bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapeutic hypothermia rescues severe acute lung injury (ALI) by IL-1β-induced neutrophil extracellular traps (NETs) formation through down-regulation of Gasdermin D
Authors: Shimada Kenichi , Nosaka Nobuyuki , Martinon Daisy , Crother Timothy R , Arditi Moshe
Published: January 2023

ARDS is a serious ALI defined by the acute onset of non-cardiogenic pulmonary edema and hypoxemia. We have previously published that NLRP3 inflammasome is required to develop hypoxemia in LPS plus mechanical ventilator (MV) two-hit-induced ALI. We initially observed that, despite equal neutrophil infiltration, severe alveolar leakage and hypoxemia were not observed in Il1r1KO mice, confirming that NLRP3 inflammasome is critical in this two-hit induced hypoxemia. However, this hypoxemia was significantly attenuated in neutrophil-depleted mice. Furthermore, we found that NETs formations were significantly attenuated in Il1r1KO mice. These data demonstrated that IL-1R signaling plays a regulating role in neutrophil functionality in the alveoli, and not their infiltration. We next examined whether NETs formation contributed to develop hypoxemia in two-hit ALI. by Pad4 inhibitor and DNase I. Both treatments improved the development of hypoxemia. In the course of experiments, we serendipitously found that body temperature is a critical factor regulating severe hypoxemia. Therapeutic hypothermia at 32°C during MV rescued development of hypoxemia with significantly attenuated alveolar leakage, reduced IL-1β secretion, and less NETs formation. 32°C condition inhibited IL-1β secretion by Mϕ as well as NETs induction. Unexpectedly, we observed similar Caspase-1 activities in LPS-primed Mϕ stimulated with ATP or nigericin at 32°C while IL-1βsecretion was significantly reduced. We found decreased GSDMD expression and unsecreted mature IL-1β at 32 °C treatment. Overall our data suggests that hypothermia suppresses ALI development by inhibiting IL-1β secretion and NETs formation through down-regulation of GSDMD.
Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression
Authors: Yu Li , Zhang Bin , Deochand Dinesh , Sacta Maria A. , Coppo Maddalena , Shang Yingli , Guo Ziyi , Rollins David A. , Tharmalingam Bowranigan , Li Rong , Chinenov Yurii , Rogatsky Inez , Hu Xiaoyu
Published: January 2023

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report widespread promoter-proximal RNA polymerase II pausing in resting macrophages, marked by broad co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.
The BCMA:Tomato Mouse – a new reporter to characterize long-lived plasma cells
Authors: Schulz Sebastian R , Wittner Jens , Kueh Andrew J. , Pal Martin , Herold Marko J , Schuh Wolfgang , Jäck Hans-Martin
Published: January 2023

Plasma cells provide humoral protection by secreting large amounts of antibodies. The heterogeneity of the plasma cell compartment has recently been demonstrated by functional studies and subpopulation markers, but the distinct properties of long-lived plasma cells (LLPC) are still unresolved.

B Cell Maturation Antigen (BCMA) has been described as an essential receptor for LLPC survival. To elucidate the mechanisms of BCMA-mediated survival we established a reporter mouse line that expresses a tdTomato fluorescent protein together with BCMA under the control of the promoter of Tnfrsf17, the gene that encodes BCMA. In this reporter model, we could demonstrate the restricted expression of Tomato and BCMA in plasma cells making this an ideal system for the identification and tracking of plasma cells. Importantly, we observed an increase in the abundance of BCMA:Tomato along with a gradual loss of CD19 expression, delineating a developmental progression from plasmablasts to mature plasma cell subsets. In contrast, plasmablasts generated in vitro display only minimal BCMA induction, confirming the independence of BCMA expression from the Blimp1 master regulator of PC differentiation.

In summary, we have developed a new plasma cell-specific reporter model that, together with an inducible BCMA-KO mouse, will allow us to investigate the BCMA-dependent signals of the generation and maintenance of LLPC.

This work was supported, in part, by the Deutsche Forschungsgemeinschaft (DFG) through research grants TRR130 to H.-M.J. and W.S. and GRK1660 to H.-M.J.
Malt1 Protease Is Critical in Maintaining Function of Regulatory T Cells and May Be a Therapeutic Target for Antitumor Immunity
Authors: Cheng Liqing , Lin Xin
Published: January 2023

The paracaspase Malt1 is a key molecule in mediating Ag receptor–induced NF-kB activation in lymphocytes, but the role of Malt1 in the function of regulatory T (Treg) cells is still unclear. Here, we reported that specific deletion of Malt1 in Treg cells would lead to Scurfy-like lethal autoimmune disease, which was caused by Treg cell dysfunction but not number loss. Interestingly, Foxp3Cre-Malt1fl/C472A mice, in which Malt1 protease was specifically inactivated in Treg cells, also displayed spontaneous inflammatory disorders, with severe hair loss and skin hyperplasia. Consistently, Foxp3Cre-Malt1fl/C472A mice showed enhanced antitumor response because of their decreased function and infiltration of Treg cells, as well as reduced CD8+ T cell exhaustion. Gene expression profiling analysis revealed dysregulated expression pattern of Treg effector genes upon Malt1 deletion or its protease inactivation. Together, our data unraveled a critical role of Malt1, especially its protease activity, in maintaining homeostasis and function of Treg cells.
Defining heterogeneity and biological signature in human effector memory CD8+ T cells expressing different levels of IL-7 receptor alpha by high-dimensional analysis
Authors: Shin Min Sun , Kim Dongjoo , Park Hong-Jai , Dong Xuemei , Koumpouras Fotios , Kang Insoo
Published: January 2023

