bluuebunny/crossref_metadata_2025 huggingface dataset:
Siglec-H ablation in plasmacytoid dendritic cells alleviates acute liver injury by promoting IL-21+ CD4 T cells
Authors: Ahodantin James , Wu Jiapeng , Funaki Masaya , Flores Jair , Wang Xu , Zheng Pan , Liu Yang , Su Lishan
Published: November 2024

Siglec-H is an immune receptor specifically expressed in plasmacytoid dendritic cells (pDCs) that inhibits IFN-α production and regulates pDC maturation. Although, the role of pDCs in autoimmune disorders is well-known, the effect of Siglec-H expression in pDCs in acute liver injury (ALI) remains elusive. To investigate Siglec-H/pDCs axis in ALI, BDCA2 transgenic and Siglec-H knockout (SH KO) mice were administrated with concanavalin A (ConA) to induce a T cell-mediated ALI in combination of either pDCs depletion with anti-BDCA2 antibody or anti-IL-21R and Stat3 blockade. Here, we report that specific depletion of pDCs in BDCA2 transgenic mice exacerbated ALI. Surprisingly, SH KO mice were resistant to ALI (lower ALT, hyaluronic acid, IL-6 and TNF-α). Th1/IFN-γ response and Stat1 signaling were both inhibited in the liver of SH KO mice while intrahepatic IL-21 and Stat3 signaling pathways were elevated. Mechanistically, we identified that Siglec-H-null pDCs exhibited immunosuppressive phenotypes (CD40LowCCR9Low), resulting in a defective CD4 T cell activation and a promotion of IL-21-producing CD4 T cells in the liver. Finally, IL-21R and Stat3 inhibitions in Siglec-H deficient mice restored ConA-induced ALI. We conclude that, Siglec-H-null pDCs mediate hepatic tolerance via induction of IL-21+CD4 T cells in the liver during ALI. These findings indicate that targeting Siglec-H/pDCs axis may provide a novel approach to modulate CD4 T cell polarization and liver disease.
Liposomal clodronate attenuates inflammation in a mouse model of myocardial infarction
Authors: Boyanova Katherine , OBarr Stephen , Lambros Maria , Agyare Eugene
Published: November 2024

Heart failure (HF) is defined as the inability of the heart to pump eough blood to meet the demands of the body. HF affects over 64 million people worldwide and is the leading cause of death, yet current treatment options do not effectively decrease mortality. The relationship between HF onset and the immune system has been vastly explored, revealing that cardiac tissue undergoes harmful remodeling after a myocardial infarction (MI) that leaves a permanent scar on the heart. Prolonged inflammation and excessive macrophage infiltration lead to a larger infarct area and increased scar formation. Our lab has demonstrated that early inflammation inhibition reduces the damage caused by an (MI). Specifically, we used liposomal clodronate as a macrophage inhibitor to reduce the inflammatory response. In this study, MI was induced using a single high dose of isoproterenol and treated with liposomal clodronate 24hrs after MI. Inflammation levels were assessed through quantification of various pro- and anti-inflammatory cytokines by ELISA and macrophage depletion and polarization was tracked using flow cytometry. At 10 days, heart tissue was harvested for immunohistochemical (IHC) analysis. Our data indicated that liposomal clodronate has anti-inflammatory properties as supported by the decrease in pro-inflammatory cytokines and macrophages. Moreover, IHC analysis revealed less collagen fibers, suggesting a lower area of cardiac remodeling and retained overall structure.
Lrba-/- murine B cells show NF-κB overactivation and altered BCR signaling.
Authors: Pérez-Pérez Daniela , Flores-Hermenegildo José Mizael , Romero-Ramírez Hector , Kilimann Manfred , Santos-Argumedo Leopoldo , López-Herrera Gabriela
Published: November 2024

Lipopolysaccharide-Responsive Beige-like anchor (LRBA) is a scaffolding protein whose deficiency is associated with an inborn error of immunity error like common variable immunodeficiency. Patients with LRBA deficiency have a heterogeneous clinical presentation including hypogammaglobulinemia, reduction of B and T regulatory cells, recurrent infections, and autoimmune diseases. Functions of Lrba on T regulatory cells have been described, however, function on B cells remains unknown. This work aims to identify the function of Lrba in B-cell receptor (BCR) signaling.

Lrba-deficient mice (Lrba-/-) have a higher number of Transitional 1 B cells in the spleen. Flow cytometry assays showed reduced B cell proliferation and an increased cell death, in response to B-cell Receptor (BCR) crosslinking. Immunoblot and confocal microscopy revealed an increased activation of p50, p65, and IκBα (components of the canonical pathway of NF-κB) in Lrba-/- B cells under basal conditions with a subsequent reduced response after BCR crosslinking. Co-immunoprecipitation assays suggest an interaction between Lrba and components of BCR signaling such as PLC-γ2 and p65.

The activated phenotype of Lrba-/- B cells as well as the interaction with p65 and PLC-γ2 indicates that Lrba is involved in the regulation of NF-κB activation driven by BCR signaling, which finally impacts on survival of B cells.
Piezo1 impacts early CD4+ T cell activation that leads to increased expression of exhaustion markers and decreased effector persistence.
Authors: Santin Alicia , Myers Jay , Choi Sung Hee , Huang Alex
Published: November 2024

