bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nonencapsulated Streptococcus pneumoniae avoid innate immunity clearance through reactive oxygen species resistance
Authors: McDaniel Larry S , Thompson Courtney D , Holcomb Shelby , Keller Lance E 
Published: August 2023

Abstract
Neutrophils provide an important mechanism of clearance for Streptococcus pneumoniae. Therefore, evasion of the innate immune response is essential for pneumococci to cause disease. The pneumococcal capsule is protective against phagocytosis but nonencapsulated Streptococcus pneumoniae (NESp) lack this capsule. Despite the lack of capsule NESp effectively colonize and cause invasive and noninvasive disease. All invasive isolates of NESp express the oligopeptide transporters AliC and AliD, which are required for virulence in multiple animal models. AliC and AliD alter downstream gene expression, and it is hypothesized that one of these regulated genes is responsible for increased immune evasion of NESp. A mutant library of AliC/AliD regulated genes was created and tested for virulence in a Galleria mellonella model of infection. Genes of interest from the in vivo screen were tested for the ability to resist phagocytic killing by neutrophil-like cells. Resistance to killing through reactive oxygen species (ROS) was examined. Of the seven AliC/AliD regulated genes tested, LytFN1 and MgtC mutants were found to have significantly reduced virulence compared to wildtype. A 40% decrease in resistance to phagocytic killing was observed in both mutants, as well as significantly decreased survival during exposure to ROS from growth in 2.5 mM hydrogen peroxide. Expressing AliD in a pneumococcal background that does not natively have the oligopeptide transporter significantly increased virulence and ROS resistance. Regulation of gene expression by AliC and AliD promote pneumococcal survival and virulence by increasing resistance to ROS mediated clearance and partially complements the lack of the opsonophagocytosis resistant capsule.
Institutional funds
A gut commensal protist protects against virus-mediated loss of oral tolerance
Authors: Sanchez Luzmariel Medina , Zeng Yanlin , Siller Magdalena , Brigleb Pamela H , Sangani Kishan A , Dermody Terence S , Jabri Bana , Verdu Elena F , Meisel Marlies , Hinterleitner Reinhard 
Published: August 2023

Abstract
Loss of oral tolerance (LOT) to gluten, characterized by a T helper 1 (Th1) gluten-specific immune response, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. We hypothesized that certain gut microbes have the capacity to protect against virus-mediated LOT. By using our previously defined reovirus-mediated LOT CeD model, we discovered that the gut colonizing protist Tritrichomonas promotes oral tolerance and protects against reovirus-mediated LOT by suppressing the reovirus-induced proinflammatory program of dietary-antigen-presenting CD103+ dendritic cells. Importantly, Tritrichomonas-mediated protection against T1L-induced LOT is not attributable to differences in antiviral host responses and is independent of the microbiota. Mechanistically, we show that Tritrichomonas colonization restrains reovirus-induced inflammatory responses in dendritic cells and thus limit their ability to promote Th1 immune responses. Finally, our studies using human stool samples support a role for Tritrichomonas sp. colonization in protecting against development of CeD.
Supported by grants from NIH (T32 AI089443)
Type I and II interferons induce granulopoiesis and correlate with organ damage during infection-induced shock
Authors: Bahr Amber , Howard Jennifer , MacNamara Kate C 
Published: August 2023

