bluuebunny/crossref_metadata_2025 huggingface dataset:
Irreversible electroporation stimulates a pro-inflammatory tumor microenvironment that facilitate antigen presentation in pancreatic cancer
Authors: Imran Khan Mohammad , Brock Rebecca M. , Beitel-White Natalie , Hendricks-Wenger Alissa , Coutermarsh-Ott Sheryl L , Manuchehrabadi Navid , Davalos Rafael V. , Allen Irving C
Published: February 2023

Pancreatic cancer is now the third leading cause of cancer-related death and is projected to turn out to be the second by 2030. Pancreatic cancer is often late-diagnosed with few treatment options with extremely low survival rates. Surgical candidacy is only 15% of total diagnosed patients. Irreversible electroporation (IRE), a novel form of tumor non-thermal ablation applies high-voltage microsecond pulses to permeabilize the cell membrane that is irreversible and leads to apoptotic cell death. IRE has had strong success in clinical trials for pancreatic cancer patients. Nevertheless, little is known on the effect of IRE beyond the initiation of cell death in the tumor tissue. We hypothesize that IRE can induce a pro-inflammatory type of cell death in pancreatic cancer that can promote inflammation and anti-tumor immune system responses. We have used in vitro, ex vivo, and immunocompetent in vivo murine models to investigate alterations to pancreatic cancer and its microenvironment. Our findings reveal programmed necrotic cell death, promotion of pro-inflammatory cytokine production, and shifts in immune cell populations from a pro-tumor (such as MDSCs and T regulatory cells) to a proinflammatory types containing cytotoxic lymphocytes and neutrophils after IRE treatment. Progression-free survival was significantly longer for the treated mice. Intriguingly, IRE treatment increases IFNγ signaling and generates viable antigens for presentation to the adaptive immune system but also promotes PD-L1 expression which in turn causes immunological stalemate. Our results suggest possible therapeutic targets for use in combination with IRE, such as gemcitabine or PD-1/PD-L1 inhibitors, to improve patient survival.
Oral delivery of a microbial extracellular vesicle induces potent anti-tumor immunity in mice
Authors: Francisco Loise , Abdou Mary , Troy Erin , Ballok Alicia , Chernac Fabian Romano , Kashyap Shubhra , Argueta Shannon , Tsang Jessica , Goldberg Michael , Sizova Maria , McBride Audrey , Keats Jake , Invally Krutika , Rommel Tyler , Ponichtera Holly , Huynh Kevin , Kravitz Valeria , Ganguly Tanmoy , Bodmer Mark
Published: February 2023

The small intestinal axis (SINTAX) is a network of anatomic and functional connections between the small intestine and the rest of the body. It acts as an immunosurveillance system, integrating signals from the environment that affect physiological processes throughout the body. The impact of events in the gut in the control of tumor immunity is beginning to be appreciated. We have previously shown that an orally delivered single strain of commensal bacteria induces anti-tumor immunity via activation of innate and adaptive immunity. Some bacteria produce extracellular vesicles (EVs) that share molecular content with the parent bacterium in a particle that is roughly 1/1000th the volume in a non-replicating form. We report here an orally-delivered and gut-restricted bacterial EV which potently attenuates tumor growth to a greater extent than whole bacteria or checkpoint inhibition. EDP1908 is a preparation of EVs produced by a bacterium of the Oscillospiraceae family isolated from a human donor. Treatment of tumor-bearing mice with EDP1908 shows superior control of tumor growth compared to anti-PD-1 or an intact microbe. EDP1908 significantly increases the percentage of IFNγ producing CD8+, NK, NKT and CD4+ cells in the tumor microenvironment (TME). EDP1908 also increases tumor-infiltrating dendritic cells (DC1 and DC2) and production of IP-10 and IFNg in the TME, creating an environment conducive to the recruitment and activation of anti-tumor lymphocytes. These data point to oral EVs as a new class of immunotherapeutic drugs that harness the biology of the small intestinal axis, acting locally on host cells in the gut to control distal immune responses within the TME. EDP1908 is in preclinical development for the treatment of cancer.
‘MR1 Tetramer-based Artificial Antigen Presenting Cells Expand MAIT Cells from Human Peripheral Blood that Effectively Kill Glioblastoma Cells.’
Authors: PRIYA RAJ , BRUTKIEWICZ RANDY RICHARD
Published: February 2023

Immunotherapy for cancer treatment requires the activation of cytotoxic effector lymphocytes. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize the MHC class I-like molecule, MR1. MAIT cells play an important role in the immune response against microbial infections and can directly kill tumor cells. Although MAIT cells can be expanded ex vivo, this method is time-consuming, expensive and requires allogenic feeder layers. To overcome the limitations of conventional dendritic cell-based vaccines and ex vivo expansion of human T cells, an artificial antigen presenting cell (aAPC) approach to expand antitumor effector cells has several advantages. In this study, we explored an efficient in vitro method to amplify MR1-specific MAIT cells from human peripheral blood using aAPCs made by coating cell-sized latex beads with an antigen-loaded MR1 tetramer complex and anti-CD28 antibody. We further elucidated the cytotoxic potential of such expanded MAIT cells against three human glioblastoma (GBM) cell lines to explore their potential use as a novel immunotherapeutic tool, as the mostly lethal GBM poorly responds to conventional chemotherapy. When aAPCs were compared to the standard allogenic feeder layer-based approach for MAIT cell expansion, they were significantly more effective. Our results indicate that the aAPC-expanded MAIT cells remained functional, retained their original phenotype, secrete pro-inflammatory cytokines, and showed cytotoxicity against the GBM cell lines. Hence, MAIT cells have the potential to be a novel tool in immunotherapy approaches for the treatment of human GBM.
Seq-ing the source of radiation-induced anti-tumor immunogenicity in patient tumors
Authors: Chow Jacky Louis , Hoffend Nicholas , Gaudieri Madeline , Abrams Scott I , Schwaab Thomas , Singh Anurag , Muhitch Jason
Published: February 2023