The emergence of novel biophysical approaches has revealed that mechanosensing at the immunological synapse is critical in regulating T cell activation and function. To investigate the role of the mechanosensitive cation channel, Piezo1, on T cell function, we generated T cell specific Piezo1 knockout mice (P1cKO). Previously we demonstrated that Piezo1 restrains inflammatory Th1 and Th17 polarization in vitro. However, Piezo1 deficiency in T cells ameliorated disease progression in primary autoimmune mouse models, while completely ablating disease phenotype in adoptive transfer autoimmune mouse models. Therefore, we hypothesized that Piezo1 deficiency in T cells increases T cell receptor (TCR) signaling and activation leading to increased T cell exhaustion and apoptosis. Preliminary studies of early TCR signaling suggest that P1cKO CD4+ T cells have increased phosphorylated ZAP-70 (pZAP-70) compared to wildtype, and Piezo1 activation by Yoda-1 conversely decreased pZAP-70. After 3 days of TCR stimulation P1cKO CD4+ T cells similarly expressed activation markers CD25, CD69, and CD95 compared to wildtype, but had an increased expression of exhaustion markers LAG-3, PD-1, and TIGIT. Cell tracking in vivo revealed that P1cKO effector CD4+ T cells expand rapidly after transfer but fail to persist long term. Taken together, our data suggests that Piezo1 plays an important role during early T cell activation that ultimately restrains hyper inflammatory CD4+ T cell responses.
Development of an anti-TRBC2 antibody for flow cytometry.
Authors: Rigaud Stephanie , Kannan Arthi , Cantor Joseph , Verma Shilpi
Published: November 2024

Each chain of the αβ T-cell receptor (TCR) contains a constant (C) region and an antigen-binding variable (V) region. There are two human isoforms of the TCR β chain, TRBC1 and TRBC2, differing slightly in the constant region, and individual αβ T-cells express one isoform or the other. The ratio of TRBC1+ to TRBC2+ αβ T-cells falls within a characteristic range of healthy donors; monoclonal T-cell malignancies skew this ratio dramatically. A monoclonal antibody specific for TRBC1 (clone JOVI.1) is unable to directly identify both TRBC1+ and TRBC2+ T cell malignancies as is done for B cell cancers using k and λ-specific antibodies. Here we characterize clone SAM.2.rMAb, to our knowledge the first monoclonal antibody that selectively binds TRBC2, and demonstrate its use by flow cytometry in combination with JOVI.1 to confirm the mutually exclusive expression of TRBC1 and TRBC2 in T cells, NKT cells, MAIT cells and Treg. This new clone allows direct identification of TRBC2+ cells, is compatible with BD buffers and can be used in phenotyping panels that include TCRαβ and CD3 antibodies. SAM.2.rMAb is thus a valuable flow cytometry reagent to potentially combine with JOVI.1 in building rapid and low-cost research use only immunophenotyping assays for basic and applied research on human T-cell malignancies.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD is a trademark of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved.
Nerve- and Airway-Associated Interstitial Macrophages Regulate Anti-Tumor CD8 T Cell Immunity in Tumors in the Lung
Authors: Bartnicki Eric , Yeung Stephen , Damani-Yokota Payal , Khanna Kamal
Published: November 2024

The suboptimal response rates among cancer patients receiving T cell-based immunotherapy underscore the necessity of addressing additional facets of the immune system that may contribute to tumor promotion. Macrophages are a main immune cell subset in tumors and elucidating their immunomodulatory roles on T cell-mediated anti-tumor responses is paramount. We previously identified a novel macrophage subset known as Nerve- and Airway-associated interstitial Macrophages (NAMs), which exhibit a remarkable immunoregulatory phenotype. Thus, we investigated the hypothesis that NAMs promote tumor growth and metastasis. Using the B16F10 melanoma-based lung metastatic model, we observed that unlike alveolar macrophages (AMs), NAMs uniquely localize to tumors that form along large airways and vasculature. Moreover, tumors that were primarily infiltrated by NAMs exhibited less CD8 T cell presence when compared to tumors devoid of NAMs, suggesting that NAMs regulate anti-tumor T cell responses. Exclusive depletion of NAMs resulted in strikingly lower tumor burden in the lungs, which was associated with a significant increase in CD8 T cell recruitment to the TME; and increased presence of anti-tumor ‘CD8-DC-CD4 triad’ interactions within the TME with CD8 T cells exhibiting an exhausted progenitor-like anti-tumor phenotype. Our findings also furnish compelling evidence to redirect our endeavors towards prioritizing NAMs as a therapeutic target in lung tumors.
VH14-2 mediated nuclear speckles association contributes to radial positioning of the Igh locus and regulates VDJ recombination
Authors: Priyadarshi Saurabh , Drake Ellen , Bhat Khalid , Farooq Hammad , Kleiman Eden , Lei Xue , Liang Jie , Feeney Ann , Kenter Amy
Published: November 2024

The Igh locus undergoes large-scale contraction and repositions to the nuclear center prior to V(D)J recombination. However, little is known about the mechanisms that mediate these processes. We recently identified a chromatin loop anchor which corresponds to a highly transcribed VH gene promoter (VH14-2 Pr) (Nat Commun 14:1225, 2023). Here we report that VH14-2 deletion increased locus contraction which was expected to increase VDJ recombination. However, deletion of the VH14-2 gene severely reduced VDJ recombination. This implied that another mechanism may contribute to VDJ recombination. Strikingly, Igh loci accumulated toward nuclear periphery in Rag2-/-VH14-2-/- pro-B cells indicating the failure of radial repositioning. Thus, efficient VDJ recombination is linked to repositioning of the Igh locus and implies that the VH14-2 gene anchors the locus in the nuclear center. Highly transcribed genes often associate with nuclear speckles that tend to locate to the nuclear center. As VH14-2 is highly transcribed we asked whether it associates with nuclear speckles. We found that the Igh locus overlaps with nuclear speckles and this association was significantly reduced in Rag2-/-VH14-2-/- pro B cells as visualized using a combination of DNA and immuno-fluorescence in situ hybridization (FISH). This study identified the VH14-2 gene as a tether that anchors the locus to nuclear speckles and in turn contributes to radial positioning and VDJ recombination.
Naturally occurring polymorphisms reveal the cis-regulatory sites and transcription factors controlling follicular and marginal zone B cells
Authors: Roman Carly , Hicks Sakeenah , Gupta Mansi , Scharer Christopher
Published: November 2024