Abstract
The mechanisms that drive pathology in infection-induced shock are not well understood. The tick-borne pathogen Ixodes ovatus erhlichia (IOE) induces severe inflammation, vascular damage, liver injury, and death. Mice that lack the receptor for type I IFN, or receptors for both type I and II IFNs, improves survival by 50–70 and 90%, respectively. IOE infection induced a significant increase in circulating granulocytes that depended on both type I and II IFNs. We also observed an IFN-dependent increase in eosinophils and basophils in the bone marrow in IOE-infected C57BL/6TA (WT). Although IOE infection induced an increase in bone marrow IL-5 and GM-CSF levels independent of IFN signaling, bone marrow concentrations of IL-3 and IL-13, both known to promote eosinophil differentiation and survival, increased in an IFNAR-dependent manner. Furthermore, we demonstrate an infection- and IFN-dependent increase of CCL3 and CCL5, two chemokines that promote eosinophil activation and migration. While bone marrow and blood eosinophils and basophils were increased, we observed a specific depletion of eosinophils in WT liver at day 7 post-IOE infection, correlating with increased cell death and severe liver pathology. In contrast, basophil frequencies were increased in IOE-infected WT mice. Additionally, Ifnar-deficient and Ifnar-Ifngr-deficient mice exhibited normal eosinophil and basophil frequencies and reduced liver inflammation, similar to uninfected mice. Together, our data reveal an IFN-driven cytokine and chemokine response that correlates with rampant granulopoiesis and mortality and provide evidence that IFN-induced eosinophil activation may contribute to vascular leak and liver damage in infection-induced shock.
Supported by grants from NIH R35GM131842
Exploring the Role of TVM Cells in Age-Related CD8+ T Cell Dysfunction through ATAC-seq and RNA-seq Analysis
Authors: Thiele Daniel , Nguyen Angela , Hussain Tabinda , Barugahare Adele , Bennett Taylah , Li Jasmine , Russ Brendan , Quinn Kylie , Turner Stephen J , La Gruta Nicole L 
Published: August 2023

Abstract
Virtual memory T (TVM) cells are a unique subset of CD8+ T cells that have both classical memory and innate-like immune function. Unlike other CD8+ T cell subsets, TVM cells are highly dependent on cytokines for their homeostasis and are selectively depleted in mice lacking IL-15. In aged mice and humans, TVM cells acquire T cell receptor-associated defects, which may contribute to the decline in overall CD8+ T cell function seen with aging. TVM cells make up a large proportion of the naive CD8+ T cell pool in aged individuals, making them an ideal target for age-specific therapeutic interventions. In this study, we performed ATAC-seq and RNA-seq analysis on young and aged mouse CD8+ T cell subsets and identified several key signatures associated with age-related dysfunction specifically in TVM cells. These signatures reveal a bifurcation in the aging process whereby memory-phenotype CD8+ T cells are affected differently by the aging environment. The aged-specific TVM signature is distinct from that of conventional memory CD8+ T cells and results in the upregulation of immunomodulatory molecules such as Fcgr2b, which encodes the low-affinity inhibitory Fcγ receptor. Expression of FcγRIIB on CD8+ T cells has been shown to play a role in suppressing anti-tumor immunity and mitigating graft-versus-host disease. We show that a subset of aged TVM cells upregulate FcγRIIB independently of antigen exposure and are prone to apoptosis in vitro. Blockade of FcγRIIB has been shown to augment CD8+ T cell responses in vivo, making it a potential therapeutic target for restoring function in aged TVM cells. These findings provide insight into the mechanisms of age-related immune dysfunction and potential strategies for targeting TVM cells in the elderly.
Supported by grants from NHMRC/ARC
BMI-1 repression of the CD8+ T cell effector program is essential for memory formation
Authors: Turner Stephen John , Udupa Vibha , Bruer Thomas , Sng Xavier , O’Hara Jessie , Barugahare Adele , Bennett Taylah , Zhang Zhongming , Degli-Esposti Mariapia , Russ Brendan 
Published: August 2023

Abstract
NaïveCD8+ T cell activation results in a program of proliferation and differentiation that results in large scale changes in phenotype and function that is critical for pathogen control and establishment of immunological memory. While these changes are underpinned by structural and biochemical changes in genome regulatory elements, the mechanisms that mediate these changes are not fully elucidated. Polycomb Repressive complexes (PRC) are chromatin remodeling complexes implicated in silencing genes encoding crucial developmental regulators. We demonstrated that upregulation of PRC1 components occurs rapidly upon naïve CD8+ T cell activation and was associated with maintenance of ubiquitination at key effector transcriptional regulators. Importantly, in vitro activation of PRC1-deficient CD8+ T cells resulted in greater proliferation and more rapid acquisition of effector function compared to wildtype cells. Use of two different viral infection models demonstrated that T cell specific PRC1 deficiency resulted in a greater effector expansion and effector differentiation with establishment of dysfunctional memory T cell populations. Strikingly, even after acute infection, the resulting memory T cells exhibited phenotypic and molecular markers of T cell exhaustion and were unable to be recalled. Our data suggest that PRC1 regulation early after CD8+ T cell activation is essential for restraining early effector T cell commitment and is a key step in establishing immunological CD8+ T cell memory.
Supported by grants from National Health and Medical Research Council of Australia (Ideas Grant 2020562).
TCF-1 dictates lung tissue resident helper T cell and memory B cell development during flu
Authors: McGee Michael C , Magazine Nicholas , Zhang Tianyi , Babawale Pius , Huang Weishan 
Published: August 2023

