bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of monoclonal antibodies against murine PD-1 and PD-L1
Authors: Bu Melissa Tian , Yuan Long , Klee Alyssa , Freeman Gordon J
Published: January 2023

Blockade of the PD-L1/PD-1 pathway has proven to be a broadly effective cancer immunotherapy. FDA-approved therapeutic monoclonal antibodies (mAb) targeting the pathway have high affinity, blocking capacity, and low antibody effector activity. A number of rat anti-mouse mAbs have been used to model cancer immunotherapy in mouse models. We set forth the amino acid sequences of mAbs specific for mouse PD-1 (29F.1A12) and PD-L1 (10F.9G2) and compare their avidities, blocking capacities, biological activities, and epitope recognition with other commonly used mAbs. Of note, we found that 29F.1A12 had an approximately 100-fold better affinity and blocking capacity than RMP1-14. Further manipulation of these sequences should facilitate better modeling of immunotherapy in mouse models and the generation of novel agents.

Supported by grants from the National Institutes of Health (P01 AI56299 and P01 CA236749).
MTAP deficiency reshapes the tumor immune landscape in lung cancer
Authors: Li Ji-Min , Zhang Jun , Chang Wen-Hsin , Hsu Ssu-Wei , Chen Ching-Hsien
Published: January 2023

Methylthioadenosine phosphorylase (MTAP) deficiency occurs in a broad range of malignancies; notably, loss of this metabolizing enzyme was reported to be associated with poor survival in cancer patients. However, the mechanisms underlying tumor progression due to MTAP loss are yet to be elucidated. Utilizing RNA-sequencing, reverse phase protein arrays, and Luminex assays, we demonstrated that MTAP deficiency alters tumor-intrinsic, immune-related pathways and reprograms cytokines toward a tumor-favorable environment in both lung and kidney cancer. In addition, MTAP-knockout cells exhibited a marked increase in the immune checkpoint protein PD-L1. Upon co-culturing primary T cells with cancer cells, we found that MTAP loss-mediated PD-L1 upregulation inhibited T cell-mediated killing activity and induced several T cell exhaustion markers. In two xenograft tumor models, we showed that the average volume of tumors derived from MTAP-deficient cells is slightly greater than that of MTAP-proficient tumors. Surprisingly, a remarkable increase in tumor size was also observed in humanized mice bearing MTAP-deficient tumors, as compared to their MTAP-expressing counterparts. Following immunophenotypic characterization of tumor-infiltrating leukocytes by mass cytometry analysis, compared to MTAP-expressing tumor xenografts, MTAP-deficient tumors was found to display decreased immune infiltrates that consist of abundant immunosuppressive cells and lower portions of both T lymphocytes and natural killer cells. Taken together, our results suggest that MTAP deficiency reconstructs the tumor immune microenvironment via cytokine reprogramming and immune remodeling, promoting tumor progression and immune evasion.

This work was supported by grants funded by the Department of Defense Kidney Cancer Research Program (KCRP) W81XWH1910831 (log# KC180170) and the California UCOP Tobacco-Related Disease Research Program (TRDRP) T29IR0704.
Hematopoietic protein RELT is upregulated in human breast and lung cancers and binds the cytoskeletal protein Filamin A.
Authors: Cusick John K , Yanumula Anusri , Shyu Yennie , Wang Wenjia , Abram Alyssa , Alcaide Jessa , Zhou James , Gillespie Brittany , Senderovich Michelle , Budhiraja Anuj , Qureshi Vaniya , Bilasy Shymaa , Shi Yihui
Published: January 2023

Receptor Expressed in Lymphoid Tissues (RELT), is a human TNFR superfamily member (TNFRSF19L) expressed most prominently in the hematopoietic system. Previous reports using RELT knockout mice indicate that RELT functions in part by inhibiting the proliferation and activation of T lymphocytes. RELT is an orphan receptor that has two homologous binding partners, RELL1 and RELL2, and collectively, these three proteins are referred to as RELT family members. This study sought to identify novel protein interactions with RELT family members and to compare the expression of RELT in normal and diseased human tissues. A yeast two-hybrid screen utilizing RELL1 as bait identified the cytoskeletal protein Filamin A (FLNA) as a prospective binding partner and physical interaction between FLNA and RELT family members was confirmed by in vitro co-immunoprecipitation. A truncated mutant of FLNA disrupts the ability of RELT family members to activate the p38 pathway and blocks the ability of RELT to induce death in human epithelial cells. Western blotting revealed endogenous RELT family members are most significantly upregulated in the breast cancer cell lines MDA-MB-231 and MCF-7, as well as the lung cancer cell line H358. Preliminary immunohistochemistry results reveal a higher intensity of RELT staining in both breast cancer and lung cancer human biopsies versus non-malignant tissue. Additionally, overexpression of RELT family members enhanced apoptosis in MDA-MB-231 and H358 cells as measured by X-gal morphology and luciferase assays. Collectively, these results further our understanding of signal transduction pathways associated with RELT family members and report the novel finding that RELT is upregulated in human breast and lung cancers.