The splenic B cell compartment consists of follicular (FoB) and marginal zone (MZB) B cells. In contrast to FoB, MZB exist in a poised state and rapidly respond to blood-borne pathogens in the absence of T cell help, predominately generating a rapid wave of short-lived plasma cells. These differences in responses are driven by differential genetic and epigenetic priming of each subset. MZB epigenetic programming are enriched for bHLH, POU, Rel and RUNT motifs, whereas FoB are enriched for IRF and KLF motifs. MZB also exhibit increased expression of genes associated with Notch signaling, Myc, and mTORC1 pathways. However, it is unclear how sequence specific transcription factors directly and indirectly regulate distinct states in MZB and FoB that underpin B cell activation, differentiation, and acquisition of effector functions. To address this, we generated an F1 hybrid model which utilizes naturally occurring polymorphism present in the wild-derived mouse line Spret/EiJ compared to the reference strain C57BL/6. The monoallelic polymorphisms present in this F1 generation allows us to use the allelic imbalance to dissect how alterations in genetic features impact TF binding, epigenetic architecture, and ultimately resulting gene expression programs. MZB and FoB were sorted from naive (Spret x B6) F1’s and subject to RNA and ATAC-seq. This study aims to define the cis-regulatory mechanisms responsible for the epigenetic and transcriptional differences of FoB and MZB cells.
Opposing roles of SWI/SNF chromatin remodeling complex BAF and PBAF in Effector Differentiation during Chronic Viral Infection
Authors: Kharel Arjun , Brown Ryan , Gai Kexin , Cui Weiguo
Published: November 2024

During chronic viral infection, CD4+ T cells help in the form of IL-21 is critical for the development of highly cytolytic effector CD8+ T cells. However, the exact signaling pathways from IL-21 that regulate CD8+ T cell function remain unclear. The current dogma states that IL-21 predominantly signals through STAT3, to generate effector CD8+ T cells during chronic viral infection.

Here, we reveal a novel mechanism of IL-21 signaling via STAT4 for effector CD8+ T cell development following chronic LCMV infection. Furthermore, STAT4-mediated effector differentiation is epigenetically regulated by BAF, a SWI/SNF chromatin remodeling complex. In the absence of either STAT4 or BAF, virus specific CD8+ T cells fail to differentiate into highly cytolytic effector CD8+ T cells capable of controlling viral infection.

The effector differentiation phenotype is balanced by PBAF, another component of the SWI/SNF complex, which promotes CD8+ T cell exhaustion during chronic LCMV infection. Conditional knockout of PBAF from CD8+ T cells impairs the development of T cell exhaustion, resulting in immunopathology and mortality of chronic LCMV-infected mice. Interestingly, this phenotype is dependent on the STAT4 and BAF-mediated transcriptional and chromatin changes. Overall, our findings underscore the delicate balance between effector and exhaustion pathways, governed by BAF and PBAF complex, to dictate the outcomes of immunopathology during chronic viral infections.
Deciphering the links between microbiome and immune profiles by multi-omics analysis during aortic plaque progression and helminth-induced regression
Authors: Lin Jian-Da , Chen Yen-Ting , Chao Pei-An , Lin I-Jung , Chou Tzu-Yin , Ye Yu-Zhen , Lien Chia-Jung , Yang Chen-Hsuan
Published: November 2024

Atherosclerosis is influenced by immune cells that mediate inflammation. The gut microbiota contributes to atherosclerosis by fostering inflammation when its composition is imbalanced (dysbiosis). However, the specific gut microbiota and the immune profiles that drive this process are elusive. Our multi-omics analysis addresses this gap by using spectral flow cytometry (36 markers), CITE-seq, and 16S rRNA sequencing on dysbiosis-conditioned mice to collect comprehensive data on immune profiles and microbiota composition. We induce atherosclerosis in Nod2-/- mice, which are predisposed to inflammatory bowel disease, to evaluate factors including plaque size, immune profiles, and microbiota diversity, comparing to atherosclerotic wild-type mice. Our findings highlight the Nod2 gene in helminth-induced atherosclerosis regression and illustrate unique immune cell dynamics, such as memory T cells, Ly6C+/- monocytes, and type 2 immune responses, in both wild-type and Nod2−/− mice. We observe marked shifts in microbiota diversity during the progression of atherosclerosis and the regression induced by helminths. By constructing multi-omics networks with machine learning models, we strive to link specific bacterial orders with immune cell types during atherosclerosis progression and regression. This integrative study aims to illuminate the complex interactions within the gut-heart axis and to explore novel therapeutic potentials of helminths in the treatment of atherosclerosis.
Enhanced host protection and survival to Listeria monocytogenes infection after small molecule innate training
Authors: Kim Marie , Knight Hannah Riley , Esser-Kahn Aaron
Published: November 2024

Homeostatic training of innate immunity can improve protection against later infections. Our lab recently reported on small molecules that induce trained immunity in vitro. Here, we aim to show the protective potential of flunisolide (FN) using live infection model. We hypothesized FN training will alter hematopoiesis and innate functionality to improve outcome to L. monocytogenes. At homeostasis, FN training skewed towards GMP and MEP lineages and away from CLP lineage. To confirm FN-induced protection, mice were infected i.n. At d3 post-infection, FN mice showed lower pathogen burden in spleen, blood, and lung. Notably, systemic burden was lower in FN mice at d2 and d3. This may be due to improved immune functionality that led to faster clearance. To test this, phagocytosis and ROS levels at d3 were measured but no significant changes were seen. Further studies on lung immune functionality and earlier timepoints may show what hinders pathogen dissemination. BM at d7 after infection showed higher percentage of Lin+ in control and Lin- cells in FN mice. This suggests BM was recovering to homeostasis in FN mice, while inflammation control was active in control. Also, BM of FN mice had significantly higher percentage of CLP, suggesting earlier onset of adaptive immune response. Furthermore, FN mice showed improved survival up to d7. We showed FN can induce innate training, altering BM landscape at homeostasis and after infection, with improved response to L. monocytogenes.
T cell TRAF3 regulates IL-4R signaling through the phosphatase PTP1B
Authors: Hornick Emma , Arkee Tina , Bishop Gail
Published: November 2024