Translational studies in a small number of patients have demonstrated a clinical benefit when combining radiation and immunotherapy. These findings are supported by correlative data showing that transcriptional changes to the tumor microenvironment following radiotherapy are associated with responses. Yet, given limited access to patient specimens following treatment, few studies have addressed how radiation primes anti-tumor immunity at single cell resolution, thus limiting our understanding of the source of radiation-induced changes within tumors. Here, we acquired renal cell carcinoma (RCC) tumors from patients treated with stereotactic body radiation therapy (SBRT) followed by nephrectomy in a pilot study (NCT01892930), to evaluate radiation-induced transcriptional changes to immune and RCC cells by single-cell RNA sequencing. We hypothesized that SBRT-induced interferon (IFN) and immune-activating signals, previously detected by bulk sequencing and array platforms, would localize to the dense lymphocyte infiltrate characterized in RCC and that the transcriptional signatures in SBRT-treated RCC would reflect increased immune recognition. Single-cell transcriptomics uncovered increased frequency of mature T cells in irradiated tumors, including increased expression of IFN-gamma and granzymes, as well as PD1 and other exhaustion markers. Additionally, irradiated-RCC cells expressed more IFN-gamma receptor and were enriched for IFN signaling and antigen processing pathways. Collectively, these results indicate radiation may improve anti-tumor immunity by increasing both T cell activity and tumor cell sensitivity to pro-inflammatory cytokines and cytolysis in human malignancies.
Quantitative Immune Profiling of Human Tumor Tissues with Multiplexed ChipCytometry
Authors: Gamber Kevin , Christians Arne , Schwarz Spencer , Northcutt Adam , Forys Jason , Campbell Thomas , Winkeler Crystal
Published: February 2023

Cancer therapies that rely on manipulating or engineering immune cells have shown much promise in recent years for effectively combating a broad array of cancer types. In order to determine the effectiveness of such therapies, it is necessary to accurately profile the complement of immune cell types present in the tumor microenvironment. Methods to reliably obtain immune cell signatures require the combination of powerful resolution to discriminate cell boundaries, broad dynamic range to capture markers of varying expression levels, and accurate cell segmentation across a wide range of cell sizes and morphologies. Many methods for quantitative immune cell profiling have demonstrated shortcomings when it comes to the aforementioned attributes. To remedy these shortcomings, we present here ChipCytometry, an imaging technology for immune profiling of cells and sectioned tissues.

ChipCytometry is a fluorescence-based imaging system that utilizes multiplexed immuno-fluorescence staining in combination with high-dynamic-range (HDR) imaging to facilitate quantitative phenotyping of individual cells within tissue samples. Employing this technology to profile metastatic and primary tumors, multiple biomarkers were stained in iterative multiplex assays to profile single cells with an AI-powered cell segmentation algorithm. The HDR imaging allows for both strong and weak fluorescent signals to be simultaneously obtained without loss of sensitivity. This immune profiling was completed on 6 different cryosectioned human tumor tissues (head & neck, liver, lung, breast, colon, and pancreas).
Circulating immune cell phenotype dynamics reflect the effect of a CD200 immune checkpoint targeted immunotherapy in pet dogs with high-grade glioma
Authors: Puerta-Martinez Francisco W , Schnell Sathya , Arnold Susan , Pluhar G. Elizabeth , Olin Michael
Published: February 2023

The extent to which immune cell phenotypes in the peripheral blood reflect intratumoral immune activity prior to and early in cancer immunotherapy is unclear. Here we studied the population dynamics of immune cells and immune phenotypic changes, using clinical blood samples and a multicolor flow cytometry analysis. These samples were serially obtained from a cohort of high-grade glioma canine patients enrolled in a trial with an immunotherapy that targets the CD200/CD200R immunological synapse. This therapy consists of an alternative checkpoint inhibitor peptide (CD200AR-L) that modulates the CD200 immune checkpoint, currently being used in an adult phase I safety trial for recurrent glioblastoma trial (NCT04642937) and a canine high-grade spontaneous glioma trial (U01CA224160-01). We found evidence of a strong effect of the therapy on peripheral immune cells in responder versus nonresponder dogs. Upon initiation of therapy, immune effector cell proliferation increased in responsive patients, whereas in nonresponders immune cell populations either didn’t change significantly or an increase in immunosuppressive phenotypes was observed. Cell populations such as MDSC, regulatory T cells and exhausted T cells were predominantly higher in nonresponder dogs. These data showed that peripheral immune cell phenotypes were linked to the outcome of the immunotherapy, thus dogs with better immune cell profiles, including increased cytotoxic differentiation, had a better prognosis and longer overall survival rates. Taken together, these results suggest that variation in patient responsiveness can be explained by differences in peripheral immune cell proliferation and differentiation soon after the initiation of immunotherapy.
STAT1 associated immune checkpoint expression and immune cell localization represent adaptive immune resistance in high-grade serous ovarian cancer
Authors: Li Deyang , Vidotto Thiago , Shakfa Noor , Afriyie-Asante Afrakoma , Peterson Nichole , de Ladurantaye Manon , Mes-Masson Anne-Marie , Francis Julie-Ann , Koti Madhuri
Published: February 2023

High-grade serous carcinoma (HGSC) is a deadly malignancy leading to ~70% of the 140,000 ovarian cancer deaths each year. Resistance to platinum chemotherapy followed by recurrence and an incurable disease occurs in most HGSC patients. Contemporary immune checkpoint blockade therapies have shown minimal efficacy in this cancer. Our previous investigations established that tumour interferon (IFN) activation status and CD8+ T cell density are predictors of chemotherapy response in HGSC. Furthermore, we also showed that the IFN induced chemokine CXCL10 is a key determinant of increased survival via immune cell recruitment in the tumour immune microenvironment (TIME). Given that signal transducer and activator of transcription 1 (STAT1) is central to the feed forward loops of cellular IFN responses, we investigated STAT1 associated transcriptomic alterations and spatial profiles of immune cells in 204 pre-treatment HGSC tumours.

