with diferuloylmethane three times weekly exhibited significantly delayed disease (type 1 diabetes) onset, and dramatically decreased disease incidence and increased survival. This protection was associated with a significant decrease in IFNγ production by T cells in the spleen and pancreatic LN. Moreover, the frequencies of CD4+Foxp3+ and CD4+Foxp3+Neuropilin− cells in the pancreatic LN were not increased by this treatment. Overall, our in vitro and in vivo data show that diferuloylmethane suppresses the differentiation of the IFNγ-producing cells that are involved in type 1 diabetes development. Diferuloylmethane may, therefore, have therapeutic potential for the prevention of the development of type 1 diabetes. Experiments are ongoing to test a more effective delivery system that could enhance bioavailability, i.e., diferuloylmethane-encapsulated nanoparticles.
Prolongation of the remission period and reversal of new onset type 1 diabetes with the selective elimination of autoreactive T cells
Authors: Carroll Kaitlin , McNally Jonathan P , Elfers Eileen , Millen Scott , Hildeman David A , Jordan Michael B , Katz Jonathan D
Published: January 2023

Organ-specific autoimmune diseases such as type 1 diabetes (T1D) are principally mediated by autoreactive T lymphocytes targeting self-antigens. The rapid proliferation and concomitant genomic stress that these T cells undergo during their active immune response to self-antigens affords a unique opportunity for their elimination, since these autoreactive T cells have distinct apoptotic sensitivity as a result of intrinsic DNA damage response upon activation. This sensitivity can be harnessed with a combination of p53 potentiation and cell-cycle checkpoint abrogation (PPCA), selectively eliminating these pathogenic cells while leaving naïve and regulatory T cell populations largely untouched. Using this therapy at the onset of T1D, we are able to significantly reverse new onset T1D (≥200 mg/dL blood glucose) and prolong the remission period in the Nonobese Diabetes (NOD) mouse, representing a novel therapeutic approach with significant translational potential for not only type 1 diabetes, but also other T cell-mediated autoimmune diseases.
Gut bacteria modulate dendritic cell priming of diabetogenic T cell effector functions in a mouse model of autoimmune diabetes
Authors: Mohamed Zaynab Sidi , Lau Kenneth , Ardissone Alexandria , Wasserfall Clive , Atkinson Mark , Triplett Eric , Larkin Joseph
Published: January 2023

Increasing evidence implicates a contribution of the microbiome, within the gastrointestinal tract, in the initiation and progression of type 1 diabetes (T1D). Notably, our understanding of mechanisms whereby gut flora interaction with the host immune system can influence T1D pathogenesis remain poorly understood. Comparison of stool samples from the non-obese diabetic (NOD) mouse model of spontaneous T1D and non-obese resistant (NOR) mice revealed distinct bacterial flora communities present within the two strains correlated to a Th17 bias. Given that dendritic cells (DC) are critical in sampling luminal antigens and priming naïve T lymphocyte effector functions, we next interrogated the populations of CD11c+ DC within the lamina propria (LP) of NOD and NOR mice. Phenotypic characterization of LP DC revealed that NOR mice possessed more mature DC denoted by a higher frequency of CD11c+ MHCII+ cells and CD103+ CD86+ cells. We have previously shown that the oral administration of a gut bacterium Lactobaccillus johnsonii (LjN6.2), present in diabetes resistant rodents, was capable of conferring diabetes resistance to diabetes prone rodents while another strain Lactobacillus reuterii (LrTD1) had no effect. Notably, T1D resistance was correlated to a Th17 bias. Hence, we sought to investigate the influence of LjN6.2 on NOD and NOR DC in comparison to LrTD1 in vitro. We found that LjN6.2, but not LrTD1 mediated an increase in IL6 and IL10 cytokine production by DC. Together, these data suggest that gut bacteria have the important ability to shape the maturation and priming abilities of DC. These results could have critical implications regarding the ability of bacterial flora to regulate the generation of a diabetogenic immune repertoire.
Targeting antigen-specific B cells using BAR-transduced cytotoxic and regulatory T cells
Authors: Zhang Ai-Hong , Parvathaneni Kalpana , Yoon Jeongheon , Kim Yongchan , Scott David W
Published: January 2023