The adaptor protein TNFR associated factor 3 (TRAF3) is a multifaceted regulator of lymphocyte biology that plays key roles in modulation of the molecular signals required for T cell activation and function. TRAF3 regulates signals mediated by the T cell receptor (TCR), costimulatory molecules, and cytokine receptors, which in turn drive activation of the serine/threonine kinase Akt. The impact of TRAF3 upon TCR/CD28-mediated activation of Akt, and thus on the diverse cellular processes regulated by Akt, including CD4 T cell fate decisions, remains poorly understood. We show here that TRAF3 deficiency led to impaired Akt activation, and thus to impaired in vitro skewing of CD4 T cells into the TH1 and TH2 fates. We investigated the role of TRAF3 in regulation of signaling pathways that drive TH1 and TH2 differentiation, and found that TRAF3 enhanced activation of Signal transducer and activator of transcription 6 (STAT6), thus promoting skewing toward the TH2 fate. TRAF3 promoted STAT6 activation by regulating recruitment of the inhibitory molecule Protein tyrosine phosphatase 1B (PTP1B) to the IL-4R signaling complex, in a manner that required integration of TCR/CD28- and IL-4R-mediated signals. This work reveals a new mechanism for TRAF3-mediated regulation of STAT6 activation in CD4 T cells, and adds to our understanding of the diverse roles played by TRAF3 as an important regulator of T cell biology.
Enhancement of autophagy in macrophages via the p120-catenin-mediated mTOR signaling pathway
Authors: Kanmani Suganya , Song Xue-Min , Kanmani Paulraj , Wu Xiao-Jing , Minshall Richard , Hu Guochang
Published: November 2024

Autophagy serves as a critical regulator of immune responses in sepsis. Macrophages are vital constituents of both innate and adaptive immunity. In this study, we delved into the intricate role of p120-catenin (p120) in orchestrating autophagy in macrophages, particularly in their response to endotoxin stimulation. Depletion of p120 effectively suppressed lipopolysaccharide (LPS)-induced autophagy in both J774A.1 macrophages and murine bone BMDMs. LPS not only elevated the interaction between p120 and LC3I/II, but also facilitated the binding of p120 with mTOR. The absence of p120 led to the inhibition of LPS-induced dissociation between mTOR and ULK-1, consequently promoting the phosphorylation of ULK-1 upon LPS stimulation in macrophages. p120 depletion also heightened LPS-triggered macrophage apoptosis, as evidenced by increased levels of cleaved caspase 3. Notably, a decrease in apoptosis observed in macrophages overexpressing p120 upon LPS stimulation was reversed by inhibiting autophagy. Additionally, the ablation of p120 accentuated LPS-induced alveolar macrophage apoptosis in LPS-challenged mice. Collectively, our findings strongly suggest that p120 plays a pivotal role in fostering autophagy while concurrently hindering apoptosis in macrophages, achieved through modulation of the mTOR/ULK-1 signaling pathway in sepsis. This underscores the potential of targeting macrophage p120 as an innovative therapeutic avenue for treating inflammatory disorders.
rhl deficient-Pseudomonas aeruginosa induces enhanced host necroptosis through upregulation pqs within quorum sensing
Authors: Liu Zihao , Wen Haitao
Published: November 2024

As the leading cause of nosocomial infections, the relationship between Pseudomoans aeruginosa (P. aeruginosa) and host innate immune responses still arouses the interest in innate immunity research. Quorum sensing (QS), a communication system evolved by P. aeruginosa to mediates its density, is involved in various activities during bacterial infection. Recent study has revealed the link between QS and host innate inflammatory responses, extending the role of QS in interaction with host defense. Although previous evidence suggests PCD exists during anti-P. aeruginosa responses, it remains unclear that what role of quorum sensing plays in connection with PCD during infection. In this study, we exhibited that the deficiency of QS subsystems, rhl, dramatically increased the cell death induced by P. aeruginosa in mouse bone marrow-derived macrophages (BMMs). However, the cell death was completely blocked by using the Casp1/11−/−Casp8−/−Ripk3−/− BMMs, indicating the association between QS and host PANoptosis. Intriguingly, we discovered the rhl deficiency upregulated downstream pqs signaling in P. aeruginosa, which significantly promoted the activation of necroptosis through elevated phosphorylation of MLKL. Deletion of pqsA gene in rhl deficiency strains of P. aeruginosa successfully rescued the necroptosis in BMMs. Taken together, our data highlight the crosstalk between QS and host PCD, ae well as a novel rhl – pqs – necroptosis axis in cell death signaling pathway.
Exploring the Therapeutic Potential of Gracilaria verrucosa-Derived Polysaccharide in Metabolic Syndrome: A Comprehensive Study from Molecular Mechanisms to Microbial Correlations
Authors: Zhang Guiguo , Kim Eunyoung , Cui Jiamei , Ko Kayeon , Kang Minhyeok , Kim Hyo Jin , Yang Jiwon , Lee Yunkyoung , Unno Tatsuya , Jee Youngheun , Jeong Youjin , Li Zemin , Wang Yunpeng
Published: November 2024