Supported by California Northstate University, College of Medicine, Seed Grant.
An Emerging and Previously Unrecognized Paradigm of Cxcl12/Cxcr4 Pathway Implication in Breast Cancer Metastasis
Authors: Lagou Maria K , Sanchez Luis R , Duran Camille L , Burt Joseph , Chen Xiaoming , Lin Yu , Eddy Robert , Harney Allison S , Entenberg David , Condeelis John S , Oktay Maja H , Karagiannis George S
Published: January 2023

The Cxcl12/Cxcr4 signaling axis promotes metastasis in mouse models of breast carcinoma and is linked with increased metastatic risk in humans. Prior studies have linked Cxcl12/Cxcr4 to breast cancer cell homing and survival at metastatic sites. However, the precise mechanism via which Cxcr4+ breast cancer cells escape primary tumors, which also express Cxcl12, is poorly understood. By using a novel imaging method for visualizing chemokine gradients, we show that Cxcl12 is concentrically expressed around cancer cell intravasation doorways, known as Tumor Microenvironment of Metastasis (TMEM). Using distance analysis algorithms, we also show that TMEM-mediated Cxcl12 gradients contextually associate with Cxcr4+ breast cancer cells migrating towards TMEM doorways. As such, pharmacological inhibition of the Cxcl12/Cxcr4 axis significantly abrogates translocation of Cxcr4+ cancer cells to TMEM doorways, thus suppressing metastatic dissemination. However, the targeted elimination of Cxcr4 from breast cancer cells paradoxically results in a suboptimal response compared to pharmacologic inhibition, implying the existence of a compensatory mechanism. Using clodronate-mediated macrophage depletion, we demonstrate that, in the absence of Cxcr4 expression in tumor cells, accompanying Cxcr4+ macrophages may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, Pdgfrb+ stromal cells may express low levels of Cxcl12, but TMEM-generated Cxcl12 gradients are primarily linked to a subset of Iba1+ perivascular tumor-associated macrophages. Overall, our data reveal a new paradigm for the implication of Cxcl12/Cxcr4 in early stages of metastasis and propose an avenue for rationalized antimetastatic treatments.

Supported by Recruitment Funds RECR 3A3218
Multi-modal immune profiling of mucinous ovarian carcinoma: analysis from the Ovarian Tumor Tissue Analysis/Multidisciplinary Ovarian Cancer Outcomes Group consortia
Authors:   Ovarian Tumor Tissue Analysis Consortium, Meagher Nicola S , Hamilton Phineas T , Anglesio Michael S , Harris Holly R , Konecny Gottfried E , Schildkraut Joellen M , Talhouk Aline ,   Australian Pancreatic Genome Initiative, Friedlander Michael L , Pearce Celeste L , Pike Malcolm C , Köbel Martin , Doherty Jennifer A , Goode Ellen L , Nelson Brad H , DeFazio Anna , Ramus Susan J
Published: January 2023

Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer. Women with advanced disease (FIGO stage III/IV) have very poor outcomes, with 5-year survival of <20%. Little is known about the anti-tumor immune response in MOC, limiting potential immunotherapeutic options for patients.

Immune data from 3 platforms were analysed. A NanoString mRNA expression panel with 39 immune-related genes (n=60 MOC, n=4198 other ovarian histotypes), a NanoString mRNA plexset with PD-1, PD-L1 (n=241 MOC, n=115 upper and lower gastrointestinal (GI) cancers), and 2 multicolor immunofluorescence (mcIF) panels (n=121 MOC) for CD68, PD-L1, PD-1, CD8 and FOXP3. Data overlapped all platforms for 22 MOC. Immune marker expression by ovarian cancer histotype was measured. Uni- and multivariable Cox Proportional Hazards assessed overall survival (OS) within MOC. Hierarchical clustering looked for subgroups of patients based on co-expression of immune genes.

Compared to more common high-grade serous ovarian cancer (HGSC) and GI cancers, MOC were less immunologically active. High IGHM expression was associated with poorer OS (HR 1.31 (95% CI 1.02–1.69), p=0.033). High tumor densities of FOXP3 and PD-1 positive cells conferred poorer OS in a model with age, stage and site; however, these and PD-L1 and CD68 were associated with grade. The 39 gene NanoString and mcIF datasets revealed 4 clusters, roughly translating to immune cold, moderate and hot (groups 3,4). mcIF analysis revealed a subset with PD-L1 expression in the absence of T-regulatory cell infiltrates.

This study provides novel insights into the MOC immune landscape. Despite relatively low immune activity overall, immunotherapy could be explored for a subset of patients with MOC.

Supported by NSW Ministry of Health and UNSW Sydney, through the NSW Health PhD Scholarship Program 2017-2022; Translational Cancer Research Network top-up scholarship 2021
Upregulated Ly-6 gene expression is associated with poor overall survival in uterine corpus endometrial carcinoma patients
Authors: Rathbun Luke , Magliocco Anthony , Bamezai Anil K
Published: January 2023

The human Ly-6 (hLy-6) gene family has recently gained interest for its possible role in tumor progression. High expression of hLy-6 genes is associated with poor overall survival in pancreatic ductal adenocarcinoma patients. To expand upon this work, we have carried out in silico analyses of all known hLy-6 gene expression in other cancers using TNMplot which shows differential gene expression in cancer tissues. We have also analyzed the patient survival by Kaplan-Meier plotter after mining the TCGA database. We report that upregulated expression of several hLy-6 genes is associated with poor cancer patient survival in many other cancer types, especially uterine corpus endometrial carcinoma (UCEC). Importantly, the expression of a number of hLy-6 genes is elevated in UCEC tissue when compared to the expression in normal uterine tissue. Further analysis of tumor-specific expression of the hLy-6 gene family is needed to uncover the function of Ly-6 proteins as well as the signaling pathways these proteins trigger that endow tumor survival and poor patient prognosis. Human Ly-6 gene products can potentially be used as biomarkers for cancer detection and as tumor associated antigens to identify high-risk cancer patients and appropriately design and/or calibrate cancer treatments for such patients.
Analysis of chemokine network in primary human glioblastoma
Authors: Lachota Mieszko , Zielniok Katarzyna , Gozdz Agata , Szpak Patrycja , Kalaszczynska Ilona , Zagozdzon Radoslaw
Published: January 2023

Chemokines play an integral role in cancer immunoevasion by modulating leukocyte infiltration. They selectively increase the migration of immunosuppressive cells while impairing the infiltration of cytotoxic lymphocytes with potent antitumor activity. Some cancer-associated chemokines may also enhance tumor progression by acting as growth factors or promoting epithelial-mesenchymal transition.