Eliminating adverse humoral immune responses in an antigen-specific manner is highly desirable. However, effective means for achieving this goal is still lacking. Analogous to the chimeric antigen receptor (CAR) strategy successfully used in cancer immunotherapy, we have created a chimeric receptor comprising a protein antigen or its domain, linked with the signal transduction domains, CD28-CD3ζ. We termed this receptor as BAR for B-cell receptor (BCR)/antibody receptor. Human CD4 T cells transduced with a BAR containing OVA (OVA-BAR) proliferated in the presence of anti-OVA antibodies. The functional utility of BAR was confirmed in that human CD8 T cells transduced with a C2-BAR that contained the C2 domain of human clotting factor VIII (FVIII) demonstrated killing activity towards co-cultured FVIII C2-specific B cell hybridomas. To test the hypothesis that BAR transduced regulatory T cells (Tregs) could directly and effectively suppress the activity of specific B cells, C2-BAR and OVA-BAR transduced human CD4+ Tregs have been successfully expanded in vitro, and shown to maintain Treg phenotypic markers in terms of co-expression of Foxp3 and Helios. Functional suppression of specific B-cell activity will be tested using a murine FVIII-specific B cells in a xenogeneic model in vitro and in vivo.
Tetrandrine (TET), an immunosuppressive component of Chinese herb, induces tolerogenic dendritic cells and consequently expands regulatory T cells
Authors: Chen Xin , Oppenheim Joost J
Published: January 2023

Tetrandrine (TET) is a chemical component derived from the Stephania Tetrandra, a traditional Chinese medicine with anti-inflammatory and immunosuppressive properties. The underlying mechanism of its immunosuppressive effect remains incompletely understood. There is now compelling evidence that CD4+Foxp3+ regulatory T cells (Tregs) play an indispensable role in the maintenance of immune homeostasis by actively suppressing the activation of effector immune cells. It is also well established that the dendritic cells (DCs) with immunosuppressive property, termed tolerogenic DCs (Tol-DC), are instrumental for the generation and expansion of Tregs. Therefore, we examined the effect of TET on the differentiation of Tol-DCs and its effect on Tregs. We found that, after pre-treatment with non-toxic concentrations of TET, LPS-activated human monocyte-derived DCs failed to mature. TET-treated DCs (TET-DC) expressed high levels of IL-10 and low levels of IL-12, and expressed high levels of ILT3 mRNA, indicative of a typical Tol-DC phenotype. The allostimulatory effect of TET-DC was markedly reduced. Importantly, co-culture with TET-DC, Foxp3 expression by CD4 cells was increased ~2-fold. Intra-peritoneal (i,p.) injection of TET into normal mice, TET treatment also increased the number of functional suppressive Tregs which produced ~4-fold higher IL-10 than Tregs isolated from control mice. Taken together, our study revealed that TET had the capacity to promote the generation of Tol-DC and consequently to expand Tregs. Therefore, induction of immunosuppressive network of Tol-DC and Tregs represent a novel mechanism underlying the anti-inflammatory effect of this Chinese herb-derived naturally occurring compound.
CTLA-4-targeted proteins in differentiating CD8+ T lymphocytes
Authors: Brunner-Weinzierl Monika C , Wissing Josef , Arra Aditya , Pierau Mandy , Klawonn Frank , Jänsch Lothar , Lingel Holger
Published: January 2023