Human effector memory (EM) CD8+ T cells have been divided into a small number of subsets based on the expression of a single or a few cell surface molecules. Previously, we showed the presence of human EM CD8+ T cell subsets expressing IL-7 receptor alpha chain (IL-7Rα) high and low that have different characteristics like the expression of cytotoxic molecules, cytokines, co-stimulatory molecules, and DNA methylation. However, it is unknown whether IL-7Rα can serve as a key molecule in defining a comprehensive landscape of heterogeneity in human EM CD8+ T cells as determined by high-dimensional single cell analysis. Here we addressed this question by performing Cytometry by Time-Of-Flight (CyTOF) and single-cell RNA-seq (scRNA-seq) analysis on human EM CD8+ T cells and analyzing such data using computational algorithms. IL-7Rα had diverse, but organized, expressional relationship in EM CD8+ T cells with molecules related to gene regulation, effector function, survival, and migration, which rendered an immune landscape defining heterogeneous cell subsets. The differential expression of these molecules likely has biological implications as we found in vivo signatures of transcription factors and homeostasis cytokine receptors, including T-bet and IL-7Rα, as well as the possible alterations of EM CD8+ T cell heterogeneity defined by these molecules in lupus patients. Our findings indicate the existence of heterogeneity in human EM CD8+ T cells as defined by distinct but organized expression patterns of multiple molecules in relationship to IL-7Rα and possible biological significance of differential expression of such molecules in modulating downstream events and pathologic conditions.
Nonencapsulated Streptococcus pneumoniae causes Bacteremia in C1q-Deficient Mice
Authors: McDaniel Larry S , Bradshaw Jessica L
Published: January 2023

Since the introduction of the pneumococcal conjugate vaccines, the frequency of infections by nonencapsulated Streptococcus pneumoniae (NESp) has steadily increased. We previously demonstrated that NESp expressing AliC and AliD instead of the canonical capsule synthesis genes in the cps locus are able to survive killing in a whole blood bactericidal assay. Also, NESp expressing AliC and AliD are associated with invasive pneumococcal disease (IPD). The purpose of this study was to determine if NESp expressing AliC and AliD evade complement-mediated killing. Flow cytometry was used to examine binding of C3b and factor H (FH) to NESp in vitro. We used a chinchilla model of otitis media (OM) and mouse models of infection to examine complement interaction with NESp in vivo. Deletion of aliC and aliD resulted in reduced expression of choline binding protein AC (CbpAC), a variant of pneumococcal surface protein C (PspC). There was a four-fold increase in C3b deposition on a CbpAC mutant compared to the wild-type. Deletion of cbpA significantly reduced virulence in OM in which complement is known to play a role. When C1q-deficient mice were intravenously challenged with the wild-type strain, 60% of the mice failed to survive compared to 100% survival of mice challenged with the CbpA deficient mutant. Further, the wild-type NESp burden in the blood of C1q-deficient mice reached 1×107CFU/ml, and bacteremia persisted for at least five days. In contrast, the CbpAC mutant was not detectable in the blood by 48 hours post-infection. Overall, the ratio of CbpA and deposited C3b on the NESp surface appears to be crucial for NESp to counteract complement deposition and evade host response. Thus, NESp are able to initiate bacteremia and persist within the host.
Identification of a phagosomal F-actin structure that evades dendritic cell immunity to Cryptococcus gattii
Authors: Jamil Khusraw , Polyak Maria J , Feehan David D , Surmanowicz Philip , Li Shushun , Ogbomo Henry , Stack Danuta , Olszewski Michal A , Ganguly Anutosh , Mody Christopher H
Published: January 2023

Hypervirulent Cryptococcus gattii is a major cause of life-threatening cryptococcosis in immunocompetent individuals and responsible for the ongoing outbreak in the Pacific Northwest. This deadly fungus is known to subvert dendritic cell (DC) maturation and concomitant T cell immunity via immune evasion mechanisms that are poorly understood. Here, we show that primary human DC can phagocytose endemic yeasts but trafficking to the late phagolysosome is blocked by retention of a filamentous actin (F-actin) cage surrounding the phagosomes. Structural studies by super resolution microscopy revealed a novel highly branched F-actin cage that physically interfered with lysosomal fusion. Mechanistically, we demonstrate that C. gattii F-actin cage promotes immune evasion by silencing the canonical RelA signaling of the NF-κB pathway required for DC costimulation and T cell activation. Disruption of the F-actin cage through targeted inhibition or by TNF-α signaling reprogrammed quiescent DC to immunocompetent antigen-presenting cells (APCs) and revealed the existence of a negative feedback loop between periphagosomal F-actin aggregation and pro-inflammatory NF-κB signaling. Collectively, our results have uncovered a unique mechanism of DC immune subversion by organisms such as hypervirulent C. gattii and revealed the potential clinical significance of the immunomodulatory function of phagosomal F-actin in host-pathogen interaction.
CD8 T cell immunosurveillance and elimination of HIV target cells in the female reproductive tract
Authors: Joag Vineet , Mitchell Jason , Fife Brian T , Masopust David
Published: January 2023