Here, we acquired fresh human glioblastoma (GBM) tissues to define chemokine expression patterns in GBM tumors by integrating bulk and single-cell RNA sequencing of both tumor and healthy brain tissues along with Luminex secretome profiling of GBM primary cell cultures. Extensive mapping of GBM-associated chemokines has enabled us to gain insight into intratumoral chemokine-mediated cell-to-cell communication. Among others, we identified CCL7 and midkine (MDK) to play a crucial role in the formation of the hostile tumor microenvironment (TME) in GBM. We hypothesize that these chemoattractants can facilitate the GBM tumor growth by recruiting M2 macrophages, promoting epithelial-mesenchymal transition, and activating the prosurvival PI3K-Akt pathway. Indeed, high expression of both chemokines was linked to poor overall survival.

Additionally, by integrating the data, we further identified the cellular source of the most abundantly secreted chemokines in GBM tumors. Since immune cells require TME infiltration to mediate an appropriate antitumor response, impaired tumor infiltration is one of the critical limiting factors for cancer cell therapy. Our chemokine atlas provides novel targets to enhance adoptive cell therapies by improving the potential of effector cells for tumor homing and anticancer activities in GBM.

This research was funded by the National Science Centre, Poland (2020/37/B/NZ6/02191), the Polish Ministry of Science and Higher Education (grant number DI2018 020548), and the National Centre for Research and Development (STRATEGMED3/307326/6/NCBR/2017).
Leucine-rich repeats and immunoglobulin-like domains protein 1 is highly expressed on tumor-infiltrating T cells and may play a role in modulating T cell activation
Authors: Ta Hieu M , Roy Dia , Zhang Keman , Dong Juan , Gilmour Cassandra , Yoo Seong-Keun , Alban Tyler , Makarov Vladimir , Parthasarathy Prerana Bangalore , Chan Timothy , Wang Li
Published: January 2023

Immune checkpoint receptors (ICRs) regulate T cell-mediated immune responses to self - and neo-antigens, thereby play an important role in the development of autoimmunity and immune responses to infections. In cancer, ICRs such as PD-1 and CTLA4 impair immuno-surveillance against neoplastic cells, while other costimulatory molecules like ICOS, OX40 could augment the immune responses. Immunotherapy that targets either inhibitory or costimulatory immune checkpoint have become the breakthrough therapy for cancer treatment. However, the efficacy of this class of therapy is limited to a fraction of patients, pointing the need to overcome non-redundant immuno-inhibitory pathways or discover new immuno-costimulatory pathways. This study identified the expression of Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) in activated CD4+ and CD8+ T cells. LRIG1 is a type-I transmembrane protein with 1092 amino acids (120 kD) and ubiquitous tissue expression patterns. We show that LRIG1 expression is not detectable on naïve CD4+ and CD8+ murine T cells but is induced upon T cell receptor (TCR) stimulation in vitro, and in tumor infiltrating lymphocytes (TILs) from multiple murine models. We also detected LRIG1 expression on CD4+ and CD8+ TILs in several types of cancer patients such as non-small-cell lung cancer, breast cancer, melanoma, B cell lymphoma. LRIG1 was originally identified as a tumor suppressor gene in a variety of tissues, but its expression and function have not been explored in immune cells. Our data suggest that LRIG1 is highly expressed in TILs in multiple cancer types and might play a role in regulating anti-tumor immune response

This project is also supported by fundings 2R01CA164225 (NCI), 1R01CA223804 (NCI), and American Cancer Society (ACS) RSG-18-045-01
CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells
Authors: Byrne Katelyn T , Kim Samuel I , Arora Charu , Verginadis Ioannis I , Cassella Christopher R , Markosyan Nune , Koumenis Constantinos , Vonderheide Robert H
Published: January 2023

Cytolytic CD8+ T cells are a major mediator of immune-induced tumor rejection, but many patients have primary or acquired resistance to CD8+ T cell immunity. Reduced major histocompatibility complex I (MHC I) expression contributes to resistance, including treatment-refractory pancreatic ductal adenocarcinoma (PDAC). Interrogating interventions in PDAC will reveal novel mechanisms regulating sensitivity to immunotherapy. Using a genetically engineered mouse model of PDAC, we previously reported that agonistic anti-CD40 and dual immune checkpoint blockade (ICB) induces CD8+ and a CD4+ T cell responses mediating tumor rejection. Here, we utilized CRISPR− Cas9 to disrupt the dominant MHC I (H-2Kb) allele expression in PDAC cell lines, precluding direct antigen presentation to CD8+ T cells. Injection of MHC I-deficient tumor clones revealed normal tumor growth at baseline, and significantly delayed tumor growth in response to CD40/ICB in a CD4+ T cell-dependent manner (p < 0.0001 vs. vehicle-treated mice). There was no direct contribution of tumor rejection by CD8+ T cells, despite the generation of a tumor-specific response (24.5% vs. 1.98% in vehicle-treated mice, p<0.003). CD4+ T cells upregulated IFN-γ production (52.3% vs. 11.9% in vehicle-treated mice, p<0.0001), and host IFN-γ expression was required. No changes were observed in the expression of cytotoxic molecules by CD4+ T cells after CD40/ICB, and depletion of myeloid cells did not dampen treatment efficacy. Thus, CD4+ T cells mediated PDAC rejection via non-canonical mechanisms after treatment with CD40/ICB. This study provides critical insight into novel mediators of tumor clearance, and opportunities to exploit CD4 T+ cells as regulators of tumor immunity.