RNA-sequencing based whole transcriptomic profiling revealed that higher STAT1 expression significantly correlated with higher immunomodulatory gene expression, including immune checkpoints and activators, in both chemotherapy sensitive and resistant tumours. Findings were independently validated in a cohort of 379 HGSC tumour RNA-Seq profiles from The Cancer Genome Atlas Network ovarian cancer dataset. Multiplex immunofluorescence based spatial profiling of CD8+ T cells, FoxP3+ T regulatory T cells, CD68+M1, and CD163+ M2 macrophages and expression of PD-L1, PD-1, IDO1 immune checkpoints was performed. Findings from our study provide evidence for IFN mediated adaptive immune resistance in the HGSC TIME and will potentially inform the design of rational chemo-immunotherapy approaches.
Induction of Innate Immune Visibility of Neuroendocrine Tumors Potentiates Immune Responses and Tumor Rejection
Authors: Zhu Mingrui , Huang Yi , Bender Matthew , Girard Luc , Kollipara Rahul , Eglenen-Polat Buse , Naito Yujiro , Savage Trisha , Huffman Kenneth , Koyama Shohei , Kumanogoh Atsushi , Minna John , Johnson Jane , Akbay Esra
Published: February 2023

Natural killer (NK) cells serve as an important role in suppressing tumor growth by directly attacking malignant cells. These cells recognize the tumor cells through moleclar patterns associated with oncogenic transformation. NKG2D ligands (NKG2DLs) are one of these markers present on tumor cells which can activate NK cells and T cells by interacting with NKG2D receptors on these lymphocytes. Majority of tumors express NKG2DLs. Interestingly, tumors carrying neuroendocrine features such as small cell lung cancer (SCLC) and neuroblastoma express very low level of ligands for NK cell activating receptor NKG2D (MICA, MICB, ULBP1, ULBP2, ULBP3), which may help them escape from innate immune surveillance. Further characterization of SCLC immune environment showed reduced antigen presentation, reduced infiltration of total immune cells specifically NK cells, as compared to NSCLC, a more immunogenic tumor. Transcriptional inducers of NKG2DLs did not induce NKG2DL in SCLCs due to hypo-acetylation of MICA/B region. Histone deacetylase (HDAC) inhibitors were able to induce NKG2DL on tumor cell surface. Restoring NKG2DL in preclinical SCLC and neuroblastoma models either by ectopic expression or HDAC inhibitors stimulated direct NK cell killing in co-culture assays. NKG2DL stimulation also caused potentiation of antibody directed therapies such as CD3-EpCAM in neuroendocrine tumors. In vivo studies showed suppressed tumor growth and metastasis in an NK and CD8 T cell dependent manner when NKG2DLs are stimulated. We conclude that: restoring innate immune visibility can target neuroendocrine tumors to both innate and adaptive immune cytotoxicity.
Deficiency of RIPK3 (receptor-interacting protein kinase 3) in antigen-presenting cells dampens the pro-inflammatory T cell response following antigen presentation.
Authors: Pilon Celeste , Lesage Sylvie , Chabot-Roy Geneviève , Levine Jerrold S , Rauch Joyce
Published: February 2023

STUDY OBJECTIVE: We have previously shown that RIPK3 (receptor-interacting protein kinase 3)-deficient mice are protected from the development of an induced murine model of systemic lupus erythematosus (SLE). The development of SLE autoantibodies in this model is associated with the generation of an antigen-specific T cell response. We hypothesize that RIPK3-deficient antigen-presenting cells (APCs) are impaired in generation of a robust T cell response, thereby impacting induction of SLE in our murine model.

METHODS:

We performed a complete cellular immunophenotyping on mice deficient in RIPK3, compared with wild type C57BL/6 mice. We also assessed the function of RIPK3-deficient APCs in an in vitro antigen presentation assay using ovalbumin (OVA) and OVA-specific OT-II T cells.

RESULTS:

No major differences in immune cell composition and phenotype were identified between naïve RIPK3-deficient and WT mice. However, RIPK3-deficient bone marrow-derived dendritic cells (BMDCs) were found to produce lower levels of pro-inflammatory cytokines (interleukin [IL]-6 and tumor necrosis factor [TNF]-α) following lipopolysaccharide (LPS) stimulation. Moreover, presentation of OVA by RIPK3-deficient BMDCs resulted in a lower proportion of interferon (IFN)-γ producing OT-II T cells, compared to antigen presentation by WT BMDCs. In contrast, T cell proliferation was similar whether antigen was presented by RIPK3-deficient or WT BMDCs.

