bluuebunny/crossref_metadata_2025 huggingface dataset:
An engineered viral protein rewires signaling pathways to overcome T cell suppression.
Authors: Zheng Yating , Gu Zehui , Shudde Claire , Piper Taylor , Wang Xinyu , Aleck Grace , Zhou Jiajia , Chanda Monica , Trinch Lilliana , Zou Weiping , Courtney Adam
Published: November 2024

Cancer can evade destruction by depriving T cells of cytokines, such as IL-2, that cause JAK-STAT signaling required for T cell functionality. We hypothesized that the direct activation of STAT proteins in a cytokine-independent manner could restore the signals needed by T cells. Because STATs are regulated by phosphorylation, we predicted that enforced recruitment of kinase activity to STATs could activate them directly in the absence of cytokine. Tyrosine-protein kinase-interacting protein (TIP), expressed by Herpesvirus saimiri, co-opts LCK kinase by recruiting LCK to non-canonical substrates, including STATs. We evaluated whether a minimal region of TIP could be isolated from the full-length viral protein and combined with STAT binding sites derived from cytokine receptors. Using this platform, we demonstrate that LCK kinase activity can be recruited to targeted STAT proteins to activate them. We determined that a STAT5 activator (aSTAT5) allowed CD8+ T cells to remain viable in the absence of IL-2 and retained their capacity to produce IFNg and kill cancer cells ex vivo. We therefore incorporated aSTAT5 into tumor-specific T cells (TST) and transferred them into tumor-bearing mice. We determined that aSTAT5 TST caused potent tumor regression and reduced tumor outgrowth due to improved T cell persistence and functionality. Our findings highlight that T cells can be improved to resist the miscommunication that cancer causes by targeting intracellular signaling pathways.
Immune experiences alter tolerance in T cell-mediated autoimmunity.
Authors: Rollins Meagan , Bhela Siddheshvar , Weyu Eyob , Nelson Christine , Quarnstrom Clare , O'Flanagan Stephen , Smith Olivia , Sofianou Xenia , Seelig Davis , Fife Brain , Masopust David , Vezys Vaiva
Published: November 2024

Autoimmune diseases afflict >20M Americans and alarmingly the incidence of autoimmune diseases is increasing particularly in children. The hygiene hypothesis (HH) proposes that there is an inverse correlation between microbial exposure and subsequent development of autoimmunity, i.e., the “cleaner” the environment, the higher the probability that autoimmunity develops. Critically, how microbial exposures and infections alter self-antigen (Ag), a critical component of immune tolerance, is unknown. Our data show that immune-experienced mice have decreased self-specific CD8 T cell priming and organ pathology mediated by resident cells in lymph nodes (LN). Resident Ag-bearing LN stromal cells isolated from immune-experienced mice induced less self-specific CD8 T cell proliferation in vitro indicating changes in self-Ag. The HH determines that environmental stressors are important drivers of autoimmunity, yet the human immune experience is poorly modeled in SPF models of tolerance and autoimmunity. Therefore, we used mice exposed to natural mouse pathogens, "dirty" mice showed a decrease in T cell activity to self-Ag. The cohousing of NOD mice with pet shop mice resulted in zero mice developing autoimmune diabetes. Overall, our data suggest novel mechanisms by which self-antigen is altered in lymph nodes due to previous immune experiences addressing a critical mechanism behind the hygiene hypothesis, intersecting TCR reactivity with environmental impact.
Intricate mechanisms trigger scattered crypt IEC apoptosis mediated necrotizing enterocolitis pathogenesis in neonatal mice
Authors: Subramanian Saravanan , Bu Heng-Fu , Chou Pauline , Wang Xiao , De Plaen Isabelle , Tan Xiao-Di
Published: November 2024

Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal inflammatory disorder among premature infants. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive epithelial necrosis in intestinal villi and mucosa inflammation remain unclear. We developed a novel triple-transgenic mouse model, namely, 3xTg-iAPcIEC (inducible apoptosis phenotype in crypt-IEC) using doxycycline (Dox)-inducible tetO-rtTA system and vil-cre technology, for inducing tissue-specific overexpression of Fasl in IECs. We found that scattered crypt IEC apoptosis caused intestinal crypt inflammation and villous necrosis resembling NEC in neonates. This pathological progression initiated a set of NEC-associated intricate cellular and molecular pathophysiological effect including increase in Rip3 and Ifng, infiltration of CD8+ T cells, and dysbiosis with Gram-positive bacteria. Mechanistically, scattered crypt IEC apoptosis-induced IFN-γ and RIP3 activation were noted to mediate both intestinal crypt inflammation and mucosal villous necrosis, whereas CD8+ T cells and dysbiosis with Gram-positive bacteria contribute to mucosal villous necrosis. Notably, we observed that blocking any of these events protects NEC development in 3xTg-iAPcIEC mouse pups, underlining their central roles in the NEC pathogenesis. Our findings may advance knowledge in preventing or treating this devastating disorder in humans.
Case report: Use of emapalumab-lzsg for secondary hemophagocytic lymphohistiocytosis in a patient with chronic granulomatous disease
Authors: Patel Raveena , Gunderson Carly , Ishikawa Caren , Alqahtani Mashael , Cadilla Adriana , Douglas Dorothea
Published: November 2024

Background: Chronic granulomatous disease (CGD) is an immunodeficiency marked by infections due to NADPH oxidase defects. A hyperinflammatory response predisposes to granuloma formation and hemophagocytic lymphohistiocytosis (HLH), a syndrome defined by uncontrolled cytokine release. We present an adolescent with undiagnosed CGD who developed secondary HLH, requiring the use of emapalumab-lzsg, a human anti-interferon-gamma antibody. To our knowledge, this is the first report of emapalumab-Izsg use in a patient with secondary HLH with CGD.