Funded by the Parker Institute for Cancer Immunotherapy.
Mycolic acid nanoparticle vaccination leads to antigen persistence and unique differentiation of mycobacterial lipid antigen-specific T cells
Authors: Morgun Eva , Zhu Jennifer , Bobbala Sharan , Aguilar Melissa Salinas , Wang Junzhong , Conner Kathleen , Cao Liang , Seshadri Chetan , Scott Evan A , Wang Chyung-Ru
Published: January 2023

Tuberculosis remains a serious global epidemic and with the rise of multi-drug resistant strains, an efficacious vaccine solution is imperative. Vaccines currently being developed for Mtb utilize protein antigens, which target MHC-restricted conventional T cells, overlooking the potential of Mycobacterium tuberculosis (Mtb) lipid antigens such as mycolic acid (MA), a key lipid found in Mtb cell wall. Mycobacterial lipids are presented by group 1 CD1 molecules (CD1a, b, c) to cognate T cells. Group 1 CD1-restricted T cells can be identified in patients with TB and have been shown to provide protection in Mtb infection. Using biocontinuous nanospheres (BCNs), a type of self-assembled nanostructure able to load both hydrophobic and hydrophilic molecules, we have created a vaccine containing MA. We found that MA BCN is able to effectively activate CD1b-restricted MA-specific T cells in vitro and in vivo. Interestingly, we discovered that MA persists within lung alveolar macrophages for at least 6 weeks after intratracheal vaccination with MA BCN. Antigen archiving was in part due to the encapsulation of MA within BCN. Nanoparticle vaccinations carrying lipid antigens may thus lead to persistent depots of antigen that could offer long-lasting immune response and protection.

Supported by grants from NIH (5R01AI145345-03, 1F30AI157314-01, 5T32GM008152-35)
Duration of peripheral blood cellular immune responses induced in dogs in response to a modified live attenuated vaccine for Ehrlichia chaffeensis
Authors: McGill Jodi L , Madesh Swetha , Hove Paidashe , Ganta Roman R
Published: January 2023

Ehrlichia chaffeensis (ECH) is an obligate intracellular gram-negative bacterium and a frequent cause of severe and fatal tick-borne infection in people in North America. The canine is a common incidental host and a useful model for vaccine development and assessing immune responses to ECH infection. We recently established mutagenesis methods for ECH and demonstrated that immunization of dogs with an ECH mutant with a functional disruption in the ECH_0660 gene protected the vertebrate host from both tick-transmitted and intravenous wildtype ECH challenge when given 4 weeks after immunization. In the current study, we evaluated the duration of immunity offered by the ECH_0660 gene mutant live attenuated vaccine (MLAV) against wildtype infection challenge. Groups of 12 dogs were immunized with the ECH MLAV and then challenged 2, 4, 8 and 12 months later via tick-transmitted or blood-transmitted infection with wildtype ECH. Animals were monitored for 4 weeks after challenge and then sacrificed for evaluation of tissue pathology and bacterial burden. Immunization with the MLAV induced ECH-specific T cell responses, detectable by ELISPOT for IFNγ; and differentiation of IFNγ- and/or TNFα-producing ECH-specific T effector memory CD4 T cells (CD45RAneg CD45RO+ CD62Lneg). Antigen-specific recall responses were detectable in the peripheral blood of most (27/32) vaccinated dogs for 4 months after immunization, but only in 5/24 of the dogs after 8 months. This result suggests that a single MLAV vaccination induces cellular immune responses and differentiation of memory helper T cells. Future work will investigate the correlations between cellular immunity, humoral responses and bacterial load.

Supported by grants from NIH (R01 AI152418)
Resident CD8+ T-cells drive dysplastic repair of aged lungs following viral pneumonia
Authors: Narasimhan Harish , Goplen Nicholas P , Cheon In Su , Sun Jie
Published: January 2023

Global studies have established age as the most critical risk factor in determining morbidity and mortality following respiratory viral infections such as influenza and SARS-CoV2. Previous studies from our group identified non-resolving inflammation in aged lungs post influenza infection, primarily driven by the persistence of tissue resident memory (Trm) CD8 T-cells. We also observed sustained impaired lung function in aged COVID19 convalescents, which negatively correlated with levels of pulmonary CD8 Trm cells. While critical for acute antiviral responses, we have demonstrated that this population is pathologic rather than protective following viral clearance during aging, evidenced by improved outcomes upon its depletion.

Upon severe lung damage during viral infection, epithelial progenitor cells characterized by cytokeratin 5 (Krt5) expression are mobilized for repair. However, these cells are known to be incapable of differentiating into functional ATI and ATII cells, instead persisting as dysplastic epithelial scars, a process known to be exacerbated with age. Thus, in addition to mortality during acute infection, aged individuals also experience adverse long-term outcomes due to dysfunctional repair of the lung.