Gracilaria verrucosa-derived polysaccharide (GVPS) is recognized for its health benefits, yet a comprehensive exploration of its effects, particularly on the gut-liver-adipose tissue axis in metabolic syndrome, is lacking. In this study, we unraveled GVPS's protective mechanism on intestinal barrier inflammation in an intestine-mimicked co-culture model through RNA-seq analysis. Additionally, we explored its impact on the pathophysiology of a diet-induced obese (DIO) model and characterized GVPS's structure. As a result, GVPS alleviated inflammation and enhanced gut permeability in the inflamed co-culture model through partial regulation of mucin synthesis. In DIO mice, GVPS reduced body weight, adipose tissue weight, blood glucose, triglyceride, and inflammatory cytokine levels, while ameliorating obesity-induced tissue dysfunction and including gut microbiome alterations, suggesting potential modulation of the gut-liver-adipose tissue axis through increased short-chain fatty acid. Further, correlation analyses revealed core microbes (Lactobacillus and Bacteroides) and core genes (Gnb4, Poc1b-galnt4, and Cxcr3) influenced by GVPS. GVPS was revealed to be a long-chain polysaccharide with highly branched and helical domains composed of more than 90% galactose and with a molecular weight of 8.1 × 104 g/mol. Taken together, our findings highlight GVPS as a promising therapeutic agent for metabolic syndrome, unraveling its molecular mechanisms and microbial associations.
Naturally occurring CD4+CD25+ T regulatory cells induced during burn injury exhibit decreased apoptosis.
Authors: Fazal Nadeem
Published: November 2024

The aim of this study was to characterize the molecular pathways of cell signaling of the immune system affected by burns. The adherent PP (Peyers Patches) and MLN (Mesenetric Lymph Nodes) cells were obtained from burn and burn-plus-peritonitis animals and cocultured with CD4+CD25- T cells from sham rat PP and MLN for 24-72 hours. At several time points during coculture, non-adherent T cells were separated and checked for CD25 expression and their apoptosis (Propidium iodide and Annexin V labeling followed by flow cytometry). The T cell apoptotic behavior was evaluated via measurements of mitochondrial morphology (whole cell confocal microscopy using mitochondria-specific stain), mitochondria membrane potential change (dye uptake), and mitochondrial cytochrome C release (Western blot analyses).This might imply that potential inhibitory effects of the Treg cells might be reinforced by a decrease in their apoptotic rate. We evaluated whether an apoptotic rate decrease in the induced Treg results from the actions of burn and burn-plus-peritonitis related antigens. Furthermore, we evaluated if there was difference in the extent of potential anti-apoptotic response in the induced Treg cells in the burn vs. burn-plus-peritonitis injured animals. A greater decrease in Treg cells’ apoptosis in the burn/peritonitis group might be able to contribute to a prolongation of their “suppressive” effect on the CD4+ helper T cells in the combined injury condition.
Skull bone marrow serves as a niche of adaptive immunity for CNS immunosurveillance
Authors: Park Jang Hyun , Kipnis Jonathan
Published: November 2024

The central nervous system (CNS) has been considered as an ‘immune-privileged’ organ owing to limited graft- and cancer-rejection, as well as the absence of lymphocytes and lymphatics. However, recent findings unveiled that the immune system actively discerns the status of the CNS through the glymphatic system and meningeal lymphatics. There is a diverse immune repertoire in the CNS borders presenting antigen, affecting behaviors, and defending CNS tissues. Recent studies discovered the channels in the skull bone marrow (SBM), exchanging immune cells and cerebrospinal fluid (CSF). Consequently, molecular exchange and immune cell activation occur in the CNS border, including SBM, like mucosal tissues. Nevertheless, the mechanism responding to CNS antigens and the maintenance of tolerance is unclear. In this study, we noted greater activation and differentiation of T/ B cells in the SBM than dura mater and other BMs. SBM displayed premature features of lymphoid structures, encompassing Tfh, activated B cells, and antibody-secreting cells. We also observed that Tfh cells directly interacted with B cells and induced differentiation. Moreover, our observations indicated that skull T/B cells respond to CNS antigens, but not microbial/food antigen. Thus, our study suggests that the SBM is a niche for adaptive immune cells in response to CNS antigens. The SBM may sustain heightened inflammatory states to promptly counteract tumorigenesis and pathogen invasion, protecting CNS.
Preclinical studies to determine myelin peptide-specific nanoparticle tolerance mechanisms and clinical translation
Authors: Podojil Joseph , Cogswell Andrew , Chiang Ming-Yi , Xu Dan , Ifergan Igal , Kakade Sandeep , McCarthy Derrick , Elhofy Adam , Miller Stephen
Published: November 2024

Infiltration of autoreactive T cells, subsequent tissue destruction, and the release of additional self-epitopes resulting in epitope spreading underly pathogenesis in many autoimmune diseases. Treatment with Ag-containing biodegradable poly(lactide-co-glycolide) nanoparticles, i.e. tolerogenic immune-modifying particles (TIMP/CNPs), has been shown to be safe and efficacious for the induction of Ag-specific tolerance in murine models of autoimmunity, and in a Phase Ib/IIa clinical trial for celiac disease. The present studies identified novel pathways required for Ag-specific TIMP-induced tolerance, the ability of treatment to modulate spread epitope-specific CD4+ T cell activation, and the functionality of TIMPs containing human myelin-derived peptide epitopes (CNP-102) in a pre-clinical mouse model of multiple sclerosis (MS). Following treatment, myeloid cells phagocytose TIMPs, undergo apoptosis, and Ag-specific tolerance therapy increases both FoxP3+ regulatory T cells and IL-10+ Tr1 cells via a STING/IFNAR-dependent pathway. TIMP treatment also modulates spread epitope-specific T cells associated with disease progression, but not encapsulated within the TIMP, i.e. tissue-specific tolerance. Therefore, treatment activates various Ag-specific regulatory T cell subsets capable of inhibiting disease-associated autoantigens not encapsulated within the TIMP via release of immunoregulatory cytokines, and CNP-102 has proven efficacious in preclinical mouse models of MS.
High-Fat Diet-Induced Gut Microbiota Alterations Worsen Experimental Autoimmune Encephalomyelitis Severity
Authors: Shahi Shailesh , Rux Allison , Ghimire Sudeep , Lehman Peter , Meyerholz David , Mangalam Ashutosh
Published: November 2024