Case Presentation: A 16-year-old male with Chron’s disease presented with six months of fevers. Imaging showed reticulonodular disease and granulomas throughout the lungs. Respiratory status deteriorated, requiring intubation and ECMO. Blood cultures grew Burkholderia cepacia. Flow cytometry was abnormal and genetic tests confirmed CGD. Further clinical and lab derangements led to HLH testing. HLH was confirmed and treated with steroids, etoposide, and intravenous immunoglobulin. With no clinical improvement, emapalumab-Izsg was recommended. Six-week course of emapalumab-Izsg led to disease regression, with decline of CXCL-9, a marker of active disease.

Conclusion: Emapalumab-Izsg has been used in primary HLH while utility in secondary HLH remains undefined. The standard of care for secondary HLH includes immunosuppressive and chemotherapeutic therapies. We propose that emapalumab-Izsg be strongly considered an option in secondary HLH.
Alveolar macrophage (AM) Toll-like Receptors (TLRs) signaling drives inflammation and pathogenic neutrophilic infiltrate during neonatal murine Influenza virus infection
Authors: Rao Abhishek , Kumova Ogan , Onufer Abigail , Pascasio Judy , Carey Alison
Published: November 2024

Like human neonates, murine neonates are highly susceptible to influenza viral (IV) infection. AMs are tissue-resident immune cells which are activated through TLRs to initiate immune cell recruitment to the site of infection, critical for viral control. However, bystander damage can also occur in the delicate alveoli. Therefore, we hypothesized that AM TLRs contribute to neonatal murine IV pathogenesis via neutrophil recruitment. To determine the relative contribution of AM TLR signaling, we utilized the Cre-flox system to generate AM-specific TLR deletions (LysMCre-MyD88flox). Three-day-old transgenic mice were infected with influenza type A virus strain PR/8/34 (H1N1) and tracked for survival. LysMCre-MyD88flox neonates demonstrated improved survival (70%) compared to wild type (WT) (33%). Moreover, LysMCre-MyD88flox neonates had decreased inflammatory cytokine concentrations in the broncho-alveolar lavage (BAL) compared to WT, specifically IL-18, IL-6, TNF-α and myeloid recruiting chemokines MCP-1 and CXCL1. Flow analysis of the BAL revealed lower neutrophils in the LysMCre-MyD88flox group, consistent with the cytokine concentrations. To test the pathogenic role of neutrophils during IV infection, infected WT neonates were neutrophil-depleted and had improved survival (60% vs. 24%, isotype group). These findings suggest AM TLRs increase IV pathogenesis in neonatal mice and that TLR-blockade is a potential therapeutic target in this vulnerable population.
Influenza virus (IV)-induced Type I Interferons Disrupt Alveolar Epithelial Barrier Function in the Developing Neonate
Authors: Onufer Abigail , Chang-Mell Joshua , Cort Laura , Rao Abhishek , Mdluli Nonto , Pascasio Judy , Carey Alison
Published: November 2024

Type I interferons (IFN-I) limit IV replication in adults. However, IV-infected murine neonates lacking a functional IFN-I receptor (IFNAR-/-) have significantly improved survival and reduced lung pathology relative to wild-type (WT) neonates, indicating IFN-I toxicity in neonates. We hypothesized that IV-induced IFN-I signaling in type II alveolar epithelial cells (AT2s), the primary cell type of IV infection and initiator of host response, contribute to neonatal viral pathogenesis. Therefore, we performed RNA-Seq on AT2s purified from IV-infected IFNAR-/- and WT neonates at 1-, 2-, and 3-days post-infection (DPI). Pairwise comparisons highlighted differentially expressed genes (DEGs) dependent on genotype and timepoint, with DEGs highest between infected IFNAR-/- and WT neonates at 2 DPI and significant pathway upregulation of organ repair. Therefore, we hypothesized neonatal IV-infected IFNAR-/- AT2s possess improved barrier integrity early in infection. Indeed, genes associated with barrier dysfunction (il1b, tgfb1) increased in WT neonates over time, while tight junction gene cldn8 is higher in IFNAR-/- neonates. In addition, IF-stained IFNAR-/- lungs have improved expression of tight junction molecules, ZO-1 and occludin, in comparison to WT neonates at 6 DPI, a time of peak mortality. This suggests that early transcriptional differences in neonatal IFNAR-/- AT2s contribute to increased barrier integrity, mitigating later IV-induced damage and lung pathology.
The role of CXCR4-CXCL12 in the development and migration of human natural killer cells
Authors: Eisman Shira , Koenigsberg Batya , van den Haak Frederique , Pedroza Luis , Hegewisch-Solloa Everardo , Shannon Michael , Mace Emily
Published: November 2024

Natural killer (NK) cells play a critical role in controlling viral infection and malignancy and undergo differentiation from multipotent progenitors in secondary lymphoid tissue. Despite dependence on the microenvironment for differentiation, little is known about the cell-cell interactions that promote NK cell precursor trafficking and maturation in tissue. We have identified the chemokine receptor CXCR4 and its ligand CXCL12 as regulators of the cellular interactions between human NK cell precursors and stromal cells that support NK cell differentiation. Using in-vitro NK cell differentiation, we found that treatment of CD34+ precursors with CXCR4 antagonist AMD3100 leads to a lower efficiency of NK cell generation. Since previous work demonstrates the association of cell migration with NK cell development, we interrogated the effect of AMD3100 on NK cell migration. Using live cell imaging we found that AMD3100 treatment of NK cells or progenitor cells results in altered migratory properties and decreased arrest of cells on CXCL12+ stromal cells. Additional experiments using high resolution microscopy and flow cytometry suggest that integrins are interacting with CXCR4 in NK cells. Our data suggests that the CXCR4-CXCL12 interaction is critical for the migration and proper development of NK cells. Future studies will focus on the necessity for CXCR4-CXCL12 in specific key transitions in development and interrogate the cell biological mechanisms of this process.
N-glycan signature of neutrophil function in a Staphylococcus aureus skin infection model by mass spectrometry imaging
Authors: Bergamin de Castro Tialfi , Ntiamoah Abena , Wu Meng-Jen , Archer Nathan , Scott Alison
Published: November 2024