Abstract
Influenza (flu) infections are a significant cause of annual human morbidity and mortality. The efficacy of seasonal flu vaccines depends on the accuracy of the prediction of the circulating strains for the upcoming season. To develop flu vaccines that are broadly effective and more sustainable, induction of a cross-reactive memory immunity is desirable. Flu infection induces cross-reactive tissue resident memory T (Trm) and B (Brm) cells which rapidly respond to reinfection. Tissue resident helper T (Trh) cells are a population of T follicular helper (Tfh)-like CD4+ Trm cells that promote the development of Brm cell responses following flu infection. The transcription factor TCF-1, encoded by Tcf7, is critical in the development of both memory T cells and Tfh cells following viral infections. However, the role of TCF-1 in Trh cell development has not been investigated. Utilizing a Tcf7fl/flCD4Cre conditional knockout mouse model in conjunction with intravascular labeling and influenza A peptide MHC Class II tetramers, we found that TCF-1 is required for the differentiation of flu-specific Trh cells, as well as optimal Brm development following flu infections. Most significantly, upon heterosubtypic reinfection, the Trh population in the Tcf7fl/fl control mice expanded while it became undetectable in the Tcf7fl/flCD4Cre mice, coinciding with a severely impaired lung resident germinal center B cell and CD138+ plasma cell response. Together, our data indicates that TCF-1 is critical in the development of lung tissue resident helper cells and tissue resident memory B cell response following flu infections. This work has implications for the development of universal flu vaccines.
Towards molecular mechanisms for pathogenic Th17 cell differentiation in ocular autoimmunity
Authors: Bass Victor , Mattapallil Mary J , Xu Biying , Caspi Rachel , Shih Han-Yu 
Published: August 2023

Abstract
CD4+ T helper (Th) cells play critical roles in coordinating proper immune responses by differentiating into effector subtypes specialized against different types of pathogens, while dysregulated Th cells can drive autoimmune disease by triggering inappropriate immune responses. Pathogenic Th17 (pTh17) cells, which combine behaviors of classic Th1 and Th17 effector cells by producing both interferon-gamma (IFNg) and interleukin-17A (IL17A), have been implicated in autoimmune diseases. Our goal is to map regulatory networks and identify novel regulators underlying pTh17 differentiation and activation using the murine model experimental autoimmune uveitis (EAU), in which ocular autoimmunity is driven by pTh17 cells.
To track pTh17 differentiation, we bred a double reporter mouse with IL17A-cre/tdTomato-flox tracer and IFNg-YFP. We sorted both Tomato+YFP+ and Tomato+YFP- CD4+ T cells from eyes and spleens after EAU induction for single-cell RNA-seq. Clustering of single-cell transcriptomes revealed distinct transcriptomes between eyes and spleen. In the eye, we identified clusters similar to pTh17 and non-pathogenic (np)Th17 populations defined in a myelin autoimmunity model. The pTh17 cluster contained both double positive and Tomato single positive cells, suggesting a heterogeneous pathogenic population. In contrast, the npTh17 cluster only contained single positives. Differential gene expression analysis showed that pathogenic and non-pathogenic clusters utilize distinct sets of transcription factors. We are further characterizing epigenetic differences between pTh17 and npTh17 to better understand how distinct regulatory networks shape pTh17 differentiation.
Supported by the NEI Intramural Research Program fund (ZIAEY000569-01)
Divergent roles for STAT4 in shaping effector differentiation of ILC1 and NK cells during gut inflammation
Authors: Sciume Giuseppe , Mazej Julija , Laffranchi Mattia , Candelotti Arianna Maria , Gadina Massimo , Santoni Angela , Scarno Gianluca 
Published: August 2023