CONCLUSION:

Our results suggest that RIPK3 deficiency in APCs impacts antigen presentation to T cells, identifying a possible mechanism by which RIPK3-deficient mice are protected from induction of SLE.
The Unfolded Protein Response reveals eIF2α phosphorylation as a critical factor for direct MHC class I antigen presentation.
Authors: Nagamine Brandy S , Dolan Brian P
Published: February 2023

Modifying direct Major Histocompatibility Class I (MHC I) antigen presentation is an attractive target for the treatment of various conditions including autoimmune diseases, chronic viral infections, and cancers. Disease perturbations alter the cellular environment, influencing the presentation of peptides to CD8+ T cells. The unfolded protein response (UPR) is a signaling pathway activated in response to unfolded proteins within the ER. Previous studies have established the relationship between the UPR and MHC I antigen presentation, but the exact mechanisms remained unknown. In this study, we utilized small inhibitory molecules to individual UPR pathway targets to investigate the effect on global MHC class I levels and specific peptide presentation. Preventing PERK pathway signaling and the resulting phosphorylation of eIF2α by GSK2656157 treatment did not change MHC I antigen presentation. However, treatment with salubrinal which has the opposite effect of GSK2656157, decreased both antigen presentation and overall cell-surface MHC I levels. Treatment with 4μ8C, an inhibitor of the IRE1α UPR activation pathway which prevents splicing of Xbp1 mRNA, also diminished MHC I antigen presentation. Unexpectedly, 4μ8C treatment led to an increase in eIF2α phosphorylation in addition to blocking IRE1α signaling. Given that salubrinal and 4μ8C increase phosphorylation levels of eIF2α while decreasing antigen presentation, we conclude that UPR signaling through PERK and the subsequent phosphorylation of eIF2α, results in a modest decrease in direct MHC I antigen presentation. These data suggest that the PERK pathway alters host antigen presentation centering on translational control by eIF2α.
Roles for STAT5 in homeostasis and function of CD103+cDC1s in non-lymphoid organs
Authors: Zhou Yifan , Babcock Rachel L. , Chrisikos Taylor T. , Patel Bhakti , Medik Yusra B. , Kahn Laura M. , Li Haiyan S. , Watowich Stephanie S.
Published: February 2023

Type 1 conventional dendritic cells (cDC1s) have critical roles in inducing adaptive immune responses and mediating immune tolerance. Signal Transducer and Activator of Transcription 5 (STAT5) was found to be crucial for monocyte-derived DC development; however, its role in non-lymphoid tissue CD103+cDC1s remained largely unknown. We evaluated the role of STAT5 by employing myeloid-specific Stat5-deficient mice,LysM-Cre +Stat5f/f and CD11c-Cre+Stat5f/f mice. Both strains show deficiencies in the numbers of CD103+cDC1s and alveolar macrophages (AMs) in lung, suggesting ineffective maintenance of lung homeostasis. Consistent with this concept, CD11c-Cre+Stat5f/f mice show granulocyte and monocyte accumulation in lung, accompanied by a reduction in the number of CD4+T, CD8+T, and B cells. Moreover, CD11c-Cre+Stat5-deficient mice have increased numbers of alveolar histiocytes, which are also enlarged, as well as accumulation of amorphous and eosinophilic material in alveolar spaces, alveolar histiocytosis, and proteinosis. In addition to the lung, liver CD103+cDC1s were also decreased by Stat5 depletion, however colon CD103+cDC1s were not affected. To examine cell-autonomous roles for STAT5, we generated Stat5-deficient CD103+cDC1s using our established bone marrow culture system, and tested in vitro-differentiated CD103+cDC1s for antigen presentation activity. These assays suggest Stat5 deficiency impaired CD103+cDC1s antigen cross-presentation ability. Taken together, our study showed that STAT5 is required for homeostasis and function of CD103+cDC1s in non-lymphoid organs.
MR1 expression and surface translocation in airway epithelial cells from COPD or smoker lungs is altered following exposure to IFNγ or MAIT cells.
Authors: Huber Megan E , Larson Emily , Lust Taylor N , Heisler Chelsea M , Harriff Melanie J
Published: February 2023

MAIT cells, a highly prevalent subset of T cells restricted by the MHC Class I-like molecule MR1, play a key role in early response to bacterial infection. Chronic obstructive pulmonary disease (COPD) pathogenesis is associated with airway inflammation, increased infiltration by CD8+ T lymphocytes, and infection-driven exacerbations. Cigarette smoke (CS), a leading cause of COPD, leads to impaired immune function of airway epithelial cells (AECs) and decreases surface expression of classical MHC Class I molecules. However, the impact of CS and COPD on MR1 expression and airway MAIT cell function is unknown. We have demonstrated that exposure to CS alters MAIT cell responses to primary AECs from healthy, COPD, or smoker lungs. Here, we examined whether CS impacts expression of MR1 mRNA or surface translocation of MR1 in these primary AEC populations as a possible mechanism for this dysregulation. Short-term treatment of AECs with CS or infection with S. pneumoniae (SP) did not alter MR1 expression in any of the donor AEC groups. However, incubating CS-treated or SP-infected AECs with MAIT cells increased MR1 expression. This upregulation may be mediated through IFNγ signaling pathways, as treatment with IFNγ alone resulted in MR1 upregulation. Our data further suggest MAIT cell- and IFNγ-mediated MR1 upregulation is altered in COPD cells. Additionally, we observed increased baseline surface expression of MR1 protein in smoker AECs and reduced surface translocation upon addition of exogenous ligand. COPD- and CS-mediated dysregulation of MR1 expression could impact MAIT cell function in airways, resulting in inflammation or reduced early response to bacteria, contributing to lower airway colonization and COPD exacerbations.
Thymic epithelial cells require the autophagy-related protein Vps34 for CD4+ T cell selection
Authors: Postoak Joshua , Yang Guan , Wu Lan , Song Wenqiang , Xiao Shiyun , Manley Nancy , Van Kaer Luc
Published: February 2023