Staphylococcus aureus (Sa) is a public health concern due to the rise of antibiotic-resistant strains. In humans, elevated risk is associated with Tumor Necrosis Factor (TNF) inhibition. Neutrophils expressing TNF receptors (TNFR1 and TNFR2) respond to Sa skin infection in a mouse model. TNFR2 activation induces PAD4 (peptidylarginine-deiminase-4) which leads to neutrophil extracellular traps (NET) synthesis, increasing host defenses. Alterations in PAD4 and N-glycans are related to increased skin infections and the spatial glycosylation signature during the skin response to Sa is unknown. We compared N-glycan signatures in neutrophil region between wildtype (WT), TNFR1 KO, TNFR2 KO and PAD4-/- groups. Mice were intradermally injected with Sa and skin samples collected for analysis by mass spectrometry imaging. We sprayed PNGaseF on prepared FFPE sections to cleave the N-glycans, sprayed with CHCA matrix, and evaluated in positive ion mode at 50mm spatial resolution on a timsTOF Flex (Bruker Daltonics). Differential N-glycan signatures were identified using a histology-guided approach limiting the comparisons to the PMN region. PAD4-/- showed higher intensity of N-glycans than all groups. However, specific N-glycans were higher in TNFR1 KO than PAD4-/- and TNFR2. A lower profile was found in TNFR2 KO vs. WT. We concluded that each group has a specific N-glycan signature which can be attributed to unique immune responses to Sa infection and NET formation.
role of muscle regenerative macrophages in adjuvant-mediated immune response skewing to seasonal influenza vaccine
Authors: Noureddine Moataz , Chang Lauren , Laghlali Gabriel , Warang Prajakta , Prellberg Mathew , De Geest Bruno , Coughlan Lynda , Schotsaert Michael
Published: November 2024

MF59 (AddaVax-like) is a clinically approved nanoemulsion adjuvant with an unclear mechanism of action. Here we introduce a novel approach to investigating the role of muscle regeneration in adjuvanted vaccine response. We found that AddaVax adjuvanted quadrivalent inactivated flu vaccine (QIV) induced a Th2 immune response with IL-4, -5, and -13 expression in the lungs 5 days after H1N1 lethal challenge. In contrast, IMDQ-PC (TLR7/8 agonist) induced a Th1 lung response and higher IgG2a titers. In BMDM cultures, we found AddaVax unlike IMDQ-PC to require IFNγ co-incubation for the induction of iNOS+ proinflammatory M1 phenotype. Since IFNγ is crucial for muscle regeneration, we hypothesized that AddaVax triggers macrophage-dependent muscle regeneration which leads to an enhanced Th2 vaccine response. We found that AddaVax adjuvanted QIV led to an increase of Arg-I+ M2 macrophages in the muscle 4 days post-injection, similar to late-stage Th2 response in regeneration. Using IgG titers, Our hypothesis was further supported by finding AddaVax response to be diminished in aged mice where regeneration is impaired, but rescuable by depletion of circulating monocytes and reconstitution with young BMDMs. Using in-depth characterization and selective depletion, we will investigate the crosstalk between muscle regeneration and AddaVax response allowing a better understanding of the potential muscle role in inhibiting the Th1 response necessary in viral infections, and cancer vaccines.
Hands-on gene recombination as an active learning tool for TCR and BCR assembly
Authors: Tritz Zachariah
Published: November 2024

Active learning approaches within STEM been shown superior to didactic lectures. I queried whether active learning for the difficult concept of B and T cell receptor (B/TCR) recombination promoted material retention in subsequent examinations compared to a lecture. A 2020 publication by Stranford et al. put forward an active learning tool for this topic using pieces of paper to stand in for genes, allowing students to physically recombine these “genes” with scissors and tape to show recombination of BCR and TCRs. I used an adapted version of this activity in an undergraduate immunology course at University of Wisconsin–La Crosse during the Fall 2023 semester. One section of the course (N=34) received a lecture on the topic while an experimental section of the course (N=26) participated in the activity. A questionnaire assessed student enjoyment of the activity and their perception of whether they learned better with an activity than they would have with a lecture. During the following class period, a multiple choice activity was used to determine student understanding of the material. Students that self-reported better understanding of the material with an activity did perform significantly better than their peers (p=0.045), despite the fact that these students do not have a significantly higher overall course grade (p=0.19). This suggests that a hands-on activity can be an effective way to promote student retention of content related to antigen receptor recombination.
Integrating biomedical sciences (Physiology, Biochemistry, and Cell biology) into pharmaceutical sciences curriculum
Authors: Fazal Nadeem , Graefen Bahar
Published: November 2024