HIV infection is thought to establish in a single founder CD4 T cell in the genital mucosa, however, an effective CD8 T cell-based vaccine must enable timely surveillance of all mucosal CD4 T cells within the first few days after HIV exposure. The HVTN 502 (STEP) vaccine trial employed a CD8 T cell based vaccine that while promising in animal studies, did not protect human vaccinees from mucosal HIV acquisition. We hypothesize that the quantity of mucosal HIV-specific CD8 T cells is a critical determinant of tissue immunosurveillance and was insufficient to adequately survey and eliminate HIV target cells in the STEP trial. To test our hypothesis, we applied intravital mucosal imaging to visualize how efficiently CD8 T cells survey CD4 T cells in the murine uterus. Based on CD8-CD4 T cell contact rates, a density of 4 (0.8%) HIV-specific mucosal CD8 T cells/mm2 would be required to survey all mucosal CD4 T cells within 2 days. However, vaccinees in the STEP trial had ELISPOT readings of ≥55 spot-forming units/106 blood leucocytes or approximately 0.09% mucosal HIV-specific CD8 T cells, which is 10 fold lower than the required density. Surveillance efficiency was directly correlated with the number of CD8 T cells and was independent of target cell numbers. Upon antigen recognition in the context of MHC-I on CD4 T cells, CD8 T cells formed kinapses with CD4 T cells in an LFA-1/ICAM-1 dependent manner, facilitating tethering and prolonged tracking. Direct killing of CD4 T cells by single or multiple CD8 T cells occurred by 3h after antigen presentation. We conclude that CD8 T cells are capable of rapid tissue surveillance and killing of CD4 T cell targets, and that sub-optimal numbers of HIV-specific CD8 T cells may have contributed to the failure of the STEP trial.
Dissecting the role of endogenous epithelial IL-10 using a small intestinal organoid model
Authors: Nguyen Huong D. , Stadnyk Andrew W.
Published: January 2023

IL-10 is widely appreciated as a potent anti-inflammatory cytokine, acting on leukocytes in mucosal immunity. Far less attention has been paid to the impact of IL-10 on epithelial cells, which make up the crucial barrier interface between the host mucosa and the external environment. Furthermore, most studies look into the effects of exogenous IL-10, disregarding the possible presence and function of endogenous IL-10 in the epithelium. Using ex vivo organoids we aimed to dissect any role for endogenous epithelial IL-10. We discovered that cells in small intestinal organoids (enteroids) derived from C57BL/6 mice produced IL-10 constitutively throughout development. IL-10 mRNA increased considerably on day 3, the time when enteroids begin “budding” to create crypt-like structures. In addition, a second IL-10 increase was detected at day 6 when enteroids are typically “mature”. Immunofluorescent staining for IL-10 on day 4 enteroids was localized to Paneth cells. As gut epithelial cells are constantly exposed to bacteria, we added LPS at the start of enteroid cultures and observed that IL-10 staining was significantly increased. To further investigate the role of endogenous IL-10, we grew enteroids from IL-10 gene knockout mice. IL-10 deficient enteroids develop to morphologically resemble wild-type enteroids. We are proceeding to examine any difference in permeability, cell lineages and apoptosis between the two enteroid genotypes. In conclusion, IL-10 is present in the developing small intestinal epithelium and while development is IL-10-independent, whether IL-10 impacts subcellular components is under investigation.
Spatial transcriptomics with single cell resolution
Authors: Braubach Oliver
Published: January 2023

CO-Detection by indEXing (CODEX®) is ultra-high plex immunohistochemistry that relies on a DNA-based tagging approach, whereby antibodies are labeled with specific oligonucleotide tags (barcodes), and dye-oligonucleotides (reporters) are iteratively hybridized and dehybridized across multiple cycles. CODEX® can detect dozens of antigens simultaneously, in situ and at single cell resolution. Single-cell RNA sequencing, on the other hand, provides information about hundreds to thousands of mRNA transcripts, but without spatial context. The purpose of our study was to enrich spatially resolved CODEX® data with gene expression data obtained from CITE-seq experiments performed on the same tissues. CITE-Seq concurrently measures single-cell antigen and gene expression data, and it is possible to combine these results with CODEX® data by means of Spatially-resolved Transcriptomics via Epitope Anchoring1. We are testing this approach in intact tissues and cell spreads in order to expand the depth our spatial analysis platform. The combination of CODEX® and single cell gene expression data provides an all-in-one solution for deeply multiplexed spatial analyses of protein and gene expression with single cell resolution.
A universal multiplex assay format for ultra-sensitive cytokine quantification in human serum and plasma
Authors: Lambert Jeremy , Tobos Carmen , Rissin David
Published: January 2023

Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal multiplex ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte.

Anchor antibodies specific for a peptide tag were microprinted in microwell plates. The peptide tags were conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal.

This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human serum and plasma in a multiplex assay measuring IL-5, IL-6, IL-10, IL-22, TNFa. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) >0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies.