We identified that Krt5 cysts typically associate with CD8 Trm cells, suggesting potential immune-epithelial crosstalk. Furthermore, upon depletion of pulmonary CD8 Trm cells, we observed a reduction in Krt5 cysts, increased ATI and ATII levels, and improved lung function. Hence, we hypothesize that the interactions between CD8 Trm cells and Krt5 cysts are responsible for sub-optimal repair and aim to manipulate this crosstalk to promote functional repair of the aged lung following viral pneumonia.

Supported by grants from NIH - AI147394, AG047156, and AG069264 and Mayo Clinic CBD Research Fund
Restriction of viral replication, rather than T cell immunopathology, drives lethality in MNV CR6-infected STAT1-deficient mice
Authors: Sharon Andrew J , Filyk Heather A , Fonseca Nicolette M , Simister Rachel L , Filler Renata B , Yuen Wallace , Hardman Blair K , Robinson Hannah G , Seo Jung Hee , Rocha-Pereira Joana , Welch Ian , Neyts Johan , Wilen Craig B , Crowe Sean A , Osborne Lisa C
Published: January 2023

Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. Here, we used colonization of C57BL6/J mice with the model commensal murine norovirus (MNV CR6) to interrogate host-directed mechanisms of viral regulation, and show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In STAT1-sufficient mice MNV CR6 is asymptomatic and restricted to the colon, despite the persistence of high viral loads. However, in the absence of STAT1 viral replication is disseminated and observed in key innate and adaptive immune populations.

STAT1-deficient mice develop dysregulated inflammatory CD8+ and CD4+ T cell responses, which coincide with weight loss and severe necrosis of the spleen and liver. Despite these altered T cell responses which resemble those that mediate lethal immunopathology following other model viral infections of STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. In contrast, therapeutic administration of an antiviral compound limited viral replication, preventing viral-induced tissue damage and death despite ongoing inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa. Unlike other model viral infections, CR6 induces disease in STAT1-deficient mice via uncontrolled viral replication rather than the concomitant development of dysregulated antiviral T cell responses.

Research in the Osborne lab is supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Canada Research Chair program and scholarships from the University of British Columbia.
Tetramerization of STAT5 promotes autoimmune-mediated neuroinflammation
Authors: Monaghan Kelly Lynn , Aesoph Drake , Ammer Amanda G , Zheng Wen , Rahimpour Shokofeh , Farris Breanne Y , Spinner Camille A , Li Peng , Lin Jian-Xin , Yu Zu-Xi , Lazarevic Vanja , Hu Gangqing , Leonard Warren J , Wan Edwin C K
Published: January 2023

Signal transducers and activators of transcription 5 A and B (STAT5A & B) play a critical role in mediating cellular responses following cytokine stimulation. STAT5 proteins critically signal via the formation of dimers through SH2 domain interactions. However, tetramerization of STAT5 through dimer interactions in the N-domain, has key biological roles. STAT5 tetramers control the suppressive function of regulatory T cells (Tregs), the expansion of CD8+ T cells, and the terminal differentiation of NK cells. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knock-in (DKI) mouse strain, in which dimer function is preserved, we found that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. The mild EAE phenotype observed in DKI mice correlates with impaired extravasation of pathogenic Th17 cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrated that STAT5 tetramerization mediated by GM-CSF signaling, a key EAE-associated cytokine, regulates the production of the chemokine CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a novel GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune-mediated neuroinflammation.

This work was supported by NIH grant P20 GM109098 and WVU Institutional Funds to E.C.K.W.; Praespero Innovation Award Program to E.C.K.W. and V.L.; NIH grants P20 GM103434 and U54 GM104942 to G.H.; the Intramural Research Program of the National Heart, Lung, and Blood Institute, NIH to W.J.L.; the Intramural Research Program of the National Cancer Institute, NIH to V.L. NIH grants S10 OD016165, U57 GM104942, P30 GM103488, and P20 GM103434 to the WVU Flow Cytometry & Single Cell Core Facility. NIH grants U54 GM104942, P20 GM103434 and P20 GM121299 to the WVU and Marshall University Genomics Cores. Funds from WVU Cancer Institute, WVU HSC Office of Research and Graduate Education, and NIH grants P20 RR016440, P30 GM103488, P20 GM121322, U54 GM104942, and P20 GM103434 to the WVU Microscope Imaging Facility. K.L.M. was supported through The American Association of Immunologists Careers in Immunology Fellowship Program.
Investigating immune responses to Harmful Algal Bloom cyanotoxins in protection of human health and the environment
Authors: Augustine Swinburne A J , Robillard Michelle Catherine , De la Cruz Armah
Published: January 2023

Harmful algal blooms (HABs) are a major environmental and public health concern globally that impact multiple essential areas including agriculture, drinking water, and recreational water. Although harmful blooms are caused by other organisms, those from cyanobacteria are the most common freshwater pollutant. Cyanobacteria produce secondary metabolites including cyanotoxins that pollute drinking and recreational waters and produce metabolites and cell components that can contribute to skin irritation. The aim of this study is to elucidate the specific mechanisms that lead to inflammation and irritation caused by exposure to these toxins. Multiplexing technology afforded the ability to investigate a variety of human cytokines and chemokines, and soluble receptors to measure immune responses in a single sample. 3D human tissue cultures were exposed to a variety of different toxins found in HABs. These samples were then examined using Luminex™ xMAP technology to look at 38 different cytokines/chemokines and 14 soluble receptors in each sample. We observed the expression of several cytokines/chemokines that regulate the inflammatory process in endothelial and epithelial cells and play a role in the recruitment of neutrophils to the site of inflammation. This could contribute to the rashes or skin irritation commonly reported by patients exposed to HABs. Additional observations and analyses are needed to identify the specific immune response pathways activated during exposure to these toxins. These findings could lead to a better understand of human exposure to cyanotoxins and more appropriate and effective treatment modalities for patients who exhibit rashes or skin irritations after being exposed to these blooms.