Like other healthcare and medical professional curricula, pharmacy is also struggling to incorporate basic medical sciences into pharmaceutical sciences. Enrollment issues complexed with disappearing admission test like (PCAT) have challenged the instructors to make sure that the first- and second-year pharmacy students are thoroughly prepared with foundational science backgrounds before they start learning clinical and experiential subjects. At the College of Health Sciences and Pharmacy Chicago State University, we prepared and tested ourselves for the same challenge. Immunology taught as a core subject in Pharmacy year-2 was one of the subjects chosen for addressing this issue. Immunology is a rapidly developing biomedical science that delves into the wondrous complexities of the immune system; an extraordinary defense mechanism system evolved to safeguard organisms from diverse threats. At the core of immunology, it overlaps basic cell biology and has an intricate network of cells, tissues, and molecules that need to be introduced to pharmacy students when teaching about the basics of the subject. Retrospective data was analyzed where Immunology was bundled with a 4-credit Microbiology courseas compared to a 2-credit Immunology course which was shifted to Pharmacy year 2. The data suggested that student’sperformance was enhanced when Immunology was offered as a standalone course in Pharmacy Year-2.
Cardiac resident macrophage affects doxorubicin-induced cardiomyopathy
Authors: Jeon Sae-Bom , Park Sahng Wook , Lee Seung-Hyun
Published: November 2024

Doxorubicin is an anticancer drug commonly used in patient with lymphoma or breast cancer, and its main side effect is cardiotoxicity by damaging cardiomyocytes. While the mecahnisms of doxorubicin-induced cardiotoxicity are poorly understood. Recent studies demonstrated that the cardiac resident macrophages play essential roles in regulating homeostasis of cardiomyocyte, promoting electrical conduction, and eliminating dead cardiomyocytes and debris. In this study, we showed that cardiac resident macrophages were involved in cardiomyocyte death in a mouse model of doxorubicin-induced cardiotoxicity. Mice were injected with 20 mg/kg of doxorubicin and their hearts were harvested to determine the composition of immune cells in the heart. Using FACS analysis, we observed that the number of CD45+Ly6GintCD11b+F4/80+ macrophages per mg was significantly reduced in the hearts of doxorubicin-injected mice. These cells are cardiac resident macrophages with a CCR2- MHCⅡ+ phenotype. Also, The TUNEL assay confirmed that macrophage death observed in the cardiac tissue of mice 1 day following doxorubicin administration. Finally, we treated iPSC-derived cardiomyocytes and macrophages with doxorubicin and observed cell death, and found that macrophages were more sensitive to doxorubicin than cardiomyocytes. These results suggest that cardiac resident macrophages are sensitive to doxorubicin, which can affect the death of neighboring cardiomyocytes and lead to cardiotoxicity.
Homeostatic Type I IFN Response at the Placenta Contributes to Sex-biased Neurodevelopmental Outcome
Authors: Kim Eunha , Lai Jenny , Sun Yanyi , Kim Minjin , Choi Gloria , Lee Alice , Huh Jun
Published: November 2024

The maternal-fetal interface, placenta, plays pivotal roles in fetal development. Recent evidence shows that the placenta contributes to the sex-biased prevalence of some developmental disorders such as neurodevelopmental disorders. Commencing an exploration into the molecular intricacies of fetal sex-specific transcriptional changes within the placenta, our study leveraged single-cell RNA sequencing. We uncovered a diminished expression of interferon (IFN)-responsive genes (ISGs) in male fetus-associated trophoblasts compared to female, which have pivotal roles in the process of spiral artery remodeling. We further found maternal NK cells associated with male fetus placenta showed a reduction in the interferon production pathway. The introduction of maternal immune activation (MIA), which induces neurodevelopmental abnormalities in male offspring, it reversed these sex-specific disparities in the placenta. In pursuit of unraveling the potential link between sex-specific ISGs expression and neurodevelopmental outcomes in MIA offspring, we selectively removed IFN alpha and beta receptor subunit 1 (IFNAR1) in a trophoblast-specific manner. Blunting IFNAR1 in the placenta protected male offspring from MIA-induced behavioral abnormalities. Our data suggests the intricate landscape of sex-specific IFN response at the maternal-fetal interface and underscore its role in shaping the sex-bias observed in MIA-induced neurodevelopmental phenotypes.
Heightened infiltration of immune effector cell types in the hippocampal regions in the APP/PS1 mouse model of Alzheimer’s disease.
Authors: Maynes Mark , Ayasoufi Katayoun , Wolf Delaney , Tritz Zachariah , Jin Fang , Hansen Michael , Johnson Aaron
Published: November 2024

Alzheimer's disease (AD) is among the most common neurodegenerative diseases in which a role for immune cells in its etiology is only beginning to be realized. AD has two molecular pathological hallmarks: the presence of amyloid beta (Aβ) plaques and tau tangles within neuronal cells. The amyloid plaques can be modeled in mice by inserting transgenes that enhance human Aβ to create plaques inside the mouse brain. Amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) are two transgenes inserted into C57BL/6 mice that results in Aβ plaques in the hippocampus and cortex. This mouse is known as the APP/PS1 mouse. Immune cell infiltration into the brain is associated with aged APP/PS1 mice. However, comprehensive immune profiling has not been performed in this model to outline immune cell effectors potentially involved in neuropathology. Therefore, we employed spectral flow cytometry to investigate the immune cell profile in 24-month-old APP/PS1 mice compared to an age matched control. There are increased numbers of CD8 T, γδ T, and NK cells expressing perforin and other activation related surface markers. Using small animal MRI imaging, we also observed enlarged ventricles mice indicative of brain atrophy in APP/PS1 mice when compared to wildtype age matched control. These results provide insight into the regional immune cell infiltration of the brain, setting the stage for further analysis of immune mechanisms of neuropathology in experimental AD.
Dysfunctional CD8 T cells in tumors are addicted to antigen
Authors: Erwin Megan , Philip Mary
Published: November 2024