The generation of a functional, self-tolerant T cell repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). The presentation of self-antigens to developing thymocytes involves a variety of cellular processes, including proteolysis, endocytosis, vesicle trafficking, and autophagy. In our current study, we focus on the class III PI3K, Vps34, which has been implicated in autophagy and vesicle trafficking. To address the contribution of Vps34 to TEC function, we have generated mice with a TEC-specific Vps34 deficiency. Conditional mutants display progressively hypoplastic thymi with reduced TEC cellularity and altered TEC subset ratio. Additionally, when TCR transgenic lines were bred to these animals we found profound defects in the positive selection of the CD4 T lymphocyte lineage but not the CD8 T lymphocyte lineage. Interestingly, cortical TECs from mutant mice display an increased abundance of unprocessed antigen bound to surface MHCII molecules indicating altered antigen processing. Collectively, these studies identify a role for the PI3K Vps34 in maintaining TEC homeostasis and contributing to the repertoire of selecting ligands processed and presented by TECs.
Allergic and anaphylactic responses to alcohol-soluble wheat gluten in female versus male Balb/cJ mice in an adjuvant-free model
Authors: Jorgensen Rick Dean , Gao Haoran , Raghunath Rajsri , Chandra Shivam , Nagisetty Siddharth , Ng Perry KW , Gangur Venugopal
Published: February 2023

Wheat is a major allergenic food capable of eliciting life-threatening anaphylaxis. Food allergies including wheat allergy are growing globally, and therefore warrant further investigation. While animal studies are being conducted to advance our understanding of wheat allergy, it is unknown whether female versus male mice differ in their allergic responses to wheat gluten. Here we tested the hypothesis that alcohol-soluble wheat gluten (ASWG) when applied over the skin will sensitize both female and male mice for oral hypothermia shock response in an adjuvant-free mouse model. Balb/cJ mice were produced and kept on a plant protein-free diet during this study. Groups of adult mice (n = 8–10) were exposed to durum wheat ASWG via the skin (once a week for nine weeks), without an adjuvant using our previously reported transdermal exposure (TDE) method. In females, TDE with the ASWG elicited a significant ASWG-specific IgE antibody response. However, in males, TDE with the ASWG did not elicit a significant ASWG-specific IgE antibody response. Oral challenge of ASWG, but not vehicle, elicited significant hypothermia shock response in ASWG skin-sensitized female mice. In contrast, no significant hypothermia shock response was observed in males upon oral challenge with ASWG or vehicle. These data demonstrate that female mice are more susceptible than males to develop an allergic response and oral anaphylaxis to ASWG in this model.
Blimp-1 expressing lung ILC2s are a distinct cell population in allergic asthma
Authors: Zhang Jinyi , He Kun , Ford B. Rhodes , Hettinga Angela , Poholek Amanda C.
Published: February 2023

Blimp-1 is a transcription factor known to have critical roles in multiple cell types including B, T and NK cells. However, its expression and function in non-NK innate lymphoid cell populations have not been well studied. To examine the expression and function of Blimp-1 in group 2 innate lymphoid cells, we analyzed several models of type 2 immunity. We found that Blimp-1 is not highly expressed in ILC2s at steady-state, however it is induced under specific stimulation conditions. Especially, Blimp-1 expression is substantially increased in house dust mite (HDM) activated mouse lung over time. In vivo and in vitro ILC2 stimulation with cytokines show that both IL-33 and IL-10 can induce Blimp-1 expression, indicating Blimp-1 is driven by multiple signals. Interestingly, HDM driven Blimp-1 expressing ILC2 have a distinct transcriptional program compared to classically-activated ILC2s treated with IL-25 and IL-33. ILC2 that express Blimp-1 predominantly lack type 2 cytokine expression, suggesting Blimp-1 plays a role independent of cytokine production and may represent a unique functional state of ILC2 cells in settings of chronic antigen including allergic disease. Our findings therefore demonstrate a previously unappreciated Blimp-1 expressing ILC2 population with distinct features that may be important for type 2 mediated disease.
Ruxolitinib ameliorates airway hyper responsiveness and lung inflammation in a corticosteroid-resistant murine model of severe asthma
Authors: Das Rupali , Bahal Devika , Hashem Tanwir , Kammala Ananth Kumar , Subramanian Hariharan
Published: February 2023

Asthma prevalence has increased considerably over the decades and it is now considered as one of the most common chronic disorders in the world. While the current anti-asthmatic therapies are effective for most asthma patients, there are 10–30% subjects whose disease is not controlled by such agents and they account for about 50% of the healthcare costs of asthma. Such asthmatics develop severe asthma (SA), a condition characterized by a dominant Th1/Th17 cytokine response that is accompanied by type 2-low endotype. As JAK (Janus Kinase) signaling is very important for the activation of several cytokine pathways, we examined whether inhibition of JAKs might lessen the clinical and laboratory manifestations of SA. To that end, we employed a murine model that recapitulates the complex immune response identified in the airways of human SA patients. To induce SA, mice were sensitized with house dust mite extract (HDME) and c-di-GMP (mucosal adjuvant) and then subsequently challenged with HDME and a lower dose of c-di-GMP. In this model, treatment with the JAK inhibitor, ruxolitinib, significantly ameliorated all the features of SA, including airway hyper responsiveness and lung inflammation; cellular influx and cytokine production as well as total IgE antibody titers. Thus, these studies highlight the JAKs as critical targets for mitigating the hyper-inflammation that occurs in SA and provide the framework for their incorporation into future clinical trials for patients that have severe or difficult-to manage asthma.
Post-Thrombotic loss of the epigenetic enzyme MLL1/KMT2a in macrophages suppresses urokinase expression and may contribute to an antifibrinolytic phenotype
Authors: Sharma Sriganesh B. , Beardslee Renee A. , Luke Catherine , Dowling Abigail R. , Henke Peter K. , Moore Bethany B. , Gallagher Katherine A. , Obi Andrea T.
Published: February 2023