This work is supported by the United States Environmental Protection Agency.
Early life adversity programs a hyperactive mast cell phenotype and induces long-lasting changes in the mast cell transcriptome
Authors: LeMon Janelle , Maradiaga Nidia , Moeser Adam James
Published: January 2023

Early life adversity (ELA) induces long-lasting chronic low-grade immune activation and increases the risk for inflammatory and metabolic disorders later in life. Our previous studies demonstrated that mast cells (MCs) which are well-established orchestrators of immune responses and as effector cells of the stress response exhibit heightened reactivity to later life stressors. Here, we used a murine model of ELA (neonatal maternal separation + early weaning; NMSEW) to determine the long-lasting programming effects of ELA on MC phenotype and function. Bone marrow-derived mast cells (BMMCs) from adult female NMSEW mice exhibited greater IgE-mediated histamine release and LPS- and IL33-induced cytokines release, compared with normally handled (NH) control mice. RNA sequencing of BMMCs from adult NH and NMSEW female mice, followed by gene pathway analysis using Ingenuity Pathway Analysis, revealed significant alterations in gene expression associated with cell migration and trafficking, glycolysis and TCA cycle regulation, lipid and cholesterol synthesis, and toll like receptor signaling. Ongoing experiments aim to characterize the long-lasting metabolic alterations induced by NMSEW through functional assays and protein expression analysis. Understanding how ELA programs MCs could identify the cellular mechanisms driving persistent MC hyper-activation and related risk for inflammatory diseases.

Supported by grants from NIH/NICHD (R01 HD072968), NIH/NIAID (R01 AI152268), and USDA (USDA-NIFA 2019-67015-29483).
Atopic dermatitis patients have increased numbers of FcɛRIα and IgE double positive monocytes and plasmacytoid dendritic cells in peripheral blood mononuclear cells
Authors: Bin Lianghua , Richers Brittany , Leung Donald Y M
Published: January 2023

Antigen presenting cells including monocytes and dendritic cells have been reported to express cell surface high-affinity IgE receptor (FcɛRI) and binds to IgE. However, the frequencies of these cells in human peripheral blood mononuclear cells (PBMCs) are ambiguous, and the biological function of these cells in human remains to be characterized. In this study, we investigated the frequencies of cell surface expression of FcɛRIα and cell surface bound IgE in PBMCs isolated from normal human controls (NC) and atopic dermatitis (AD) patients using multicolor flow cytometry methods. While the frequencies of FcɛRIα+ single positive cells have no significant differences between NC and AD, we found that the frequencies of FcɛRIα and surface-bound IgE double positive cells (FcɛRIα+ IgE+) in human PBMCs are significantly increased in AD patients (mean ±SD: 2.52% ± 1.33%) compared to NC (mean ±SD: 1.26% ± 0.79%) (p=0.003). In addition, the frequencies of FcɛRIα+ IgE+ cells are positively correlated with serum IgE levels (Spearman analysis: r=0.67, p<0.0001). The FcεRIα+ IgE+ cells in both NC and AD subjects are comprised of monocytes, conventional DCs (cDCs) and plasmacytoid DCs (pDCs). While the numbers of FcεRIα+ IgE+ cDCs have no significant difference between NC and AD, FcɛRIα+ IgE+ monocytes and pDCs are significantly increased in AD patients compared to NC subjects. Collectively, our study identified a distinct FcɛRIα+ IgE+ cell population in human PBMCs, and this population is increased in AD. Further investigation is warranted to characterize the biological significance of this particular cell population in normal humans and AD patients.

Supported by grants from Genentech (ML41620)
Obesity Inhibits Innate Immune Functions During Pseudomonas aeruginosa Pneumonia
Authors: Huizinga Gabrielle P , Warheit-Niemi Helen I , Gallagher Katherine , Moore Bethany B , Singer Kanakadurga
Published: January 2023

Obesity is a major global public health concern. Worldwide, 39% of the population is overweight and 13% of the population is obese. Obesity contributes to many diseases, such as heart disease, stroke, some cancers, and notably, type 2 diabetes (T2D). Although T2D itself is a non-communicable disease, many patients with diabetes are more susceptible to microbial infections and exhibit a higher burden of disease. Although obese animals have an increased production of myeloid cells, previous work from our labs has shown that a failure to heal diabetic wounds corresponds to elevated levels of prostaglandin E2 (PGE2) in the inflammatory macrophages that are recruited to the wound. Furthermore, past studies from our lab have demonstrated that PGE2 signaling can impair the innate immune functions of macrophages. Therefore, we hypothesized that diabetic obese mice would be more susceptible to a respiratory Pseudomonas aeruginosa strain PA01 infection due to impaired function of lung resident and recruited macrophages. Preliminary studies demonstrate that obese diabetic mice do have an increased P. aeruginosa burden in the lung 24 hours after infection. Additionally, we have determined that naïve obese diabetic mice have an increase in lung neutrophils compared to lean mice, however, after infection with P. aeruginosa, these mice have a trend of decreased neutrophil recruitment. Additionally, functional assays show that macrophages and neutrophils from obese diabetic mice have a defect in killing P. aeruginosa. These observations suggest that obese diabetic mice have not only a defect in innate immune cell recruitment, but also a defect in innate immune cell function during bacterial pneumonia.