Chronic antigen stimulation, as during cancer or chronic infection, leads to T cell dysfunction. We studied the impact of chronic antigen stimulation on T cell receptor (TCR) signaling in CD8 T cells using a genetic liver cancer mouse model. Tumor-specific CD8 T cells (TST) initially acquired proximal TCR signaling defects, but with increased antigen exposure showed both proximal and distal signaling defects. Despite impaired TCR signaling, a stable population of TST was maintained throughout tumorigenesis. We tried to rescue dysfunctional TST by removing them from tumors but found that TST failed to survive in tumor-free mice; thus, T cells are addicted to antigen, though antigen impairs TCR signaling. We investigated the mechanism of antigen-dependent TST persistence and found that throughout tumorigenesis, a subset of terminally-differentiated (TCF1-) TST within liver tumors could be found in cell cycle. The proliferation-associated distal TCR signaling pathways MAPK and mTOR were impaired in TCF1- TST, and we hypothesize that antigen-dependent persistence is NFAT-dependent. Studies are underway to determine whether all TCF1- TST can enter cell cycle in a stochastic, NFAT-driven manner, or if only a specific subset of TCF1- TST proliferate. By understanding how T cell signaling is regulated in response to chronic antigen stimulation, we can design strategies to selectively modulate TCR signaling to maintain proliferation while preventing loss of effector function.
Liver metastases reprogram neutrophils to potentiate cancer progression
Authors: Pearson Ashley , Holcomb Erin , James Jadyn , Wang Zhuwen , Kyi Justine , Huber Amanda , Green Michael
Published: November 2024

Liver metastases are associated with poor overall survival and increased disease progression, regardless of primary tumor type. Liver metastases suppress systemic antitumor immunity to limit immunotherapy efficacy on primary tumors through hepatic siphoning and deletion of CD8+ T cells, induction of T regulatory cells, and depletion of dendritic cells. However, it remains unclear how liver metastases promote the metastatic cascade and subsequent disease progression. To investigate how liver metastases reprogram the immune system to potentiate disease progression, we use a preclinical polymetastatic murine model of liver metastasis that spontaneously progresses to the lungs. Using B16F10 melanoma, 4T1 triple negative breast cancer, and LLC non-small cell lung cancer in syngeneic C57Bl/6 or BALB/c mice, we observed that the presence of liver metastases increased lung metastatic burden. We hypothesized liver metastases potentiate the formation of a tumorigenic, immunosuppressive premetastatic niche in distant sites. We conducted single cell RNA sequencing, flow cytometry, and immunofluorescence of the liver, peripheral blood, and lungs of mice with or without liver metastases. These data suggest an association between Ly6g+Ly6c- neutrophils and progression in mice with liver metastases. Future efforts will define the contribution of neutrophils to disease progression with the ultimate goal of understanding and addressing the immunosuppression promoted by liver metastases.
Optimization of T Cell Activation Protocols to Enrich Stem Cell Memory Phenotypes: Metabolic and Health Assays as Predictive Tools
Authors: Haupt Kim , Sylvester Kayla , Lauer Anthony , Karassina Natasha , Vidugiriene Jolanta
Published: November 2024

The generation of T cells with a stem cell memory phenotype (T_SCM) holds significant promise for immunotherapy due to their longevity and potent antitumor activity. This study aimed to optimize T cell activation and expansion conditions to selectively enrich for the T_SCM population. Utilizing a suite of cell health and metabolism assays, we monitored T cell cultures to identify predictive markers of T_SCM enrichment. We observed that early activation steps correlated with a surge in ATP production and alterations in cell reducing potential, indicative of metabolic reprogramming. Subsequent activation phases were marked by an upregulation in both catabolic and anabolic pathways, as evidenced by the increased turnover of key metabolites including glucose, lactate, glutamine, glutamate, branched-chain amino acids (BCAA), and pyruvate. Notably, the rate of T cell activation influenced the composition of the resultant T cell populations, with the media composition further affecting these dynamics. Our findings suggest that metabolic assays, particularly those measuring changes in ATP levels and primary metabolite flux, can serve as effective early predictors of T_SCM enrichment. These insights may guide the development of more efficacious T cell-based immunotherapies by facilitating the targeted generation of T cells with a stem cell-like memory phenotype.
Excess airway iron during severe SARS-CoV-2 infection impairs neutrophil antifungal activity enabling 2° mold infection
Authors: Saitornuang Sawanan , Lam Cuong , Riegler Ashleigh , Meehan Will , Oh Joo-Yeun , Patel Rakesh , Haas Hubert , Saini Shefal , Lapi Suzanne , Leal Sixto
Published: November 2024

COVID-19-Associated Pulmonary Aspergillosis (CAPA) is an invasive 2° mold infection caused by Aspergillus species affecting ~5% of patients with severe SARS-CoV-2 (SARS-2) infection. In this study, we hypothesized that excess airway iron released during SARS-2 infection promotes 2° infection via enhancement of fungal growth and impairment of neutrophil antifungal activity. We show that SARS-2 bronchoaveolar lavage samples (n=48) have higher levels of total iron, ferric iron, and oxyhemoglobin relative to bacterial pneumonia (n=40). Likewise, FFPE lung tissues from SARS-2 infection exhibited increased extracellular ferric iron, ferrous iron, ferritin, and hemoglobin. We show that both iron and hemoglobin enhance the growth rate of Aspergillus fumigatus via siderophore and reductive iron assimilation dependent mechanism. Utilizing lung epithelial cell transmigration model, we show that iron enhances neutrophil intracellular ROS production resulting in enhanced conidiacidal activity. In contrast, transmigrated neutrophils exposed to both heme and hemoglobin exhibit decreased ROS production and NET formation with significant impairment in hyphal killing. When both neutrophils and fungi are co-cultured with iron, heme, or hemoglobin, fungi consistently win the battle characterized by uncontrolled hyphal proliferation. These data identify both microbial and host mechanisms which may be targeted pharmacologically to mitigate the iron-dependent growth of 2° mold infection.
IL-10 regulates BCL-3 mediated adaptation to repeated TLR4 stimulation in macrophages
Authors: Bongartz Hannes , Bradfield Clinton , Gross Julia , Nita-Lazar Aleksandra , Fraser Iain , Meier-Schellersheim Martin
Published: November 2024