Multiple sclerosis (MS) is a complex neuroinflammatory disease caused by both genetic and environmental factors. Genetically, HLA class II alleles such as HLA-DR2 and –DR3 are strongly linked with MS. Among environmental factors, obesity and gut microbiota has emerged as an important risk factor in MS pathogenesis and severity. This study investigated the impact of High-Fat-Diet (HFD)-induced gut microbiota alterations on experimental autoimmune encephalomyelitis (EAE), utilizing HLA-DR3 (0301) and HLA-DR2(1501).DQ6(0602) transgenic (Tg) mice, crucial for investigating genetic-environment interactions. HFD induces obesity in HLA-DR3, HLA-DR2.DQ6 Tg, and C57BL/6 (B6) mice, exacerbated EAE severity and induced CNS inflammation, and demyelination. Fecal microbiota analysis revealed microbiome dysbiosis with a significant increase in hydrogen sulfide (H2S)-producing bacteria such as Desulfovibrio piger and Bilophila wadsworthia. Mono-colonization of D. piger and B. wadsworthia in gut microbiota-depleted B6 mice mirrored the heightened EAE severity observed in HFD-fed mice. Further, antibiotic depletion study in HFD-fed mice reduced EAE severity and fecal H2S levels, highlighting importance of H2S-producing gut bacteria in obesity-induced EAE modulation. This study identified a disease-promoting role of H2S-producing gut bacteria and underscored the possibility of targeting bacterial H2S as a potential therapeutic intervention to alleviate disease severity in obese MS patients.
IDO2 mediates inflammatory responses through both enzymatic and non-enzymatic mechanisms
Authors: Peng Weidan , Merlo Lauren , Mandik-Nayak Laura
Published: November 2024

The tryptophan catabolizing enzymes indoleamine-2,3-dioxygenase (IDO)1 and IDO2 play important immune modulatory roles in development of inflammation and tolerance. In contrast to the immune regulatory function described for IDO1, IDO2 is a proinflammatory modifier of disease in models of autoimmune arthritis and contact hypersensitivity. Using catalytically inactive IDO2 knock-in mice, we found that the enzymatic function of IDO2 was required to mediate inflammation in contact hypersensitivity but was not required for the development of autoimmunity, suggesting IDO2 acts through both enzymatic and non-enzymatic mechanisms depending on the immune context. Mechanistic studies identified the transcription factor Runx1 as a protein that directly binds to IDO2. The IDO2-Runx1 interaction is blocked by a therapeutic IDO2 monoclonal antibody, implicating Runx1 as a potential downstream mediator of IDO2 function. Runx1 has been shown to directly activate/repress genes involved in B cell activation and differentiation and indirectly affect transcription by inhibition of the NFκB pathway. Preliminary studies using luciferase reporter assays show that IDO2 does not affect Runx1’s direct transcriptional activity, suggesting that the downstream non-enzymatic IDO2 pathway is mediated by Runx1 indirect transcriptional regulation. Taken together, these data demonstrate IDO2 modulates inflammatory immune responses using both its enzymatic function and a non-enzymatic Runx1-mediated pathway.
IRF5 is required for iHPC differentiation and Inhibiting IRF5 activity rescues TLR7-Driven MAS
Authors: Lu Ailing , Thulin Natalie , Huang Amanda , Hamerman Jessica
Published: November 2024

Abstract: Macrophage Activation Syndrome (MAS) is a severe complication in rheumatic diseases, notably systemic juvenile idiopathic arthritis (sJIA), marked by uncontrolled macrophage and T cell activation causing systemic inflammation and multiorgan dysfunction. In a TLR7 overexpressing mouse model (TLR7.1), we observed spontaneous MAS-like disease with thrombocytopenia and anemia, due to a unique population of hemophagocytes called inflammatory hemophagocytes (iHPCs). We demonstrated the critical role of IRF5 signaling in iHPC development using IRF5-deficient mice. However, whether IRF5 is required for iHPC differentiation in TLR7-driven MAS and whether loss or inhibition of IRF5 function in vivo protects from disease onset and/or severity remains unknown.

Methods: We used IRF5-/- mice and IRF5 inhibitor (N5-1) in the TLR7.1 MAS model to examine the requirement of IRF5 for the differentiation and function of iHPCs. IRF5 activation was determined by imaging flow cytometry over the course of disease onset and in response to either preclinical or therapeutic dosing of N5-1.

Results: Results show decreased iHPCs in TLR7.1/IRF5+/- and TLR7.1/IRF5-/- mice. N5-1 inhibits iHPC generation, prevents hemophagocytosis, and rescues TLR7.1 mice from MAS.