This universal multiplex planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity multiplex assays for any analyte of their choice.
Cross/Tangential Flow Filtration of Antisera on an Industrial Scale
Authors: Holland Kathleen M , Fuller Cassandra Nicole
Published: January 2023

Purification of antisera, on an industrial scale, to date has been performed by SEC or affinity chromatography, both of which are costly. The method of cross/tangential flow filtration feeds solution across a filter surface rather than pushing product through it. Cross flow membranes are currently used in the manufacturing of serum proteins in order to dialyze and/or concentrate these solutions. Solvent, solutes, and particles smaller than the membrane’s pore size pass through the membrane, while larger molecules are retained and concentrated. This study was designed to apply membrane purification to increase the titers of polyclonal antisera on an industrial scale, as an alternative method to traditional chromatography techniques. Membranes with varying molecular weight cut offs were used to filter and concentrate neat antisera. The reduction of serum fluids and salts increase the concentration of antibodies, and therefore raise the titers. Concentrated antisera titers were then compared to neat antisera titers, to determine if a significant increase or decrease occurred, or if the method had no effect. This method has a marked resemblance to SEC, but without the need for buffers; other characteristics of the antisera remain unchanged. As the current availability of purified polyclonal antisera is scarce, the goal of this study was to produce a more effective product for use in immunology and medical device research and manufacturing across multiple testing platforms, by producing antisera with higher titers. This could potentially result in being able to process antisera to higher titers using lower concentrations of antigen in the host species, which is important from an industrial perspective for reducing manufacturing costs.
Early cancer detection using ctDNA technology – what is the relevance for Pacific Communities?
Authors: Sika-Paotonu Dianne , Anitelea Toni , Cockburn Alana , Guilford Parry
Published: January 2023

A novel cancer detection method known as circulating tumour DNA (ctDNA) analyses the small fragments of tumour DNA found in the blood circulation. Information relating to a cancer’s size and progression can be easily obtained from the DNA fragments found in a blood sample using specific isolation and quantification techniques. This is a relatively simple method for monitoring cancer and is being adapted to support early cancer detection efforts. Pacific Peoples living in New Zealand (NZ) and in the Pacific Region, remain disproportionately affected by some cancers. Given the increasing cancer rates worldwide and the medical situation for cancer patients in NZ and in the Pacific, the burden of cancer remains concerning as significant health inequalities exist for Pacific Peoples in particular. To support meaningful and respectful scientific knowledge translation activity with appropriate engagement promoting dialogue between researchers and Pacific communities, a Pacific Fono (gathering) event was held in the Wellington Region that included discussion of the ctDNA early cancer detection technology. Feedback from members of the various Pacific communities who attended indicated that researchers had engaged respectfully and facilitated discussion appropriately that was helpful, and also supported understanding of the ctDNA research work. Strong support was also expressed for the development of an early detection tool that involved a simple blood test methodology. The importance of taking into account the cultural context, combined with the appropriate scientific communication was shown to build meaningful connections and support respectful dialogue with Pacific communities.
Antibody-Immobilized Nanofiber Scaffolds for the Delivery of Human Tumor Antigen-Specific Cytotoxic T Lymphocytes
Authors: LEE EUNJU , Jia Joann Ng Pei , Kim Tae-Jin , Lim Siyoung , Kim Jiyoung , Kim Jungwon , Kim Jeongsoo , Kim Junghee , Park Sunghee , Lee Kyung-mi
Published: January 2023

Adoptive cell therapy (ACT) employing the transfer of ex vivo expanded effector cells is an attractive approach for cancer treatment. The successful clinical translation of ACT, however, is impeded by inadequate effector cell numbers and poor persistence after infusion. To circumvent these limitations, we propose the use of biocompatible poly (lactide-co-glycolide) (PLGA) nanofiber scaffolds as a platform to effectively deliver human tumor antigen-specific cytotoxic T lymphocytes (CTLs) directly to the tumor site. We modified the PLGA nanofiber scaffolds with monoclonal antibodies (anti-CD8 and anti-CD28 Abs) to provide adhesion and co-stimulatory signals to CTLs. We show that CTLs can be attached and gradually released from the nanofiber scaffolds. We found that the nanofiber scaffold-released CTLs are more activated, have higher CD28 expression, and exhibit higher cytotoxicity with elevated IFNg production, compared to CTLs expanded in a conventional rapid expansion protocol. Our data highlight that anti-CD8/anti-CD28 Ab-conjugated PLGA nanofiber scaffolds can provide a biocompatible biodegradable delivery platform of tumor-specific CTLs by providing T cell persistence and tumor cytolytic function in the patients. Therefore, nanofiber scaffolds incorporated with have become potentially be a more successful strategy for ACT.
A comparison of single cell capture methods and amplification strategies to pair alpha and beta
Authors: Brown Brittany E , Pan Wenjing , Byrne-Steele Miranda , Eisenhower Mary , Li Song , Hou Xiaohong , Depinet Mollye M , Han Jian
Published: January 2023