Due to its potential to initiate pathological inflammation, Toll-like receptor 4 (TLR4) mediated activation of macrophages has been studied intensely, revealing a multitude of regulatory components and mechanisms. Systematically varying dose and duration of priming and re-exposure of bone marrow derived macrophages (BMDM) to the TLR4 ligand KLA, we quantified dose-dependent adaptation and sensitization at multiple stages of the signaling response, including TLR4 internalization, MAPK activation, and nuclear translocation of transcription factors. We find that the anti-inflammatory cytokine IL-10 neither interferes with MyD88-dependent MAPK, NFkB nor TRIF-dependent IRF signaling upon restimulation after priming. Instead, IL-10 is found to affect nuclear translocation of the transcriptional regulator BCL-3 while showing a surprisingly strong dependence on the KLA exposure history. Moreover, chromatin immunoprecipitation of BCL-3 reveals that BCL-3 occupies kb sites in the TNFa and IL-6 genes in hypo-responsive macrophages in an IL-10 dependent manner.

This work was supported by the intramural program of the NIAID, NIH.
Functional Phenotype and Ontogeny of Olfr2 Expressing Vascular Macrophages in Atherosclerotic Plaques
Authors: Orecchioni Marco , Armstrong Suthahar Sujit , Nettersheim Felix , Alimadadi Ahmad , Wang Erpei , Billitti Monica , Resto-Trujillo Natalya , Roy Payel , Hedrick Catherine , Ley Klaus
Published: November 2024

Olfactory receptor 2 (Olfr2) and its human ortholog OR6A2 have emerged as a critical modulator of inflammation, with its presence detected in approximately 30% of vascular macrophages and its depletion shown to reduce atherosclerosis in mice. Single-cell RNA sequencing identified several distinct subsets of macrophages in the atherosclerotic plaque. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages. Olfr2+ macrophage number increased in response to a western diet (WD) in the aorta and they expressed a variety of pro-inflammatory cytokines, including Il1b, and Il6, as well as Ccl5, Cx3cl1, and Ccl22. By extracting differentially expressed genes from bulk RNA-seq data of Olfr2+ vs. Olfr2- vascular macrophages, we defined a signature that mapped with mouse plaque single-cell data of monocytes, aortic intima resident macrophages (MacAir) and Trem2-Gpnmb foamy macrophages. Similar results were found in human plaque, with monocytes and foamy macrophages being significantly enriched. Olfr2 expression also increased in oxLDL-mediated foamy macrophages in vitro. Adoptive transfer experiments confirmed that Olfr2+ macrophages are monocyte-derived, accumulate in the aortic plaque within 72 hours, and display enhanced proliferative potential. Collectively, our findings indicate that Olfr2 expression is boosted by diet in monocytes-derived foamy macrophage subsets that by sensing octanal promote inflammation and atherosclerosis.
γδ​​​​​​​ T17 cells are master regulators of the acute antiviral response in HSV-1 infected corneas
Authors: Rodenberg Rachel , Spadafora Domenico , Fitzpatrick Steffani , Daly Grant , Lausch Robert , Barrington Robert
Published: November 2024

Herpes stromal keratitis (HSK), caused by herpes simplex virus 1 (HSV-1), is the leading cause of infectious blindness in developed countries. Innate-acting γδ T17 cells are critical for protection against ocular HSV-1 infection. It is well established these cells can facilitate neutrophil influx into the cornea, but whether γδ T17 cells regulate other innate immune cells following ocular HSV-1 infection remains unclear. Natural killer (NK) cells also participate in the innate immune response generated against HSV-1 by directly killing infected cells through secretion of granzymes and promoting the antiviral response by production of IFN-γ. Our data strongly suggest that γδ T17 cell secretion of IL-17A promotes NK cell accumulation in the infected cornea. This is demonstrated in several ways: 1) in mice that lack γδ T cells (TCRδ-/-) there are fewer antiviral NK cells following corneal HSV-1 infection compared to wild-type (WT) mice; 2) administering IL-17A to TCRδ-/- mice restored the NK cell population; and 3) neutralization of IL-17A or 4) inhibiting γδ T17 cell influx to corneas in WT mice diminished NK cell accumulation leading to increased viral titers. In contrast, IL-17A production is enhanced in the absence of IFN-γ. In sum, this study identifies a novel mechanism by which IL-17A production by γδ T17 cells promotes antiviral NK cell responses that, in turn limit further IL-17A production via INF-γ secretion by NK cells in the HSV-1 infected cornea.
SKIN RESIDENT T CELL AND NPY1R+ NEURON INTERACTIONS DURING WOUND REPAIR
Authors: Lam Kendra , Colin Joseph , Almaguer Berenice , Jameson Julie
Published: November 2024