Objectives:

Macrophages (Mϕs) are critical in the process of subacute and chronic venous thrombus (VT) resolution. Epigenetic alterations can reprogram Mϕ function in chronic inflammatory states. We hypothesized that the chromatin modifying enzyme MLL1/KMT2a, which increases H3K4 trimethylation at NF-kβ targeted promoters during inflammatory processes, influences Mϕ-mediated post-thrombotic fibrinolysis.

Methods:

Bone marrow derived Mϕs (BMDMs) from C57bl6 mice and immortalized Mϕs (RAW264.7) were used for in vitro experiments. Small interfering RNAs (siRNAs) were transfected to achieve MLL1 silencing, and qPCR, immunoblotting, and chromatin immunoprecipitation (ChIP) assays were performed. In vivo, VT formation was induced by IVC ligation, after which BMDMs were harvested.

Results:

Analysis of procoagulant and antifibrinolytic transcripts in MLL1 silenced BMDMs and RAW264.7 cells revealed suppression of urokinase (Plau) mRNA and protein levels by >80% and >50% respectively (p<0.05) relative to controls. ChIP analysis of the Plau promoter in MLL1 knockdown cells showed decreased enrichment (~4.6 fold, p=0.0216) of H3K4me3 and suggested a functional role for MLL1 to promote Plau expression. BMDMs harvested from mice 7 days post-thrombosis showed suppressed mRNA and protein levels of MLL1 (by >65% and >55% respectively, p<0.05), PLAU (by >85% and >55% respectively, p<0.05) levels and decreased H3K4me3 enrichment on the Plau promoter (~7.2 fold, p=0.0031) relative to controls.

Conclusions:

The post-thrombotic inflammatory state induces MLL1-mediated epigenetic modifications in the bone marrow, resulting in suppression of Mϕ urokinase expression. These changes may contribute to an antibrinolytic Mϕ phenotype.
Langerhans cells rely on good neighbors to overcome gene deficiencies.
Authors: Herbst Christopher H , Bouteau Aurélie , Igyártó Botond Z
Published: February 2023

We recently identified that epidermal resident Langerhans Cells (LCs) acquire gene expression fingerprints from surrounding Keratinocytes (KCs) in the form of mRNA and protein. In the present study, we aimed to determine whether this transfer can also overcome gene deficiencies. For this purpose, using the Cre/Lox system, we specifically deleted genes for Connexin 43 (Cx43), MyD88, and MHC-II in LCs. While all three genes underwent recombination, reduced protein levels were only observed for MHC-II, whereas Cx43 and MyD88 protein levels remained unaltered. Considering that KCs lack MHC-II, but express Cx43 and MyD88 at high levels, we posit that LCs can acquire gene products from surrounding KCs to overcome their own deficiencies if those products are available.

In summary, we present evidence that cells can compensate for gene deficiencies if the surrounding cells can provide. These findings highlight the limitations of cell-specific gene deletion, and could provide an explanation as to why certain gene deletions do not lead to measurable deficiencies.
Cutaneous T cells promote distinct cellular outcomes in human keratinocytes and fibroblasts
Authors: Morawski Peter A , DeBerg Hannah , Fahning Mitchell L. , Gratz Iris , Campbell Daniel J.
Published: February 2023

Human skin is a barrier tissue home to a highly specialized immune environment in which leukocytes coordinate with local epithelial and stromal tissue in response to physical, chemical, and biological stresses. T cells are abundant in the skin and are required for both host defense to pathogens and the subsequent repair of damaged tissue. Cutaneous lymphocytes include both resident and migratory fractions, which can be identified and isolated using expression of the cutaneous lymphocyte antigen (CLA). There are multiple subsets of CLA+ T cells, each having a distinct phenotype and functional capacity, however their precise roles in contributing to host-protective and tissue-repair responses remains incompletely understood. We assessed the function of eight different CD4+CLA+ cutaneous T cell subsets from the skin and blood, including traditional T helper lineages and a novel subset of CD4+CLA+CD103+ T cells with a tissue repair phenotype, by bulk RNAseq analysis of keratinocytes and fibroblasts following administration of activated T cell supernatants. Differential gene expression and pathway analysis reveal distinct cellular outcomes in keratinocytes and fibroblasts that depend strongly on T cell source and the activity of specific cytokines. Our findings define a diverse array of immune-induced states in the skin that are dependent on the unique functional capacity of various T cell subsets, thereby constructing a comprehensive tool to map how cutaneous T cells contribute to pathological disruption, tissue repair and return to homeostasis within human skin.
The Role of Interactions of Adaptor Protein PSTPIP2 in the Development of Autoinflammatory Disease
Authors: Pavliuchenko Nataliia , Kralova Jarmila , Fabisik Matej , Brdicka Tomas
Published: February 2023

PSTPIP2 is an adaptor protein, which binds multiple regulators of neutrophil signaling to suppress inflammation. The compete loss of PSTPIP2 protein in the mouse strain Pstpip2cmo results in the development of autoinflammatory disease chronic multifocal osteomyelitis (CMO), a condition resembling human CRMO. The disease is driven by an overproduction of IL-1β and deregulated activity of NADPH oxidase. The main goal of this study is to analyze the effects of PSTPIP2 interactions on leukocyte signaling pathways, IL-1β processing, ROS production by NADPH oxidase, and auto inflammation in vivo.