The blockade of inhibitory receptors like Cytotoxic T Lymphocyte-associated molecule-4 (CTLA-4, CD152) on T cells is a potent approach to regain functionality of ineffective immune responses against tumor cells. However, the underlying mechanisms of CTLA-4 mediated attenuation in CD8+ T cells are still incompletely explored. To discover proteins targeted by CTLA-4, we used a CD8+ T cell in vitro activation system combined with iTRAQ proteomic analysis. The mass spectrometry data revealed that CTLA-4-controls central changes in phosphorylation of proteins involved in T cell signaling, RNA processing, DNA replication and microtubule polymerization. Beside other molecules, we identified and verified CTLA-4-mediated post-translational modifications of the AP-1 family transcription factor Fos-related antigen 2 (Fra-2) as well as the regulation of an inhibitor of protein translation and set up a network of proteins altered by CTLA-4. Consequently, CD8+ T cells deficient for CTLA-4-induced targets showed enhanced production of otherwise repressed effector molecules like IFN-gamma and Granzyme B and ultimately proofed to be superior in control of tumor growth. These findings delineate how CTLA-4 is able to modulate CD8+ T cell differentiation and provide new strategies to improve anti-tumor immune therapy.
Nrf2 induces IL-17D to mediate tumor and virus surveillance
Authors: Saddawi-Konefka Robert , Seelige Ruth , Gross Emilie T.E. , O’Sullivan Timothy Edward , Harismendy Olivier , Bui Jack D
Published: January 2023

Cells undergoing xenobiotic or oxidative stress activate the transcription factor Nrf2, which initiates an intrinsic “stress surveillance” pathway involving detoxifying enzymes. We recently found that the cytokine IL-17D effects a form of extrinsic stress surveillance by inducing antitumor immunity, but how IL-17D is regulated remains unknown. Here, we show that Nrf2 induced IL-17D directly in primary tumors as well as during viral infection in vivo. Expression of IL-17D in tumors and virally infected cells is essential for optimal protection of the host as il17d−/− mice experienced a higher incidence of tumors and exacerbated viral infections compared to WT animals. Moreover, activating Nrf2 to induce IL-17D in established tumors led to NK cell-dependent tumor regression. These data demonstrate that Nrf2 can initiate both intrinsic and extrinsic stress surveillance pathways and highlight the use of Nrf2 agonists as immune therapies for cancer and infection.
AK4 influences mTOR activation and the modulation of platelet-derived growth factor receptors
Authors: Larson Candice , Bautista Jonah , Camille Nelianie , Lanning Nathan
Published: January 2023

Cell growth is important for organisms; however, uncontrolled growth results in their demise. The mTORC1 pathway, when turned on, results in the following functions protein and lipid synthesis, lysosome biogenesis, autophagy, and energy metabolism. The inability to adequately turn off mTORC1 leads to cancer. Adenylate kinase 4 has shown to be both a modulator and regulator for maintaining cellular homeostasis in cancer cells. It interacts with the mitochondrial ADP/ATP exchange and promotes stress response proteins for cell proliferation. Platelet-derived growth factor receptors are also ligands that are involved in stimulating growth. They are another extracellular cue shown to drive the cell cycle. I hypothesize that when AK4 is knocked down the mTORC1 pathway will be turned off and inhibit cell growth while down regulating platelet-derived growth factor receptors on the surface of the cell. To test this hypothesis AK4 will be knocked down and the effects of cell growth as well as platelet-derived growth factor receptors will be observed quantitatively. The knockdown of AK4 expression will be modulated by siRNA and the expressions will be visualized via western blot and luminescent-based assay. I expect to find evidence to suggest AK4 in combination of platelet-derived growth factor receptors modulate a role in cancer biology.
The role of STAT3 (signal transducer and activator of transcription 3) in non-small cell lung cancer (NSCLC)
Authors: Heim Lisanne , Andreev Katerina , Balabko Ljubov , Trufa Denis Iulian , Sirbu Horia , Hartmann Arndt , Finotto Susetta
Published: January 2023

Non-small cell lung cancer (NSCLC) is associated with high mortality worldwide underlining the need for novel therapeutic approaches. Signal transducer and activator of transcription 3 (STAT3) is activated via pro-inflammatory cytokines and growth factors (e.g. IL-6, EGF, VEGF) and may promote tumorigenesis by regulating cellular senescence, proliferation and apoptosis. We recently found an upregulation of the IL-6R expression in the tumoral region of patients with lung adenocarcinoma (ADC) as compared to the tumoral region of patients with squamous cell lung carcinoma (SCC). Consistently, increased levels of pSTAT3 were observed in the tumoral region of ADC as compared to SCC patients. Moreover, stimulation of A549 lung adenocarcinoma cells with IL-6 activates pSTAT3 and promotes cell proliferation.