Supported by grants from NIH (T32 AI007528-21)
Innate type 2 immunity controls hair follicle commensalism by Demodex mites
Authors: Ricardo-Gonzalez Roberto R , Kotas Maya E , O’Leary Claire , Tenvooren Iliana , Marquez Diana , Singh Katelyn , Damsky William , Liao Chang , Schroeder Andrew , Cohen Jarish , Fassett Marlys S , Lee Jinwoo , Daniel Scott G , Bittinger Kyle , Diaz Roberto Efrain , Fraser James , Ansel Karl Mark , Spitzer Matthew , Liang Hong-Erh , Locksley Richard M
Published: January 2023

Demodex mites are obligate commensal parasites of hair follicles (HF) in mammals. Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction and aging, but mechanisms restricting Demodex outgrowth and pathogenesis are not defined. Here, we show that control over mite HF colonization of mice requires ILC2s, IL-13, and its receptor IL-4Ra, but not IL-4 or the adaptive immune system. Epithelial HF-associated ILC2s elaborate IL-13 that attenuates HF and epithelial cell proliferation at anagen onset; in their absence, Demodex colonization leads to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammatory programs leading to loss of barrier function and premature HF exhaustion over time. Humans with rhinophymatous acne rosacea, a nasal inflammatory condition associated with a high burden of Demodex, had increased HF inflammatory cells with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a critical role for skin ILC2s and IL-13, which comprise an immune checkpoint necessary to sustain cutaneous integrity and restrict pathologic infestation by colonizing HF mites.

This work was supported by the National Institutes of Health (AR007175 to R.R.R.G. and M.S.F, AR075880 to R.R.R.G, HL140868 to M.E.K., DK121476 to C.E.O., AI159229 to W.D., AR074556 to M.S.F., AI026918 and HL107202 to R.M.L.), Dermatology Foundation (R.R.R.G., M.S.F.), A.P. Giannini Foundation (R.R.R.G, M.E.K.), Robert Wood Johnson Foundation (R.R.R.G.), Howard Hughes Medical Institute (R.M.L.), University of California Tobacco-Related Disease Research Program (T29IP0554 to J.S.F.), and the Sandler Asthma Basic Research Center at the University of California San Francisco. R.E. Díaz was supported by NSF GRFP (#1650113). R.E. Díaz is a Howard Hughes Medical Institute Gilliam Fellow (#GT11377).
The role of IL-1a during early-stage Pseudomonas aeruginosa infection of the corneas
Authors: Ratitong Bridget , Marshall Michaela , Dragan Morgan , Anunciado Charissa , Pearlman Eric
Published: January 2023

Pseudomonas aeruginosa is an important cause of dermal, pulmonary and ocular disease. Our studies have focused on P. aeruginosa infections of the cornea as a major cause of blinding microbial infections. The infection leads to an influx of innate immune cells with neutrophils making up to 90% of recruited cells during early stages. We previously reported that the proinflammatory cytokines IL-1a and IL-1b were elevated during infection. Infected Il1b−/− mice developed more severe corneal disease that is associated with impaired bacterial killing as a result of defective neutrophil recruitment to the corneas. We also reported that neutrophils are an important source of IL-1b and IL-1a.

To examine the role of IL-1a in Pseudomonas aeruginosa keratitis, we inoculated corneas of C57BL/6 (WT), Il1a−/−, Il1b−/− and Il1a−/−Il1b−/− (DKO) mice with 5×10^4 ExoS expressing P. aeruginosa (PAO1 strain). We identified neutrophils and monocytes as the main producers of IL-1a which peaked at 24 hours post infection (hpi). Depletion of neutrophils or monocytes led to more severe corneal disease and higher bacterial burden. Il1b−/− and DKO mice have significantly higher bacterial burden that was consistent with impaired neutrophil and monocyte recruitment to the corneas. Surprisingly, Il1a−/− mice had the opposite phenotype with significantly less bacteria than WT mice. There were no significant differences in neutrophil recruitment. However, we hypothesize that Il1a−/− neutrophils have enhanced antimicrobial functions that resulted in more effective bacterial killing which we are addressed by RNA sequencing of neutrophils isolated from WT or Il1a−/− corneas.