Abstract
Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require the transcription factor STAT4 to elicit rapid effector responses during infections. Herein, by combining genetic and transcriptomic approaches, we revealed that STAT4 played an unexpected divergent role in regulating effector differentiation of murine ILC1 and NK cells during intestinal inflammation. Stat4 deletion in Ncr1-expressing cells led to an increased generation of cytotoxic ILC1 in the inflamed large intestine. By contrast, Stat4-deficient NK cells showed impaired terminal differentiation, characterized by lower levels of IRF-8 and KLRG1. STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation and type 2 adaptive T cells in the large intestine. Collectively our data shed light on distinctive mechanisms of transcriptional regulation driven by STAT4 in NK cells and ILC1 required for protection during intestinal inflammation.
Italian Association for Cancer Research (AIRC) [MFAG-21311], by Institut Pasteur (France), Transversal Research Program program.
Myeloid cell-cycle-related-kinase drives tumor immunosuppression in hepatocellular carcinoma
Authors: Zhou Jingying 
Published: August 2023

Abstract
Myeloid-derived suppressor cell (MDSC) comprises a heterogeneous population of immature myeloid cells featured by potent immunosuppression. We have uncovered a significant role of hepatoma-intrinsic cyclin-dependent-kinase 20 (CDK20), or cell-cycle-related-kinase (CCRK) in promoting tumor progression and immunotherapy resistance in hepatocellular carcinoma (HCC). As emerging evidence has highlighted the importance of immune cell intrinsic CDKs, we further explored the potentials of CCRK expressions and functions in different immune cells from HCC-bearing mice and patients. Of note, we uncovered specific CCRK overexpression in MDSCs but not lymphocytes, which functioned in regulating MDSC proliferation, immunosuppression, and differentiation. Intratumoral injection of bone-marrow-derived MDSCs with Ccrk knockdown resulted in impaired tumorigenicity, accompanied with increased CD8+T cell responses. Mechanistically, myeloid CCRK upregulation was functional associated with IL-6-mediated MDSC expansion and immunosuppression. Inhibition of myeloid CCRK in turn suppressed STAT3 to revert E4-binding protein 4-dependent transcriptional alterations. Using a transgenic mouse bearing Ccrk-indel mutation (Ccrkindel/indel), we found that impaired Ccrk signal reprogramed the pro-tumoral MDSCs into an antitumoral phenotype, resulting in suppression of tumor growth in our established HCC orthotopic mouse models. As we also showed CCRK overexpression in HCC patient-derived MDSCs, the findings of our study not only unravel mechanistic insights in MDSC maintenance and immunosuppression, but also offer a novel therapeutic kinase-target in HCC therapies.
The project is supported by RGC/GRF14108219.
Supported by grants from general research fund (GRF)/research grants council (RGC) of Hong Kong, 14108219.
Induction of STING/IL-29 signaling in human lung adenocarcinoma/tumor-associated neutrophil cocultures
Authors: Monterroza Lenore , Selvaraj Periasamy , Tirouvanziam Rabindra M 
Published: August 2023

Abstract
LKB1-mutant lung adenocarcinoma (LUAD), is an intractable form of lung cancer. LKB1-mutant LUAD is poor in T-cells and does not respond to checkpoint inhibitor therapy, and instead is enriched in tumor-infiltrating neutrophils (TINs). STING is a key innate sensor mediating anti-tumor responses that is inhibited in LKB1-mutant LUAD. However, the status of STING signaling and if it can be induced in TINs to antagonize tumors is unknown. Here, we developed a novel human LKB1-mutant LUAD/TIN model to tackle this question. Based on prior work from our lab (Dobosh, STAR Protoc. 2021), we grew the H1944 LKB1-mutant LUAD line at air-liquid interface on Alvetex scaffolds (Reprocell), followed by the transmigration of human blood neutrophils. The effect of the STING agonist MSA-2 was assessed in the model by a 20-plex immune mediator assay(Mesoscale). We successfully manufactured a LKB1-mutant LUAD/TIN model from H1944 cells and human blood neutrophils. MSA-2 treatment resulted in significant increases in the STING-regulated mediators IL-6, IP-10 and MIP-1beta. Most strikingly, MSA-2 treatment induced IL-29 (type III interferon) levels by 27-fold, while neither type I nor type II interferons were induced. The model showcased here mimics LKB1-mutant LUAD/TIN interplay and shows responsiveness to STING agonist treatment through induction of type III (but not type I and II) interferon and other immune mediators. Since IL-29 receptor expression is limited to epithelial and myeloid cells (including neutrophils) and IL-29 is known to mediate immunomodulatory and anti-tumor responses, future work will address if and how the large IL-29 increase observed upon MSA-2 treatment affects LUAD proliferation and TIN activation in this context.
Topology of cell-free DNA from tumour cells influences its immunogenicity in macrophages and dendritic cells in vitro
Authors: Malkin Ethan Z , Brooks Patricia , Lamorte Sara , McGaha Tracy , Bratman Scott V 
Published: August 2023