STAT3 is also important for the differentiation of both Foxp3+ T regulatory cells which suppress the anti-tumor immune response and RORγt+ T helper 17 cells, a T cell subtype expanded in lung adenocarcinoma. Indeed, ablation of STAT3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis.

However, the role of STAT3 in tumor-infiltrating lymphocytes (TIL) in NSCLC is largely unknown. We therefore will investigate the functional effects of STAT3 inactivation in T cells in a murine model of lung adenocarcinoma by using mice with conditional inactivation of STAT3 (STAT3fl/flCD4Cre). To detect STAT3-regulated genes we will isolate TIL of tumor-bearing STAT3 conditional knockout mice and control littermates.

These studies will provide new insights into the functional role of STAT3 in NSCLC and may lead to novel immunotherapeutic approaches for this disease.
Exogenous IL-33 overcomes T cell tolerance in acute myeloid leukemia
Authors: Qin Lei , Dominguez Donye , Chen Siqi , Fan Jie , Long Alan , Zhang Minghui , Fang Deyu , Kuzel Timothy , Zhang Bin
Published: January 2023

Leukemic cells can be recognized and attacked by the immune system. However, emerging studies suggest that dominant peripheral tolerance is a major mechanism of immune escape in disseminated leukemia. Therefore, novel strategies are needed to overcome the tolerance and restore the anti-leukemia T cell function for both leukemia clearance and relapse prevention. Using an established murine acute myeloid leukemia (AML) model, we have demonstrated that systemic administration of recombinant IL-33 dramatically inhibits leukemia relapse and prolongs the survival of leukemia-bearing mice in a CD8+ T cell dependent manner. Exogenous IL-33 treatment enhanced anti-leukemia activity by increasing the expansion and IFN- g production of leukemia-reactive CD8+ T cells. Moreover, IL-33 induced dendritic cell (DC) maturation and activation in favor of its cross presentation ability to evoke a vigorous anti-leukemia immune response. Finally we found that the combination of PD-1 blockade with IL-33 further prolonged the survival with half of the mice achieving complete regression. Our data establish a role of exogenous IL-33 in reversing T cell tolerance and suggest its potential clinical implication into leukemia immunotherapy.
Tumor-associated myeloid cells convert indolent prostate cancer to aggressive disease
Authors: Sass Stephanie N , Gollnick Sandra O
Published: January 2023

Difficulties differentiating indolent from progressing prostate cancer (CaP) results in ineffective treatment strategies. Tumor-associated myeloid cells (TAMC) contribute to tumor progression; yet the role TAMC play in the conversion from indolent to progressing CaP is unclear. To test whether TAMC promote CaP progression we developed a murine model using two isogenic cell lines from the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Tumorigenic C2 cells mimic progressing disease while non-tumorigenic C3 cells represent indolent disease. We report for the first time that CD11b+ TAMC isolated from C2 tumors co-injected with the non-tumorigenic C3 cells drive C3 tumor growth. In contrast, TAMC isolated from C2 tumor-bearing spleens or naïve spleens did not promote C3 tumor growth. This is the first indication that TAMC drive progression of indolent disease. Strikingly, tumor cells explanted from TAMC-induced C3 tumors were tumorigenic in the absence of additional TAMC, suggesting that TAMC induce stable changes within the C3 cells. These results have been recapitulated with the non-tumorigenic human cell line BPH-1 (benign prostatic hyperplasia). Furthermore, TAMC-induced tumor growth occurs via a TAMC secreted factor. Using a 5-day transwell system PC3M (human prostatic adenocarcinoma) TAMC were able to drive BPH-1 tumorigenesis. Future work will determine the factor secreted by TAMC and identify the molecular mechanisms responsible for tumor promotion. This work has the potential to identify critical factors responsible for the conversion of indolent to progressing disease thus laying the groundwork for novel prognostic and therapeutic strategies.
Elucidating the oncogene-driven regulatory T cell responses during melanoma tumorigenesis.
Authors: Chabanet Tamer Basel , Steinberg Shannon , Zhang Peisheng , Turk Mary Jo
Published: January 2023