Immune repertoire amplification of T cell receptors coupled to NGS provides detailed, sequence-level insight into the immune system which can be beneficial for the development of personalized therapies. However, information related to the cognate pairing of alpha and beta chains is lost once RNA extraction is performed on the bulk sample. We have previously presented low through-put (iPair) and high throughput methods for single cell pairing of the alpha and beta receptor chain. Here, we compare different methods to amplify and pair alpha and beta from single cells, as well as track CDR3 rankings from the same sample when sorted for iPair, high throughput pairing, and bulk repertoire. Template switch oligo (TSO) with 5′ cell barcoding, 3′ cell barcoding, and iRepertoire’s multiplex primers were used independently to amplify cDNA from the same cells. The cells are sorted CD14-CD4+ with 1% Jurkat cell line spiked in. Eight thousand cells from the same population were loaded onto 2 different platforms to capture single cells: a droplet-based capture and a microwell array capture. The results demonstrate that the dominant CDR3 pairs in single cell data (regardless of capture method) match the dominant CDR3 clones in bulk repertoire. All single cell methods compared accurately called the 1% Jurkat spike-in and the top paired CDR3.
Housing laboratory mice deficient for Nod2 and Atg16l1 in a natural environment uncovers genetic and environmental contributions to immune variation
Authors: Lin Jian-Da , Devlin Joseph C. , Yeung Frank , McCauley Caroline , Leung Jacqueline M. , Chen Ying-Han , Cronkite Alex , Hansen Christina , Drake-Dunn Charlotte , Ruggles Kelly V. , Cadwell Ken , Graham Andrea L. , Loke P’ng
Published: January 2023

The relative contributions of genetic and environmental factors to variation in immune responses are still poorly understood. Here, we performed a deep phenotypic analysis of immunological parameters of laboratory mice released into an outdoor enclosure, carrying susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory bowel diseases. Variations of immune cell populations were largely driven by environment, whereas cytokine production in response to stimulation was affected more by genetic mutations. Multi-omic models identified transcriptional signatures associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, exposing laboratory mice carrying different genetic mutations to a natural environment uncovered important contributors to immune variation.
Small molecule prevents inflammation in microglia by targeting the NLRP3 inflammasome
Authors: Kulkarni Amol A , Sajith Ayyiliath M , Zhang Changwen , Hao Jiukuan , Bowen J. Phillip
Published: January 2023

Uncontrolled CNS inflammation forms the pathophysiological basis of numerous disorders, including Alzheimer’s disease (AD). Evidence strongly suggests that the inhibition of the NLRP3 inflammasome is a promising strategy for the treatment of AD. A variety of natural products including curcumin, resveratrol, and isoliquiritigenin have displayed encouraging in vitro NLRP3 inhibitory activity. These natural products, however, are reported to display low chemical and/or enzymatic stability and therefore are not considered as promising leads for drug discovery. Using computer-assisted drug design methods and classical medicinal chemistry approaches, we developed a small library of tertiary sulfonylurea compounds that resembled the 3D-structure of the aforementioned natural products without the moieties responsible for the reported chemical and enzymatic instability. The compounds were synthesized, characterized, and tested for their inflammasome inhibitory activity. Preliminary studies indicated two of our compounds decreased the NLRP3 expression in a dose-dependent manner. These compounds reduced the production of inflammatory markers, such as, IL-1b, TNF-a, and caspase 1. These compounds lower the levels of inducible nitric oxide synthase and NO biosynthesis. MTT assays revealed that the sulfonylurea compounds did not affect N9 cell viability. Using computational chemistry and pharmacophore modeling, our efforts are currently on structural modifications to improve their biological activity and penetration the blood-brain barrier.
Gypenosides attenuate lipopolysaccharide-induced neuroinflammation and anxiety-like behaviors in rats
Authors: LEE BOMBI , SHIM INSOP , LEE HYEJUNG , HAHM DAE-HYUN
Published: January 2023

Neuroinflammation is considered a major factor in several neuropsychiatric disorders. Gypenosides (GPS) have pharmacological properties with multiple beneficial effects including anti-inflammatory and protective properties. In the present study, whether GPS could anxiolytic-like effects and chronic inflammation induced by injecting lipopolysaccharide (LPS) into rat hippocampus was investigated. Effects of GPS on inflammatory factors in the hippocampus and the mechanisms of these effects were also examined. Induction of LPS into the lateral ventricle caused inflammatory reactions, anxiety-like symptoms on the rats. Every day treatment of GPS for 21 consecutive days significantly increased the time spent and number of visits to the open arm in the elevated plus maze test, and significantly increased the number of central zone crossings in the open field test. GPS administration significantly reduced the freezing response to contextual fear conditioning. GPS administration significantly decreased pro-inflammatory mediators such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and nuclear factor-kappaB (NF-κB) levels in the brain. Furthermore, GPS reduced LPS-induced elevated levels of toll-like receptor 4 (TLR4) mRNA, and inhibition of brain-derived neurotrophic factor (BDNF) mRNA level. Collectively, these results showed that GPS may anxiolytic-like effects and provide a potential therapy for anxiety-like behaviors caused by neuroinflammation.
Human neural stem cells induce central nervous system specific regulatory T cells from the exTreg pool and promote repair in models of multiple sclerosis
Authors: Greilach Scott A , McIntyre Laura L , Hasselmann Jonathan , Othy Shivashankar , Ngyuen Quy , Sears-Kraxberger Ilse , Steward Oswald , Kessenbrock Kai , Cahalan Michael D , Blurton-Jones Mathew , Lane Thomas E , Walsh Craig M
Published: January 2023

Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system (CNS) and for which there is no cure. Current treatments focus on suppression of the immune system but fail to repair the resulting damage to the CNS. Neural stem cell (NSC) transplantation is a promising therapeutic strategy for treating neurodegenerative diseases through cell replacement and repair however it is unclear how these cells would mediate repair in MS. We report that human NSCs promote CNS specific T regulatory cells (Tregs) which activate endogenous repair pathways and promote remyelination in a murine model of MS. We observed remyelination, decreased inflammation and an increase in (CNS)-infiltrating CD4+CD25+FoxP3+ Tregs in EAE mice receiving an intra-spinal transplant of hNSCs. Recovery was not a result of cell replacement, as hNSCs underwent xenograft rejection, and was Treg dependent, as ablation of Tregs abrogated histopathological improvement. Treg expansion is antigen driven as hNSCs expanded CD25+FoxP3+ Tregs in-vitro when cultured with neural antigen restricted RAG2−/−2D2+ (R2D2) splenocytes but not RAG2−/−OT-II+ splenocytes. When co-cultured with B6 splenocytes, hNSCs drove the expansion of unique TCRs when compared to controls. Additionally, hNSC-Tregs also appear to derive from the exTreg pool suggesting both antigen specific expansion and antigen dependent maintenance of FOXP3 in CNS-specific Tregs. hNSC Tregs also have a unique expression profile and express transglutimase-2 which is implicated in oligodendrocyte dependent repair in the CNS.
IL-10 signaling in T cells is essential for induction of transplant tolerance
Authors: Lozano Marcos Iglesias , Bibicheff Darrel , Chicco Maria , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. We and others recently revealed new interrelations between inflammatory cytokines and interleukin 10 (IL-10) that alter the signaling outcome of the latter. In this study we investigated the role of IL-10 during transplant tolerance induction.

Using a murine full-mismatch skin transplantation model, we showed that IL-10 has a fundamental role in the protective effect of CoB. This tolerizing regimen also failed to be effective in recipient mice with T cell specific unresponsiveness to IL-10, revealing an unexpected important direct modulation of T cells by this cytokine. Accelerated rejection in these transgenic mice correlated with increased accumulation of T cells producing TNF-α, and IFN-γ, and a doubling of IL-17A secreting lymphocytes. We then investigated the mechanism behind modulation of T cells physiology by IL-10. Using in vitro generated rested-effector cells, we show that IL-10 did not reduce their signaling threshold nor their effector functions (cytokine secretion). Moreover, IL-10 did not promote the expression of inhibitory receptors. Instead, we discovered that IL-10 can completely neutralize the TLR-mediated (CD28 independent) costimulation of memory and effector T cells (a largely unexplored phenomenon). We have an ongoing RNAseq analysis to decipher this novel mechanism at the molecular level.

Overall, these results highlight the importance of IL-10 signaling for the therapeutic efficacy of CoB. A better understanding of the effects of this cytokine on T cells will reveal targets of strategies to sustain and potentially improve the clinical efficacy of CoB.
Mechanisms of transplant rejection after the establishment of donor-specific tolerance
Authors: McIntosh Christine , Miller Michelle , Wang Ying , Chen Luqiu , Zhang Zheng Jenny , Wang Jiao-Jing , Allocco Jennifer , Chong Anita S F , Alegre Maria-Luisa
Published: January 2023

Patients weaned from immunosuppressive drugs can reject their graft after years of stability, often after an infection. We have modeled this in mice: infection with Listeria monocytogenes (Lm) at the maintenance phase of tolerance can precipitate rejection of a cardiac allograft. Here, we investigated which alloreactive Tconvs may mediate rejection after Lm infection. To track the function of allospecific Tconvs, we seeded tracer TCR75 cells, which recognize I-Ab-restricted donor Kd peptide, into B6 mice prior to Balb/c cardiac transplantation with or without tolerance induction by anti-CD154 + Balb/c splenocytes. ≥35 days post-transplantation, TCR75 cells persisting in tolerant mice developed a PD-1hi CD127lo exhausted-like phenotype and expressed the anergy markers FR4 and CD73. TCR75 cells were dysfunctional, exhibiting 10-fold less recall expansion compared to memory cells and lacked IFNγ and TNF production upon re-stimulation. Intriguingly, TCR75 cells in tolerant hosts remained dysfunctional after Lm infection, suggesting that they may not participate in infection-triggered rejection. Having found that programming T cell dysfunction in tolerance required persistence of the allograft for ~3 weeks, we tested whether T cells specific for alloantigens expressed transiently after transplantation, such as donor MHC Class II, remain functional during tolerance and may mediate rejection after Lm infection. We used tracer TEa cells specific for a donor Class II Eα peptide presented on I-Ab. During tolerance, TEa cells remained PD-1lo and CD73lo, and retained recall expansion and TNF production. We conclude that functional allospecific Tconvs present during tolerance may pose a previously unappreciated risk to the graft.
Marginal zone B cells induce T cell homeostatic proliferation following antibody-mediated lymphoablation
Authors: Hasgur Suheyla , Fan Ran , Valujskikh Anna
Published: January 2023