To achieve this, we have generated mice with mutations in major interaction sites in PSTPIP2 including W232 responsible for binding to PEST family phosphatases (PEST-PTP), and the C-terminal sequence containing major tyrosine phosphorylation sites known to bind inositide phosphatase SHIP1 (Pstpip2ΔC-t). W232A mutation resulted in enhanced ROS production by neutrophils and increased IL-1β cleavage. Moreover, some W232A mutants developed CMO symptoms. However, the kinetics was delayed and severity was milder, when compared to Pstpip2cmo strain. In contrast, Pstpip2ΔC-t mutants did not show any CMO symptoms or increased IL-1β cleavage. Interestingly, partially deregulated ROS production could still be measured in Pstpip2ΔC-t neutrophils. Collectively, our data demonstrate an important function of PEST-PTP binding to PSTPIP2 in the suppression of auto inflammation, while SHIP1 binding contributes to the regulation of neutrophil oxidative burst. The obtained data give us a novel perspective on PSTPIP2 interactome and extend our knowledge of the molecular interactions regulating physiological and pathological inflammation.
Mitochondrial respiration contributes to the interferon gamma response in antigen presenting cells
Authors: Olive Andrew J , Kiritsy Michael , Sassetti Christopher
Published: February 2023

To effectively control infections, antigen presenting cells act as an integration point where signaling and metabolic pathways collaboratively determine cellular activation state and shape T cell responses. The cytokine Interferon γ (IFNγ) is key to modulating multiple macrophage functions that allow the host to respond effectively to infection. How a single cytokine like IFNγ regulates parallel immune mechanisms remains unclear, limiting our ability to target IFNγ-dependent mechanisms therapeutically. We conducted a series of forward genetic screens dissecting the networks that control IFNγ-mediated expression of the key immune molecules MHCII, CD40 and PD-L1. Our multi-screen approach enabled the identification of novel pathways that control these functionally distinct markers and uncovered a new pathway of immunometabolism that requires the electron transport chain Complex I to activate IFNγ responses. Mechanistic studies demonstrate that in both mouse and human cells the loss of Complex I results in blunted IFNγ-responses that were characterized by the reduced activation of the IFNγ receptor complex and Stat1. We developed a new model of mitochondrial dysfunction using long-term treatment with Linezolid, an antibiotic used to treat TB that blocks the mitochondrial ribosome, which recapitulated defects in the IFNγ response. Furthermore, perturbation of Complex I inhibits the capacity of both macrophages and dendritic cells to stimulate T cells. Thus these studies identified a new role for mitochondrial activity as a central point where local signals are integrated to determine antigen presentation cell function.
Somatosensory regulation of macrophage function during inflammatory pain
Authors: Hakim Sara N. , Woolf Clifford J.
Published: February 2023

Pain is a protective mechanism that alerts the organism against dangerous stimuli, much like the innate immune system. These stimuli range from noxious chemical, mechanical, and thermal stimuli to regional inflammation caused by wounds or infections. During an inflammatory pain condition, such as post-operative skin incision, the inflammatory milieu communicates directly with sensory neurons via signaling molecules to either promote or resolve the sensation of pain. The sensory neurons that initiate pain, nociceptors, release neuropeptides in the tissue they innervate which can act on neighboring immune cells and regulate their function. Here, I investigated how neuropeptides released by nociceptors during inflammatory pain regulate macrophage function. In LPS-activated macrophages, I found that the neuropeptides CGRP and PACAP but not Substance P and Galanin reduced secretion of IL-6 and TNF-α suggesting a neuropeptide-specific effect. Notably, CGRP promoted a reparative macrophage signature, by enhancing the release of IL-10 and the transcription of reparative genes such as Arginase I. When macrophages are only transiently exposed to LPS, they acquire a reparative phenotype. Interestingly, CGRP promoted Arginase I induction even further in these reparative macrophages, suggesting that CGRP could act on reparative as well as inflammatory macrophages to promote an anti-inflammatory milieu. These findings suggest that, during an inflammatory condition, when sensory neurons relay the sensation of pain, they also regulate macrophage function locally by release of CGRP and PACAP. By promoting an anti-inflammatory milieu, CGRP release may accelerate both the healing of tissue injury as well as the resolution of pain.
Neutrophil Extracellular Traps promote joint damage in hemophilia.
Authors: Kaminski Tomasz W. , Brzoska Tomasz , Tutuncouglu Egemen , Ragni Margaret V. , Sundd Prithu
Published: February 2023