Regulatory T cells (Tregs) are critical mediators of tumor immune suppression. While Tregs are found in established tumors, little is known about the kinetics and dynamics of Treg accumulation and the factors promoting it during oncogene-driven tumorigenesis. The present studies characterize Treg response kinetics and conversion dynamics during early tumor development in a model of autochthonous, tamoxifen-inducible BrafV600E Pten−/− melanoma. While microscopic skin dysplasia appeared 16 days following tumor induction, FoxP3+ Treg frequency and absolute numbers accumulated by day 26, coinciding with locally-invasive neoplasm development. BrafV600E inhibition with PLX4720 prevented Treg accumulation, suggesting that oncogenic Braf controls this process. Following adoptive transfer of CD4+ T cells specific to TRP-1, antigen-specific FoxP3+ Tregs preferentially accumulated in tumor-induced skin and draining lymph nodes (dLNs), as compared to tumor-free counterparts. In contrast, depleting Tregs prior to transfer abrogated the TRP-1-specific Treg response, suggesting a predominant role for natural Tregs. Furthermore, we observed an increase in chemokine (C-C motif) ligand 17 (Ccl17) and Ccl2 prior to Treg influx to tumor-induced skin. Oncogenic Braf regulated expression of Ccl17 and Ccl2, both of which mediated Treg migration in vitro. We are currently investigating the putative role of Ccl17/2-Ccr4 chemotactic axis in Treg recruitment to sites of Braf-driven tumorigenesis in vivo.
Reverse engineering B. Schlosseri developmental-regulated cell death programs for cancer immunotherapy
Authors: Corey Daniel M , Ring Aaron , Rosental Benyamin , McCracken Melissa , Weissman Irving L
Published: January 2023

B. schlosseri, a colonial marine species, offers a unique platform to study mechanisms underlying cellular recognition. We used adoptive transfer studies, FACs analysis, and RNA-sequencing to study the progression by which fused-colonies eliminate chimeric partners. We show the principle cell type mediating elimination of partners in a chimera is a cytotoxic Morula cell (MC), which utilizes developmental-regulated programmed cell death pathways, initiated during the ‘takeover’ phase of colony life, to eliminate partners. Adoptive transfer of FACs purified cytotoxic MCs from third party donors into recipients recapitulates events of chimeric partner elimination. Transfer of 1×10^5 allogeneic MCs eliminated 33 of 78 (42%) of recipient palleal buds and 20 of 76 (20.5%) adult zooids by day 14 post-transfer. Transfer of phagocytic populations had only minimal effects on recipient colonies. We identified 275 genes (FDR < 0.05) from global RNA-seq expression profiling that increased by expression of at least 50 fold associated with elimination of chimeric partners. These include an IL-17 family member (IL-17C), the phagocytic receptors (MerTK, AXL), and programmed cell removal factors (CALR, MFGE-8). To probe the role of specific receptors we used in vitro studies. Recombinant Il-17c promoted allogeneic cytotoxicity while MFGE-8, a phosphatidylserine binding protein, induced engulfment of apoptotic cells. Studies using chimeric phosphatidylserine binding-cytokine fusion proteins are underway. Preliminary data suggests these fusion proteins promote inflammatory turnover of apoptotic cells. These studies provide new insights by which reverse engineering cell death programs can be utilized to enhance anti-tumor immunity.
Nanovaccine-based immuno-gene therapy: Targeting microRNA-148a for reprogramming tumor-associated dendritic cells in vivo
Authors: Ma Yifan , Liu Lanlan , Yi Huqiang
Published: January 2023