Supported by grants (EP) RO1: EY18612 and (BR) F31: EY032312-01
The E3-ligase TRIM7 acts as an antiviral factor by ubiquitinating the SARS-CoV-2 membrane protein to limit apoptosis and virus replication
Authors: Gonzalez Orozco Maria J , Hage Adam , Xia Hongjie , Huante Matthew , van Tol Sarah , Giraldo Maria I , Puebla-Clark Lucinda , Menachery Vineet , Stephens Robin , Endsley Mark , Endsley Janice , Shi Pei-Yong , Freiberg Alexander , Rajsbaum Ricardo
Published: January 2023

Cytokines, such as type-I Interferons (IFN-I), are essential to mount an effective antiviral response. The production of IFN-I is regulated by multiple mechanisms, including ubiquitination of host signaling factors. However, viruses can hijack the ubiquitin (Ub) system to promote viral replication. For example, we recently reported that the E3-Ub ligase TRIM7 ubiquitinates the envelope protein of Zika virus, which increases virus binding to its receptor favoring virus entry. Previous studies suggest that TRIM7 may play dual roles during infection by promoting or inhibiting IFN-I induction depending on the conditions. Here, we used our recently generated Trim7−/− mice to study its physiological role during SARS-CoV-2 (SCoV2) infection. We found that Trim7−/− mice have increased weight loss and lung virus titers compared to WT controls upon infection with SCoV2. We also found a dysregulated innate immune response, which included increased IFN-I induction and reduced neutrophil recruitment and production of inflammatory cytokines such as IL-6 in Trim7−/− mice. We observed a higher frequency of apoptotic cells in Trim7−/− compared to WT mice, specifically in CD45− lung epithelial cells. Mechanistically, using co-immunoprecipitation studies, we confirmed previous reports that TRIM7 interacts with the viral membrane protein (M), which can cause apoptosis. Using recombinant mutants that lack ubiquitination sites on M, we found that TRIM7-mediated ubiquitination of M protects cells against death. This suggests that TRIM7 restricts SCoV2 infection via a novel non-degradative mechanism involving ubiquitination of M. Understanding the role of TRIM7 in inhibiting SCoV2 replication could help develop treatments for COVID-19.

Supported by grants from NIH R01 AI166668
Differential regulation of macrophage galactose-type lectin (MGL) orthologs by mycobacterial ligands
Authors: Paez Reina N , Browning Sarah , Files Megan , Varghese Alyssa , Naqvi Kubra F , Huante Matthew B , Strong Emily , Lee Sunhee , Endsley Mark , Endsley Janice J
Published: January 2023

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for approximately 10 million new infections and 1.5 million deaths each year. Pattern recognition receptors (PRRs) expressed on the surface of macrophages play an important role in orchestrating the immune response to pathogens. Amongst these, the C-type lectin receptors (CLRs) are an important PRR system that is increasingly understood to mediate innate immunity to mycobacteria. We recently described a new anti-bacterial and anti-inflammatory role for the macrophage galactose-type lectin-1 (MGL-1) in a murine model of experimental TB. MGL is a type II CLR that is known to recognize galactose and N-acetylgalactosamine monosaccharides. We observed that exposure to Mtb activated expression of both murine MGL orthologues, MGL-1 and MGL-2. The specific pathogen-associated molecular patterns that activate MGL orthologue transcription, however, have yet to be determined. Here we demonstrate that expression of MGL-1 and MGL-2 are differentially regulated by cytokines, TLR agonists, and mycobacterial ligands. MGL-1 expression is activated by dexamethasone, IL-4, IL-10, and the mycobacterial cell wall glycolipid Trehalose 6,6′-dimycolate. In contrast, MGL-2 expression is activated by TLR2/6 and TLR2/1 agonists and total lipid extracts of mycobacteria. Use of a MyD88 inhibitor further showed that molecular regulation of MGL-1 and MGL-2 transcription, following exposure of macrophages to mycobacteria, is TLR pathway-dependent. These results identify overlapping and unique host immune pathways that differentially activate MGL orthologues following exposure to Mtb and could be targeted through immune modulation.
Recombinant Fasciola hepatica fatty acid binding protein (Fh15) as a novel anti-inflammatory biotherapeutic in an acute gram-negative non-human primate sepsis model
Authors: Espino Ana M , Rosado-Franco Jose J , Armina-Rodriguez Albersy , Marzan-Rivera Nicole , Burgos Armando G , Spiliopoulus Natalie , Mendez Loyda B
Published: January 2023

Due to their phylogenetic proximity to human, non-human primates are considered an adequate choice for basic and pre-clinical model of sepsis. Gram-negative bacteria are the primary causative of sepsis. During infection, bacteria continuously release the potent toxin lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to death. Our previous research has demonstrated in vitro and in vivo using a mouse model of septic shock that Fh15, a recombinant variant of the Fasciola hepatica fatty acid binding protein, acts as an antagonist of TLR4 suppressing the LPS-induced pro-inflammatory cytokine storm. The present communication is a proof-of concept study aimed to demonstrate that a low-dose of Fh15 suppresses the cytokine storm and other inflammatory markers during the early phase of sepsis induced in rhesus macaques by i.v. infusion with lethal doses of live E. coli. Fh15 was administrated as isotonic infusion 30 min prior to the bacterial infusion. Among the novel findings reported in this communication, I) Fh15 significantly prevented bacteremia, suppressed LPS levels in plasma and the production of C-reactive protein and procalcitonin, which are key signature of inflammation and bacterial infection, respectively, II) reduced the production of pro-inflammatory cytokines, and III) increased innate immune cell populations in blood, which suggest a role in promoting a prolonged steady state in rhesus macaques even in the presence of inflammatory stimuli. This is the first report demonstrating that a F. hepatica-derived molecule possesses potential as anti-inflammatory drug against sepsis in an NHP-model.

This research was supported by the National Institute of Allergy and Infectious Diseases Grant/Award Number: 1SC1AI155439-01, Office of Research Infrastructure Program of NIH (ORIP-NIH) 2016-2021, Grant/Award Number: P40OD12271; National Institute on Minorities Health and Health Disparities, Grant/Award Number: 5R25GM061151, G12MD007600 and R25GM061838.