Antibody-mediated lymphoablation is commonly used in clinical transplantation. Using mouse model of heart transplantation and murine anti-thymocyte globulin (mATG) lymphoablation, we previously showed that B cell-derived IL-1β and IL-6 are critical mediators of homeostatic T cell proliferation. While marginal zone (MZ) B cells are best known as important sensors and amplifiers of inflammation, their role in depletion-induced lymphopenia has not been previously addressed. The goal of this study was to test the contribution of distinct B cell subsets to proinflammatory cytokine production and T cell homeostatic reconstitution. B6 (H-2b) recipients of BALB/c (H-2d) heart allografts were treated with mATG (d. 0, 4). At d. 8 posttransplant, the proportion of IL-1β, IL-6 and TNF-α secreting cells was significantly higher within MZ vs follicular (FO) B cell subset. Depletion of both FO and MZ B cells with anti-CD20 mAb in addition to mATG (d. -3, 11) significantly diminished T cell reconstitution and prolonged heart allograft survival compared to mATG treatment alone. Notably, specific MZ B cell depletion with anti-VLA4 and anti-LFA1α mAbs (d. -4, -2) resulted in similarly impaired T cells recovery along with improved heart allograft survival. Consistent with these findings, mATG treated Notch2flox/flox CD19Cre/+ mice, which lack MZ B cells but contain normal numbers of FO B cells, had delayed T cell recovery and prolonged heart allograft survival compared to WT recipients. These results show the critical role of MZ B cells in stimulating homeostatic T cell proliferation in lymphopenic heart allograft recipients and suggest that transient removal of MZ B cells may improve the efficacy of lymphoablative induction therapies.
The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity and promotes natural killer cell memory by regulating c-Myc and mitochondrial respiration
Authors: Dong Han , Adams Nicholas , Xu Yichi , Cao Jin , Chen Xi , Romee Rizwan , Sun Joseph Chai-Yuen , Glimcher Laurie H
Published: January 2023

Natural killer (NK) cells are critical mediators of host immunity to pathogens. Though historically being considered as a part of the innate immune system, NK cells exhibit adaptive features and can ‘remember’ stimuli like inflammation or viral infection and hence are capable of responding more robustly upon re-encountering the stimulation. Successful design of an NK cell-based therapy would ideally rely on harnessing NK cell memory, the key drivers and regulators of which remain poorly understood. Here, we demonstrate that the endoplasmic reticulum stress sensor IRE1α and its substrate transcription factor XBP1 drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mTOR pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. NK cells with c-Myc haploinsufficency phenocopied IRE1α deficiency, while c-Myc overexpression largely rescued the proliferation defect in IRE1α−/− NK cells. Like c-Myc, IRE1α also promotes oxidative phosphorylation in NK cells. Combined, our study identifies an IRE1α-XBP1-cMyc axis in NK cell immunity; and based on this work, we are currently investigating the role of IRE1 in NK cell memory using novel transgenic mouse models and small molecule drug treatment in primary human cells. We are also evaluating the therapeutic benefit of manipulating IRE1 function in CAR-NK cells to treat cancer. Our ongoing study should advance the current knowledge of NK cell memory and also open new opportunities in cancer therapy for patients with otherwise extremely poor prognoses.
cGAS-STING pathway mediates antitumor immunity of tumor cryoablation in syngeneic murine Rhabdomyosarcoma
Authors: Alshebremi Mohammad A , Myers Jay T , Kingsley Daniel , Huang Alex Yee-Chen
Published: January 2023

Tumor cryoablation, a medical procedure that utilizes ultra-cold temperatures to destroy in situ local tumor tissues, has been established for cure of many tumor types and for stimulation of the immune system. The mechanism of immune response to tumor cryoabltion are not well defined. To understand the molecular pathway of immune activation by cryoablation, we performed comprehensive analyses on the impact of tumor cryoablation on mononuclear phagocytic lineage cells and the antitumor immunity. Here, we show that cryoablation of a murine Rhabdomyosarcoma (RMS) tumor, an aggressive sarcoma affecting children and young adults, was able to induce the regression of tumor and the upregulation of the critical immune cytokines/chemokines profile and maturation markers of CD11c+ DCs. Moreover, cryoablation of RMS tumor initiates cGAS-STING signaling activation and type I interferon production in CD11c+ DCs, which are consequential in pro-inflammatory tumor microenvironment (TME). We also report that antitumor effects of cryoablation were more inhibitive, but not completely abrogated, in STING-deficient mice comparing with wild-type mice, indicating that both host-derived and tumor-intrinsic cGAS-STING signaling plays an important role in antitumor effects of tumor cryoablation. Taken together, our results demonstrate that tumor cryoablation induces antitumor immunity via cGAS-STING signaling in syngeneic Rhabdomyosarcoma (RMS) tumor, and this approach may help guide future immunotherapy to improve cancer treatment outcomes.
BRD4 inhibitors synergize with anti-PD1 in rescuing T cell exhaustion in acute myeloid leukemia by increasing TCF1 expressing CD8+ T cells
Authors: Romine Kyle Alexander , Cho Hyun-jun , Flynn Patrick , Kosaka Yoko , Byrd Kaelan , Lind Evan F
Published: January 2023