Conclusions: These findings underscore the critical role of IRF5 in iHPC development, suggest exploring IRF5 inhibitors in preclinical models and understanding their safety and efficacy could pave the way for treating or preventing MAS.
YTHDF2 promotes tumor immune evasion by inhibiting CD8+ T cell-mediated anti-tumor immunity
Authors: Ma Shoubao , Xiao Sai , Caligiuri Michael , Yu Jianhua
Published: November 2024

YTHDF2 is a well-known m6A reader protein that binds and destabilizes m6A-modified mRNA. We have identified YTHDF2 as a novel regulator of antitumoral polarization in tumor-associated macrophages (TAMs). However, whether YTHDF2 regulates tumor immune evasion remains unknown. Here, we found that loss of YTHDF2 inhibited tumor growth and prolonged survival in syngeneic B16-OVA melanoma and MC38 colon cancer mouse models. Mechanistically, Ythdf2 deficiency-mediated immune remolding was multifactorial, involving promoting the recruitment and polarization of TAMs via chemoattractant CX3CL1, as well as impairing the tumor glycolysis metabolism and enhancing the mitochondrial respiration of tumor-infiltrating CD8+ T cells, thereby enhancing CD8+ T cell effector functions. We screened a small compound that can target YTHDF2 and promote its degradation. YTHDF2 degrader showed a potent anti-tumor effect and better efficacy when combined with anti-PD-L1 antibody therapy. Collectively, YTHDF2 is implicated as a regulator that orchestrates immune evasion and is a promising target to enhance cancer immunotherapy.
Increased DC trafficking and T cell memory in lymphangiogenic melanoma model during immunotherapy response
Authors: Foley Colleen , Potin Lambert , Swartz Melody
Published: November 2024

High vascular endothelial growth factor-C (VEGFC) and lymphatic vessel density in the tumor microenvironment (TME) are associated with lymph node (LN) metastasis, immunosuppression, and poor prognosis in melanoma and other solid tumors. Paradoxically, high VEGF-C also renders immunotherapy more effective in mouse models and correlates with survival after immune checkpoint therapy in patients. Here, we asked how VEGF-C may alter the systemic anti-tumor immune response in melanoma to drive response to immunotherapy, focusing on immune cell trafficking and memory T cell development during immunotherapy response. We used a B16F10 tumor model transduced to either overexpress VEGFC (B16-VEGFC) or a control vector (B16-Control) with a model of immunotherapy using transferred antigen-specific pmel CD8+ T cells activated in vivo by gp100 peptide and CpG adjuvant vaccination.

We found that immunotherapy resulted in increased antigen-specific T cells in the B16-VEGFC vs. B16-Control tumors, and these antigen-specific T cells were more central memory. We also found more bystander activation and proliferation of endogenous CD8 T cells. In the B16-VEGFC tumor-draining LN, we observed increased CD103+ DC trafficking. Overall, VEGFC tumors promote more prolonged immunity after immunotherapy by supporting T cell memory development and enhancing ongoing immune activation and recruitment through increased DC trafficking to the LN and recruitment of newly activated and circulating T cells.
Identifying and Targeting Immunotherapeutic Resistance in Pediatric Brain Cancer
Authors: Sarkar Omar Sadi , Donninger Howard , Farge Justin , Chew Lewis , Huang Michael , Raj Ashok , Yaddanapudi Kavitha
Published: November 2024

Although mortality from childhood cancer is decreasing, a cure remains elusive for certain tumors. Survival rates for patients with high-grade medulloblastoma are dismal. Thus, the need for new therapeutic strategies remains an urgent unmet need. Within the tumor microenvironment (TME), suppressor cells curb the immune system, including myeloid-derived suppressor cells—a heterogeneous population of immature myeloid cells that play a role in cancer progression and metastasis. Two subsets of these cells—granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs)—promote this immunosuppressive milieu by inhibiting anti-tumor T cell activity. In our study, we identified M-MDSCs as a biomarker for high-grade, metastatic pediatric cancers including medulloblastoma. Our data indicated that medulloblastoma M-MDSCs are induced by high levels of the soluble factors GM-CSF and IL-6 produced by HTB-186 medulloblastoma cells. M-MDSCs from medulloblastomas are highly immunosuppressive and exert their suppressive function by inducing reactive oxygen species (ROS). Our data indicate that GM-CSF from medulloblastoma cells induces ROS production in M-MDSCs via the coordinated activation of p47phox, a critical subunit of the NOX2 NAPDH oxidase, by STAT3 and NF-κB, resulting in inhibition of T cells leading to immune suppression in the TME. These results pave the way to new therapeutic approaches for targeting immunosuppression in pediatric medulloblastoma.
Establishing an immunocompetent mouse model of CAR T anti-tumor efficacy for studies of therapeutic resistance
Authors: Rouya Christopher , Mohrs Katja , McCarthy Craig , Roselli Emiliano , Lin John , Kirshner Jessica , Dilillo David
Published: November 2024

Chimeric antigen receptor (CAR) T cell therapies redirect a patient's T-cells to recognize tumor antigens, and are an effective clinical strategy in the treatment of several hematological malignancies. However, targeting of solid tumors remains challenging, in part due to features of the solid tumor microenvironment that impair the expansion, persistence, and function of CAR T cells. To date, most studies employ xenograft models, thus limiting a mechanistic understanding of CAR T biology in the context of an intact immune system. To model the resistance of solid tumors to CAR T therapy and dissect how endogenous immune populations impact this process, we have generated an immunocompetent mouse model of CAR T therapy. Murine CARs recognizing human CD20 have been engineered with an extracellular scFv domain and intracellular CD3z signaling and 4-1BB costimulatory domains. Using flow cytometry and live-cell imaging assays, we demonstrate potent, antigen-dependent in vitro cytotoxicity and cytokine release by huCD20-targeting CAR T cells against solid (MC38) or heme (EL4) tumor cells engineered to express cell-surface huCD20. Moreover, huCD20 CAR T cells exhibit in vivo efficacy against both EL4-huCD20 and MC38-huCD20 tumor models, while CAR T that recognize an irrelevant antigen do not affect tumor growth. These results highlight a platform to explore resistance mechanisms and potential immune cell interactions that may impair CAR T therapy directed against solid tumors.
Identifying cancer-specific T cells for TCR T-cell immunotherapy through engineered proteins
Authors: Zhang Rongyu , Ng Rachel , McKasson Michaela , Chour William , Webster Andrew , Choi Jongchan , Xie Jingyi , Trimble Cornelia , Ribas Antoni , Heath James
Published: November 2024

The identification of cancer-specific T cell receptor (TCR) sequences is crucial for advancing cancer immunotherapies. Recent studies and clinical trials have revealed a vulnerability in monoclonal T cell therapy, where cancer cells can evade the immune response through the loss of HLA heterozygosity and low antigen heterogeneity. To address this challenge, cocktail T cell therapy using TCRs that target multiple HLAs and antigens has been proposed to enhance the efficacy of adoptive T cell transfer therapy.