Immunosuppressive tumor-associated dendritic cells (TADCs) are a major obstacle for developing effective cancer vaccines. However, reprogramming TADCs in vivo remains to be difficult due to their poor responsiveness to TLR stimulation. Herein, we have identified a dysregulated miR-148a/DNMT1/ SOCS1 axis as a unique mechanism for dampened TLR3 stimulation in TADCs, in which tumor-elevated miR-148a increased immunosuppressor SOCS1 through targeting DNMT1, thereby abolishing TLR3-stimulated TADC maturation. However, targeting miR-148a with miR-148a inhibitor (miR-148ai) effectively recovered the sensitivity of TADCs to TLR3 stimulation through modulating the miR-148a/ DNMT1/SOCS1 axis. More importantly, the codelivery of miR-148ai and poly I:C (TLR3 agonist) with nanovaccines not only synergistically induced TADC maturation in vivo, but also ameliorated tumor immunosuppression, thereby leading to robust anti-cancer immunity and dramatic tumor suppression. This study proposes a nanovaccine-based immuno-gene therapy with the integration of miR-148a inhibition and TLR3 stimulation as a novel therapeutic approach to boost anti-cancer immunity by reprogramming TADCs in vivo.
microRNA-155 is a master regulator of dendritic cell function in breast cancer
Authors: Fan Daping , Wang Junfeng
Published: January 2023

In anti-tumor immunity, dendritic cells (DCs) capture, process and present tumor antigens to T cells, and initiate a tumoricidal response. However, DCs are often found dysfunctional due to their exposure to the tumor microenvironment (TME), leading to tumor escape from immune surveillance. The mechanisms underlying the regulation or dysregulation of DC function in the context of tumor has not been well elucidated. Here a vital role of microRNA-155 (miR-155) in regulating the function of DCs in breast cancer is reported. miR-155 expression is closely correlated with the maturation status, migration ability and cytokine production of DCs, as well as their ability to activate T cells. We further demonstrate that miR-155 regulates DC migration through epigenetic modulation of CCR7 expression. Moreover, IL-6 and IL-10, two cytokines abundant in the TME, are found to impair DC maturation by suppressing miR-155 expression. Animal studies show that lack of miR-155 diminishes, while forced expression of miR-155 enhances, the effectiveness of DC-based immunotherapy for breast cancer. These findings suggest miR-155 is a master regulator of DC function in breast cancer, and boosting the expression of a single microRNA, miR-155, may significantly improve the efficacy of DC-based immunotherapies for breast cancer.
NKT ligand loaded E6/E7 Antigen expressing B cell & Monocyte based Vaccine effectively controls growth of heterogeneous tumors containing MHC Class I deficient cells by reversal of NK cell anergy
Authors: SEO HYUNGSEOK , JEON IN-SU , Kang Chang-Yuil
Published: January 2023

MHC Class I downregulation or deficiency has been reported in advanced tumor microenvironments of clinical cases. Malignant transformation and in vivo selection of highly metastatic cells show MHC Class I loss neoplasm, which contributes to the evasion of tumor from the host immune system. These tumor microenvironments are major impediments for cancer immunotherapy, thereby imposing restriction of cytotoxic T lymphocytes. Besides, it has been reported MHC Class I low states induce NK cell anergy, which interrupt NK cell mediated antitumor immune responses. To overcome these limitations, we designed NKT ligand loaded, E6/E7 Ag expressing b cell & monocyte based vaccine. We found our vaccine elicited NKT, NK cell and Ag specific CTL response, and effectively eradicated solid tumors. We also found a negative correlation between tumor growth and MHC Class I expression level. When the MHC Class I expression level was lowered, the major antitumor effector cell is changed from CTL to NK cells. Thus, we generated a MHC Class I knock out tumor cell line for testing our vaccine effects on MHC Class I deficient situation to mimic actual clinical cases. We established heterogeneous tumor models, which contains various percent of MHC Class I KO tumor cells. The data show our vaccine eliminated heterogeneous tumors, which contains up to 10% MHC Class I KO cells, and effectively eradicated of MHC Class I KO cells via NKT, NK cell. Moreover, our data show this NK anergy in MHC class I KO tumor-bearing mice was reversed by our vaccination via NKT cell producing cytokines. Taken together, our vaccine induced diverse immune responses overcoming immunoresistant tumor microenvironment and is expected to effectively cure advanced tumor patients in clinical trials.
Combination of IL-12 and radiotherapy induced regression of large orthotopic liver cancers through modulation of immune cells in tumor microenvironment
Authors: Wu Chia-Jen , Tsai Yi-Ting , Lee I-Jung , Wu Ping-Yi , Tao Mi-Hua
Published: January 2023