Acute myeloid leukemia (AML) is an aggressive myeloid lineage cancer with limited treatment options. Single-agent treatments with targeted inhibitors have resulted in some disease remissions, however, resistance almost always develops, prompting development of combinatorial treatment strategies. Studies have shown that AML patients exhibit markers of immune suppression such as increased levels of exhausted T cells (PD1/LAG3+) as well as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs)1. Inhibitors targeting the BET family, which are histone readers, have been shown to increase IFNy and IL-2 production in T cells by inhibiting BATF—a known negative regulator of TCF1, as well as decrease PD-L12–4. We, therefore, hypothesized that combining BRD4i and anti-PD1 would synergize by modulating both tumor intrinsic biology (though MYC reduction) and boosting T cell activity. We have characterized a FLT3-ITD/TET2/Lys-Cre mouse model of AML and observed significantly increased numbers of PD1+/TIM3+/TCF1− T cells, Tregs, and MDSCs when compared to healthy mice. T cells derived from mice with AML have significantly reduced T cell proliferation upon co-stimulation with CD3 and CD28. Ex vivo treatment with BRD4i + aPD1 significantly increased T cell proliferation in AML mice and increased TCF1 expression in a dose dependent manner. In vivo treatment with BRD4i + aPD1 showed enhanced reduction of leukemic blasts, reduced PD-L1, and increased CD8+ T cell infiltration into the bone marrow. Our work highlights the efficacy of this novel drug combination in targeting tumor intrinsic and extrinsic factors in AML and identifies a potential novel role for BRD4 in regulating TCF1 expression in T cells.
Treg dysregulation contributes to pathogenesis of Myelodysplastic syndrome
Authors: Camacho Virginia , Matkins Victoria , Patel Sweta , Anderson Ashlyn , Landuy Ashley E , Ferrell Paul Brent , Maynard Craig L , Harrington Laurie E , Turnquist Heth Roderick , Welner Robert S
Published: January 2023

A strong clinical correlation exists between a history of autoimmunity and Myelodysplastic syndromes (MDS), but immune etiology remains unclear. However, a hallmark of disease is that Regulatory T cells (Tregs) are significantly altered in MDS patients. Thus, Tregs can be used as prognostic indicators of severity. Additionally, dysregulated CD4 T cell responses are causally linked to inflammatory disorders in MDS patients, yet the cellular dynamics that underlie these responses is poorly understood. We now show that Treg dysfunction mimics the pre-leukemic phenotypes of MDS including increased HSC proliferation and myeloid-skewed differentiation. Furthermore, we show that mice with Treg-restricted MDS driver mutations, TET2 and ASXL1, develop an inflammatory disease with unstable Tregs that secrete inflammatory cytokines and lose immunosuppressive capacities. TET2 and ASXL1 deficiency in Tregs leads to hyperproliferation of CD4+Foxp3+ T cells with a conversion into IL-17-producing cells. Importantly, CD4 and CD8 T cells are also altered in MDS and show impaired effector function as well as hallmarks of T cell exhaustion and anergy leading to expansion of malignant clones. Together, this indicates that T cell abnormalities and lack of immune function effectively propel leukemic evolution. Our findings indicate that the pro-inflammatory environments of MDS deteriorate T cell stability and accelerate the expansion of hematopoietic stem/progenitor cells carrying MDS-associated pre-leukemic mutations. Therapeutic intervention targeting both Tregs and effector cells in pre-malignant diseases such as MDS are likely to prevent progression to less treatable acute myeloid leukemia.
Arginase-1 production by myeloid cells in tumor microenvironment induce inflammasome activation and activate PI3K pathway in tumor-associated macrophages in head and neck cancer
Authors: Momen-Heravi Fatemeh , Dubey Sunil K
Published: January 2023

Tumor-associated macrophages (TAMs) express cytokines and mediators that can suppress T cell recruitment and activation in the tumor microenvironment, support tumor growth, and promote resistance to therapy. We found that Arginase-1 is produced in high quantities by TAMs and tumor cells in head and neck cancer tumors with different background mutations. High levels of Arginase-1 were found in tumor samples and the circulation of patients and animal models of head and neck cancer.

Arginase-1 is released into the cellular microenvironment and packed into the exosomes in a metabolically active form. Mechanistically, Arginase-1 further propagated the anti-inflammatory macrophage phenotype in the tumor microenvironment and induced the epithelial-mesenchymal transition (EMT) by activation of STAT3 and beta-catenin. Arginase-1 released from the tumor further induced activation of the autophagy cascade and the PI3K pathway in TAMs. Mechanistic results showed that STAT3 activation by Arginase-1 containing exosomes has a positive feedback effect on the expression of Arginase-1, Il-1β, and lead to inflammasome activation. The silencing of STAT3 negated the effect of Arginase-1 containing exosomes and led to modulation of the inflammasome activation in the head and neck cancer tumor microenvironment. These results indicate a mechanistic role of TAMs and Arginase-1 containing exosomes in the tumor microenvironment by activation of autophagy and inflammasome, and induction of the EMT. The mechanistic links presented in this study regarding the role of arginase in head and neck cancer pathogenesis could be targeted as therapeutic strategies.