Hemophilic arthropathy (HA) is the pathophysiology resulting from recurrent joint bleeding in hemophilia. HA leads to joint damage, chronic pain and poor quality of life. Despite advances in the treatment to prevent joint bleeding, HA continues to be a major morbidity affecting hemophilia patients and the mechanism contributing to HA remains elusive. Recent evidence suggests that joint-bleeding may promote the release of erythrocyte and tissue-derived damage-associated-molecular-pattern molecules (DAMPs) that can trigger the sterile inflammation in the joints, however, the innate immune pathways remain unclear. We used a model of knee joint injury-induced HA in FVIII-total knockout (F8TKO) mice and samples from hemophilia patients diagnosed with HA. In vivo multiphoton-excitation fluorescence imaging of injured synovial cavity in live F8TKO or control mice was used to assess NETs formation within joint capsule. Imaging-flow-cytometry and ELISA assays were used to evaluate circulating NETs in HA patients and mice with knee-injury. Bleeding severity scoring, histology, IHC and confocal imaging of joints were conducted to assess the joint injury in mice. F8TKO but not control mice manifested knee-joint injury and severity of bleeding 5-days post knee-injury. Knee-joint injury was associated with increased neutrophil accumulation and NETs shedding within the synovium of F8TKO mice. Circulating NETs were abundant in the plasma of patients diagnosed with HA and F8TKO following injury but not plasma of controls. These findings are the first to suggest that NETs contribute to pathogenesis of HA. Currently, study is underway to identify the innate immune pathways that promote NETs shedding, leading to joint-damage in hemophilia.
In vitro co-culture approach reveals multi-modal control of dendritic cell functions by nociceptors
Authors: Hanc Pavel , von Andrian Ulrich H
Published: February 2023

Nociceptors – sensory neurons that respond to noxious stimuli by eliciting the sensation of pain or itch – are increasingly beginning to be appreciated as controllers of the immune response. Notably, dendritic cells (DCs) – myeloid cells of the immune system – were previously found to be impacted by nociceptors, however, while some functional outcomes of the interaction have been described, our mechanistic understanding of the underlying molecular communication network is rudimentary at best and a bigger picture pertaining the modalities and outcomes of the interaction is missing entirely.

Consequently, here, we have established a novel in vitro co-culture system for DCs and nociceptors, which allowed us to explore in detail the modalities and outcomes of their communication. We show that DCs and nociceptors engage in a tight physical interaction and that nociceptors have the ability to attract DCs by production of the chemokine CCL2. Additionally, nociceptors enhance pro-inflammatory cytokine production by activated DCs in a contact-dependent, neuropeptide-independent manner, and nociceptive activity induces membrane depolarization and calcium flux in the interacting DCs. Next, using RNA sequencing, we identified genes that are controlled by nociceptors in steady-state DCs in the absence of immune stimulation, and uncovered a nociceptor-induced program of enhanced sentinel function, characterized by an increased expression of pro-IL1b but no overt immune activation of DCs both in vitro and in vivo. Finally, we identified the CGRP family of neuropeptides as nociceptor-derived signals mediating this effect.
Using CRISPR/cas9 in primary natural killer cells to study antibody dependent cellular cytotoxicity signaling.
Authors: Dahlvang James D. , Sangala Jules A. , Hart Geoffrey T.
Published: February 2023

In contrast to conventional natural killer (NK) cells, adaptive NK cells lack expression of the Fc receptor γ chain and transcription factor PLZF. A higher proportion of adaptive NK cells is correlated with protection from malaria. Adaptive NK cells have shown enhanced degranulation in response to opsonized targets, as measured by CD107A and interferon-γ (IFN-γ) expression. We hypothesize that people with a higher frequency of adaptive NK cells are protected from malaria because of the enhanced degranulation of adaptive NK cells. However, it is unclear why adaptive NK cells degranulate better than conventional NK cells. To answer this question, we developed a CRISPR/cas9 protocol to ablate genes associated with the antibody-dependent cellular cytotoxicity (ADCC) signaling pathway and evaluated the effect of these ablations on NK cell degranulation. Individual gene targets included the Fc receptor γ chain, CD3ζ, Syk, ZAP70, and PLZF. Combinations of these targets were also ablated. After ablating gene(s) of interest, mutant NK cells were incubated with an opsonized target and evaluated five hours later for CD107A and IFN-γ expression. As a control, we found that the combined ablation of the Fc receptor γ and CD3ζ chain severely diminishes NK cell degranulation. Our results from other gene targets will be shown. Furthermore, details of our CRISPR/cas9 method will be shared. The method is the first to effectively ablate multiple primary NK cell targets using the Amaxa 4D nucleofector. On average, we obtained 82.8% protein ablation across all targets. Overall, this work will give insight into why adaptive NK cells function better at ADCC and help the field effectively ablate genes in primary NK cells.
HMGB1 dimerization could protect nuclear DNA damage and cell death from oxidative stress
Authors: Kwak Man Sup , Rhee Woo Joong , Lee Yong Joon , Kim Hee Sue , Kim Young Hun , Kwon Min Kyung , Shin Jeon-Soo
Published: February 2023

Oxidative stress can induce covalent disulfide bond formation between protein-protein thiol groups and generate hydroxyl free radicals that damage DNA. HMGB1 is a DNA chaperone and damage-associated molecular pattern molecule. As a redox-sensitive protein, HMGB1 contains three cysteine residues: Cys23, Cys45, and Cys106. In this study, we focused on the relationship between HMGB1 dimerization and DNA stabilization under oxidative stress conditions. HMGB1 dimerization was positively modulated by CuCl2 and H2O2. Mutation of the Cys106 residue blocked dimer formation. Treatment of HEK293T cells with CuCl2 and H2O2 enhanced the oxidative self-dimerization of HMGB1, whereas this dimerization was inhibited in mutant HMGB1C106A cells. Furthermore, we performed a bimolecular fluorescence complementation assay to visualize Cys106 oxidation-induced HMGB1 dimerization in live cells exposed to oxidative stress and were able to reproduce the dimerization effect of HMGB1 in fluorescence resonance energy transfer analysis. Interestingly, dimerized HMGB1 bound to DNA with higher affinity than monomeric HMGB1. Dimerized HMGB1 protected DNA from damage due to hydroxyl free radicals and prevented cell death. In conclusion, dimerized HMGB1 may play a regulatory role in DNA stabilization under oxidative stress.