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide with few therapeutic options. Radiation is wildly used for cancer treatment, but local relapse often occurs. Interleukin 12 (IL-12) has potent antitumor activities by activating natural killer cells (NK) and CD8+ T cells, reprogramming myeloid cells to an immune active phenotype, and inhibiting new blood vessel formation. We hypothesized that combined radiation and IL-12 treatment may change tumor suppressive microenvironment and induce antitumor immune responses. Our data showed that only combination of radiation and intratumoral injection of adenovirus encoding IL-12 (Ad/IL-12) led to tumor regression in mice with large subcutaneous or orthotopic HCC, and significantly prolonged their survival. Mechanistic studies revealed that combination therapy enhanced antigen (Ag) presentation ability of DCs by increasing MHC class II, CD40 and CD86 expression. Combination therapy also significantly reduced numbers of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and impaired their suppressive functions by reducing reactive oxygen species (ROS) production. Accordingly, combination therapy significantly increased the cell numbers of tumor-infiltrating CD8+ T cells and their expressions of CD107a and TNF-α. The cell numbers of tumor-infiltrating NK cells was not affected, but the expression of activation markers was significantly increased after treatment. Together, our results demonstrate that combination therapy effectively against advanced HCC through activating tumor-infiltrating NK cells and tumor-specific CD8+ T cells, enhancing Ag presentation ability of DCs while reducing the suppressive functions of MDSCs.
NK cells are not lytic and act exclusively as helper cells in the gp96-mediated immune response
Authors: Sedlacek Abigail L , Kinner Lauren B , Binder Robert J
Published: January 2023

Through release from necrotic cells or extraneous administration, immunogenic Heat Shock Proteins (HSPs) in the extracellular environment elicit T cell responses with specificity to the peptides that the HSPs chaperone. Hence, immunogenic HSPs carry the antigenic fingerprint of the cell from which they are isolated. Thus, tumor derived gp96 (a prototypical immunogenic HSP) is being investigated as a potential immunotherapy. CD4+ and CD8+ T cell responses are elicited following immunization with gp96 and are dependent on a cross-priming antigen presenting cell (APC). While a role for natural killer (NK) cells has been postulated, their function in the gp96-mediated immune response is unknown. In these studies, we investigated how NK cells are activated following immunization with gp96 and what role they play in the ensuing immune response. We show that NK cells are indirectly activated by gp96 matured APCs. Following immunization with tumor-derived gp96, we show that while NK cells are essential during the effector phase of the anti-tumor immune response in vivo, they do not exhibit increased target cell lysis ex vivo. Alternatively, they produce IFNγ and are necessary for T cell mediated target lysis and optimal APC maturation. We’ve compared gp96 activated NK cells to those indirectly activated by other mediators (LPS and IL-2) and determined that gp96 activated NK cells present a unique phenotype where IFNγ production is observed, but other classical functions associated with activated NK cells, i.e. increased expression of granzyme B, do not occur. Collectively, these data indicate that gp96-activated NK cells act exclusively as helper cells to mediate the anti-tumor immune response.