In pursuit of this goal, we have developed peptide-bound major histocompatibility complex (pMHC) proteins in the form of single-chain-trimers (SCTs). This innovative platform allows for the rapid and high-throughput identification and isolation of cancer-targeting CD4+ and CD8+ T cells targeting a wide array of HLAs and cancer antigens. The identified antigen-specific TCRs are subsequently sequenced, validated for functionality, and assessed for potential therapeutic applications.

We applied this technology to PBMCs extracted from patients with HPV-16-related precancerous conditions in a clinical trial. We successfully identified cancer-specific TCRs, which are currently undergoing pre-clinical validations for their safety and efficacy in CRISPR-mediated TCR T cell therapy.

In summary, the application of the SCT technology holds significant value for fundamental immune-oncology studies and contributes to the advancement of immunotherapy for cancer.
Sirt2 inhibitors protect chronic inflammatory disease by improving gut epithelial barrier integrity
Authors: Hou Dan
Published: November 2024

Inflammatory Bowel Disease (IBD), as a chronic inflammatory disease, triggers a global health threat with poorly understood mechanisms driving its progression. Unraveling these mechanisms and identifying new therapeutic targets are crucial for improved treatments. Prior studies revealed conflicting roles of Sirtuin 2 (Sirt2) in IBD: genetic knockout of Sirt2 exacerbates IBD, while pharmacological inhibition diminishes its severity. This contradiction hinders Sirt2 inhibitor development for IBD treatment. Our investigation using distinct Sirt2 inhibitors (TM and AGK2), a PROTAC Sirt2 degrader, and genetic knockout mice addressed this puzzle. Both TM and AGK2 showed protective effects in an IBD model induced by DSS, excluding off-target effects. Mechanistically, these inhibitors suppressed ARF6-mediated E-cadherin endocytosis, fortifying intestinal epithelial integrity and the gut barrier, protecting against IBD. Interestingly, the PROTAC Sirt2 degrader, similar to Sirt2 genetic knockout, didn’t offer protection in IBD. This discrepancy suggests that Sirt2 small molecular inhibitors only partially but not all hinder Sirt2's activities, stressing the need for substrate-dependent inhibitors in IBD treatment. Overall, our study provided an interesting example that genetic knockout and pharmacological inhibition were not consistent, and this mechanistic understanding further support Sirt2 as a promising target for treating IBD.
Multi-Omics Profiling of Tumor Infiltrating CD8+ T Cells Specific to Neoantigens and Tumor-Associated Antigens in Skin Cancer
Authors: Kong Lingyang , Huang Alexander , Jiang Ning
Published: November 2024

The objective is to characterize the immunological response towards skin cancer, particularly Merkel cell carcinoma (MCC), by conducting a detailed multi-omics analysis of CD8+ T cells infiltrating tumors, specifically those recognizing neoantigens and tumor-associated antigens. We utilized a high-throughput DNA-barcoded Tetramer-associated T cell antigen receptor sequencing (TetTCR-SeqHD) technique to profile T cell receptor (TCR) specificity, gene expression, and surface protein expression at the single-cell level. Our methodology included a neoantigen prediction pipeline using patient sequencing data, optimizing the identification of individualized neoantigens. Initial analyses were performed on melanoma patient samples, extracting tumor-antigen-specific T cells from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs). Our results show distinct TCR clonotypes and their dynamics, providing insights into the immune landscape and disease progression in skin cancer. These findings highlight the potential of multi-omics profiling in understanding the intricate interactions between the immune system and tumor cells, offering promising avenues for advancing skin cancer therapeutics and personalized medicine. Through this research, we aim to contribute significantly to the field of cancer immunology, demonstrating the profound implications of multi-omics approaches in deciphering the multifaceted immune responses within the tumor microenvironment.
Type 2 innate lymphoid cells fuel breast cancer development by inhibiting anti-cancer immunity
Authors: Naef Pascal , Rodriguez Jacob , Savage Hannah , Adam Isam , Villalta Armando , Lawson Devon , Kessenbrock Kai
Published: November 2024

The tumor microenvironment is a crucial regulator of breast cancer (BC) development. It is composed of acellular and cellular factors, among others immune cells like type 2 innate lymphoid cells (ILC2s). The molecular mechanisms of how ILC2s regulate BC immunity remain largely unknown.

To reveal the phenotype of ILC2s in BC, we used the polyoma middle T antigen transgenic mouse model, mimicking luminal BC development. ScRNA sequencing and flow cytometry experiments showed that ILC2s (expressing Gata3, Rora, Il2ra, and Il7ra) accumulated during disease development and that the ST2, cKit, and Neuropilin-1 expression on ILC2s increased with tumor burden. Additionally, PMA/Ionomycin restimulated ILC2s expressed stable interleukin-5 and -13 levels. We detected the same ILC2 phenotype in the tumors of BRCA1mutp53-/- mice, which mimic the basal BC subtype. To assess the functional relevance of ILC2s in BC, we co-injected ILC2s and the BC cell line VO orthotopically into FVB mice and measured the tumor growth. While 85% of the mice that were co-injected with VO cells and ILC2s developed a tumor, 50% of the VO-only recipients rejected the tumor cells. ILC2-co-injection induced an increased infiltration of Gr-1+CD11b+ neutrophils, which could potentially inhibit anti-cancer immunity.

In summary, we show that ILC2s accumulate, get activated, and promote an immune-inhibitory environment. Further experiments will reveal if ILC2s can be used as targets to treat BC patients.