Wound repair involves complex cellular interactions to induce proper healing. The transmission of pain signals and immune cell function occurs in unison during tissue repair. The cells and factors responsible for this neuroimmune crosstalk are important to delineate. Knowing γδ T cells and NPY play key roles in wound repair, we aimed to ascertain if γδ T cells and NPY1R+ neurons exhibit crosstalk. We identified that NPY1R+ T cell and neuron subsets including γδ T cells reside in the skin, and that there are γδ T cell-neuronal interactions in the epidermis and dermis of both non-wounded and wounded skin. We then compared dermal interactions in obese and lean mice in wounded and non-wounded conditions, as a model for diabetic neuropathy. There are decreased NPY1R+ neuron-γδ T cell interactions in wounded and non-wounded mice fed a high-fat diet for 14 weeks, indicating obesity and type 2 diabetes negatively impact crosstalk during wound healing. Last, to determine the impact of the γδ TCR on NPY1R+ neuron interactions in wound healing, we compared epidermal T cell-neuron interactions in TCRd-/- mice during wound repair. We found a decrease in interactions between αβ T cells and NPY1R+ neurons in TCRd-/- mice, suggesting γδ T cells communicate with NPY1R+ more frequently during wound repair. Further work will help uncover the factors and receptors that γδ T cells and NPY1R+ neurons utilize to communicate during wound healing, and the consequences of disrupting this relationship.
Regulation of IL-1β-mediated inflammatory responses by neural networks
Authors: Hepler Tyler , Hashimoto Okito , Tynan Aisling , Tracey Kevin , Chavan Sangeeta
Published: November 2024

Sickness behavior associated with inflammation impairs quality of life and increases morbidity and mortality. However, the neural mechanism that mediates inflammation-associated changes are poorly understood. Here, we identify the bed nucleus of the stria terminalis (BNST) as a critical node in mediating responses to cytokines. Administration of recombinant interleukin-1β (rIL-1β) significantly increases serum IL-6 (control vs. rIL-1β; 22.6±4.7 vs 1,487±341.4 pg/ml; p=0.0079). A significant decrease in core body temperature (pre vs. post; 38.1±0.2 vs. 36.1±0.4oC; p<0.0001) and activity level (pre vs. post; 0.2±0.03 vs. 0.03±0.01 A.U.; p=0.0002) is also observed with rIL-1β administration. We visualized rIL-1β-responsive neurons using activity-dependent cell labeling, and identified distinct neuronal populations in the BNST that are active in response to IL-1β. Selective activation of IL-1β-responsive neurons using a chemogenetic approach is sufficient to induce significant increase in serum IL-6 levels (control vs. IL-1β; 26.2±74.1 vs. 272.5±8.3 pg/ml; p=0.01). Reactivation of IL-1β-responsive neurons also induces significant decrease in core body temperature (pre vs. post; 37.6±0.3 vs. 36.1±0.6oC; p=0.02) and activity levels (pre vs. post; 0.1±0.01 vs. 0.06±0.01 A.U.; p=0.02). Our data defines a neural population in the BNST that is sufficient for mediating IL-1β-associated responses, revealing its role in the coordination of inflammatory responses.
Clinical implications for CAR T cell persistence and functional phenotype in patients treated with CD19 CAR T cell therapy.
Authors: Bochnacka Oliwia , Quinn Suzanne , Dalheim Annika , Scurti Gina , Hossain Nasheed , Nishimura Michael
Published: November 2024

Understanding the critical relationship between cancer and the immune system has led to novel immune therapies including chimeric antigen receptor (CAR) T-cell therapy. In the clinic, CAR T cells have been most effective against hematological malignancies. CD19 is expressed in almost all stages of B cell development as well as in most B cell malignancies, making it an ideal antigen to target using CAR T cells. Despite the promising efficacy of CD19 CAR T cell therapy, there remains a subset of patients who are resistant to therapy. At Loyola University Chicago, we initiated a CD19 CAR T cell clinical trial in 2020. A total of 6 patients were treated on protocol, with a 50% durable complete response rate. Importantly, no treated patients experienced severe side effects. Unique to the CD19 CAR T cells, is the incorporation of a CD34 marker gene (CD34t) in the CAR retroviral construct used for T cell transduction. The use of CD34t allows us to track infused CAR T cells in the blood. With the ability to distinguish CD19 CAR T cells from the endogenous T cell population, we have developed an immunofluorescence staining panel of 39 functional cell surface proteins to study the treated patient blood samples. We found that CD19 CAR T cells persist regardless of clinical outcome. We are studying the changing CAR T cell phenotype in patients with varying clinical outcomes to identify phenotypic trends that may have clinical implications and could serve as relevant clinical biomarkers.
In vitro and in vivo proof-of-concept studies of extracellular protein degradation using novel M6PR-targeting chimeras (MTACs).
Authors: Molz Lisa , Liu Hu , Zhu Nanqun , Karra Srinivasa , Tozzo Effie
Published: November 2024

The mannose-6-phosphate-receptor (M6PR) traffics proteins from extracellular and intracellular locales to late endosomes/lysosomes for degradation and is ubiquitously expressed. This natural endogenous function of the mannose-6-phosphate-receptor (M6PR) may be harnessed for degradation of soluble circulating proteins and cell specific degradation of membrane proteins.