Abstract
Activation of DNA-sensing pathways can induce an anti-tumour immune response, but the mechanisms by which tumour-derived cell-free DNA (cfDNA) activates pro-inflammatory signaling is not well-understood. In particular, the topological features of cfDNA that contribute to DNA sensor activation in phagocytes are unknown. As such, we investigated how tumour-derived cfDNA topology influences DNA sensor activation in macrophages and dendritic cells. We treated a murine colon adenocarcinoma cell line (MC38) with chemotherapy (topotecan or staurosporine) for varying durations to induce cfDNA release. We then harvested cfDNA, separated the protected (membrane-bound) and accessible (non-membranous) fractions, and quantified nuclear and mitochondrial cfDNA abundance. The cfDNA fractions were then added to murine RAW 264.7 macrophages or bone marrow-derived dendritic cells (BMDCs), and a panel of inflammatory markers was measured by RT-qPCR.
We found that accessible cfDNA from MC38 cells treated with chemotherapy for 48h increased Il6 expression in RAW 264.7, but other inflammatory markers were not elevated. Interestingly, both nuclear and mitochondrial cfDNA abundance directly correlated with Il6 expression. Surprisingly, cfDNA did not induce pro-inflammatory signalling in BMDCs. These results demonstrate that tumour-derived cfDNA immunogenicity depends on its mechanistic origins, abundance, and the immune cell type to which it is exposed. We are currently examining the impact of other cfDNA biophysical properties on macrophage activation, as well as elucidating the DNA sensing pathways responsible for inducing Il6 expression. Our findings will help uncover the role of cfDNA in mediating anti-tumour immunity.
E.Z. Malkin is supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-175873) through CIHR. S.V. Bratman is supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre and the Dr. Mariano Elia Chair in Head and Neck Cancer Research at University Health Network and the University of Toronto.
Leukemic Environment Alters Neutrophils Worsening Disease Progression
Authors: Goel Paran , Kuznetsova Valeriya , Franceski Alana , Luca Francesca , Costa Amanda , Crown Brittany , Welner Robert 
Published: August 2023

Abstract
Acute Myeloid Leukemia (AML) is the abnormal differentiation of hematopoietic stem cells in the bone marrow into malignant blasts. The leukemic marrow environment plays an important role during disease progression, by shaping the immune landscape interacting with these blasts. Mounting evidence suggests neutrophils are complicit within tumors by enhancing tumorigenesis and immunosuppression; however, little is known about how these cells contribute to AML even though neutrophils comprise half the marrow cellularity.
Tumor-associated neutrophils are found in an overactivated state, constantly releasing pro-inflammatory cytokines and granular proteins to promote tumorigenesis. Therefore, we investigated the neutrophil phenotype in our AML chimeric mouse model. We observed neutrophils in AML were also in an activated state. More importantly, sorted leukemic-exposed neutrophils were more susceptible to degranulation upon stimulation. Next, our single-cell RNA data showed that neutrophils within the AML environment had higher expression of ELANE, a gene encoding neutrophil elastase (NE). Increased NE levels have been associated with increased tumor burden in many cancers. Although NE levels increase inflammation which drives tumorigenesis, it may also directly increase leukemic blast proliferation. By targeting neutrophils in our AML chimeric model using anti-Ly6G antibody, we found decreased frequency of leukemic blasts in the neutrophil depleted mice. Also, there was a reduced frequency of the Lin-ckit+ CD16/32+ leukemic stem cells. We hypothesize that over-activated neutrophils drive AML progression in the BM by releasing NE, providing a new avenue for disease intervention.