Here we describe the design and development of novel M6PR targeting chimeras (MTACs). MTACs are heterobifunctional molecules containing Avilar’s novel, potent, small molecule M6PR-binding ligands conjugated to ligands that bind a target protein of interest for degradation. For proof-of-concept studies, MTACs were designed to target the extracellular protein IgG, the second most abundant circulating protein with a long plasma half-life. Reduction of pathogenic IgG autoantibodies is a clinically validated strategy for treating multiple debilitating autoimmune diseases. In vitro characterization of tridentate and monodentate MTACs revealed potent biochemical binding to M6PR and IgG, and MTAC-mediated cellular uptake and degradation of IgG via the endolysosomal pathway in K562 and LNCaP cells. In vivo MTAC-mediated depletion of human IgG was demonstrated using a heterologous rat PK/PD model.
Vgd1 and Vgd3 T cells are abundant gd T cells populations in high-grade serous ovarian carcinoma, non-small/small cell lung cancer, and show a heterogeneous g-chain usage
Authors: Chaurio Gonzalez Ricardo , Payne Kyle , Anadon Carmen , Lopez-Bailon Luis , Yu Xiaoqing , Mine Jessica , Bari Shahla , Plappert Noah , Perez Bradford , Innamarato Patrick , Melendez Joanna , Eysha Mostafa , Semidey-Hurtado Jonathan , Carter Bethany , Kroeger Jodi , Antonia Scott , Rodriguez Paulo
Published: November 2024

γδ T cells, in contrast to the conventional MHC-restricted αβ, recognize and kill tumor cells in an MHC-unrestricted manner, highlighting their potential for anti-tumor therapies. However, the phenotypes of γδ T cells infiltrating human tumors and their role in anti-tumor immunity remain poorly understood. To assess this, we characterized by Immuno-Seq/iRepertoire the repertoire of γδ-chains used by intra-tumoral γδ T cells in human cancers from FACS-sorted T cells in freshly dissociated high-grade serous ovarian cancers (HGSOCs), non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). Our analysis confirmed that δ1 and δ3 TILs are abundant in all diseases. Interestingly, γδ TILs show heterogenous Vγ-chain repertoires, although TCRVγ9 was the dominant γ-chain. We then encapsulated and run 10X (single-cell) analyses, with sorted, bar-coded, δ1 plus δ2 TILs. All tumors showed distinctive clusters of stem-like cells expressing TCF7, IL7R and CD27 (δ1stem); plus effector T cells expressing high levels of granzymes, IFNG and perforin and NK receptors (δ1Eff). Importantly, we found low expression of PD-1, with only a small cluster of δ1 TILs with TOX expression in lung cancer. Therefore, our data indicate that: 1) γδ T cells quasi-universally infiltrate human carcinomas; 2) δ1 T cells outnumber δ2 T cells in tumors; 3) δ1 TILs use a variety of Vγ chains, with dominant Vγ9 and 4) γδ T cell tumor infiltration is associated with better outcome.
NKG2A expression on tumor reactive CD8 T cells is induced by IL-12
Authors: Fesneau Olivier , Rosales Wesley , Samson Kimberly , Rajamanickan Venkatesh , Haugh William , Huang Jianguo , Duhen Thomas
Published: November 2024

Targeting the NKG2A/HLA-E pathway is a promising strategy to restore CD8 T cells functions, but several aspects of the biology of NKG2A+ CD8 T cells remain to be understood to fully benefit from it. Here, we characterized the NKG2A+ tumor infiltrating CD8 T cells (CD8 TILs) from patients with head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). We showed that NKG2A was expressed by 10 to 15% of CD8 TILs, most of them resembling the previously described CD39+CD103+ (DP) CD8 TILs. scRNA-seq analysis revealed that NKG2A was upregulated as cells differentiated into DP CD8 TILs and acquired cytotoxic properties. NKG2A- and NKG2A+ DP CD8 TILs shared a significant TCR repertoire overlap, suggesting local upregulation of NKG2A. Using RNAscope we demonstrated that NKG2A+ DP CD8 TILs were enriched at the invasive margin and in the tumor bed where they might interact with HLA-E-expressing tumor cells. In contrast to previous results, we showed that CD4 T cells and APCs were required for NKG2A induction and this was dependent on CD40/CD40L interaction and IL-12 secretion. Altogether, our study provides evidence that NKG2A is expressed by tumor reactive CD8 TILs. It also highlights the dual role of IL-12, promoting cytotoxic T cell responses while inducing the expression of immune regulatory markers. The latter might negatively impact the anti-tumor immune response and should be taken into consideration in the future.
Analysis of transcriptional changes associated with anti-tumor effects of iNKT immunotherapy
Authors: Ferguson Sarah , Gumperz Jenny , Brand Joshua , Dinh Huy
Published: November 2024

iNKT cells are innate T cells that can dramatically influence outcomes of immune responses, and that are associated with anti-tumor immunotherapeutic effects in murine models and human clinical trials. Using a human xenograft model of EBV-driven B cell lymphoma, we found that administering a combination of human CD4+ iNKT cells and monocyte-derived DCs markedly reduced tumor mass, while equivalent doses of iNKT cells or DCs alone had little anti-tumor effect. NanoString array analysis of tumor and spleen from mice given a combination of iNKT cells and DCs identified a number of gene expression changes in both tissues compared to untreated lymphoma-bearing mice, suggesting that the interaction of iNKT cells with DCs activates gene expression pathways that promote anti-tumor responses. Ongoing RNA-seq analyses are aimed at further investigating transcriptional changes resulting from iNKT-DC interactions. Gene expression signatures of DCs and iNKTS cultured alone or mixed and immediately lysed will be compared to those of iNKT cells and DCs co-cultured for 24 hours. Differential gene expression analyses will be performed to identify specific transcripts that are up or down-regulated after iNKT-DC co-incubation. Transcriptional changes resulting from iNKT-DC interactions will be compared to those induced in iNKT cells or DCs alone after exposure to other activating stimuli. Together, these analyses will identify candidate pathways that promote anti-tumor outcomes.