bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel stem cell treatment to combat pediatric inflammatory response during cardiopulmonary bypass surgery
Authors: Asbury Julie Marie , Anderson Alanna , French Beth
Published: August 2023

Cardioplegia and exposure to the heart-lung machine required in cardiopulmonary bypass induce a systemic inflammatory response. Adverse effects cause longer ICU stays, higher medical costs, and worsened mortality and morbidity, especially in neonatal and pediatric populations. nCPCs have been shown to have regenerative and anti-inflammatory properties when introduced in animal studies as a treatment. Surgeons at UMB performed cardiopulmonary bypass surgery on non survival porcine subjects with a control and treatment group of 6 porcine subjects in each. NDMU tested and analyzed serum samples of the subjects for immunological markers or interleukins. The results revealed that the treatment does seem to have an effect on the pro-inflammatory cytokines of TNF-α, IL-1β, and IL-6. TNF-α, IL-1β, and IL-6 concentrations decreased significantly from 3124 to 1480 pg/mL, 8319 to 2714 pg/mL, and 18,011 to 10,825 pg/mL, respectively. Furthermore, the treatment also has an effect on IL-10, an anti-inflammatory cytokine, noted by an increase from 289 to 328 mg/mL. Overall, the treatment seems to have a greater effect on the macrophage immune response, while it seems to have little to no effect on the neutrophil immune response. Surgeons are currently working on survival porcine subjects. Moreover, H&E staining and RT-qPCR of samples will be performed to determine the localization of damage and relative expression of the cytokines of interest.
Modified adenovirus induces antigen specific T cells and effector memory T cells in mice
Authors: Sieling Peter A , Golovato Justin , Niazi Kayvan , Rabizadeh Shahrooz
Published: August 2023

We engineered a novel adenovirus with a modified capsid for use as an integrated anticancer gene therapy and to circumvent pre-existing adenovirus immunity. The modified adenovirus, SynBAd, when administered intravenously, induced antigen-specific CD8 T cell immunity in the liver of mice (C57BL/6) and a splenic population of effector memory T cells at levels equivalent with those induced by human Adenovirus serotype 5 (Ad5). Although the virus elicited equivalent liver and splenic T cell responses, SynBAd did not elevate liver enzymes as commonly seen with Ad5. SynBAd bound factor X with similar affinity to Ad5, though it localized preferentially to the spleen relative to Ad5, suggesting greater systemic immune stimulation. CD8 T cells in the liver and spleen increased with SynBAd administration, indicating that the virus acts as a potent mobilizer of T cells. Although SynBAd is a potent immunogen, antibodies generated in mice immunized with Ad5 only weakly neutralized SynBAd infection in vitro, suggesting that preexisting Ad5 antibodies will not dramatically influence SynBAd efficacy. Localization of the adenovirus after administration, activation of antigen specific T cells, and mobilization of effector memory T cells, suggest that this approach will promote an antitumor immune response that could be used in multiple tumor indications.
IMPACTing autoimmunity through dual targeting of antigen presenting cells and T cells via inhibitory receptor agonism
Authors: Grebinoski Stephanie J , Lutz Stephen , Cianci Michael , Borah Minasri , Stanlie Andre , Goydel Rebecca , Visweswaraiah Jyothsna , Higginson-Scott Nathan , Otipoby Kevin , Cipolletta Daniela
Published: August 2023

Autoimmune diseases are triggered by a critical break in peripheral tolerance, thereby allowing the immune system to target and destroy host tissues. Tolerance mechanisms, such as inhibitory receptor (IRs) on T cells, can greatly influence autoimmune disease progression and outcomes, representing an opportunity for therapeutic intervention. Indeed, evidence in both animal models and patients suggests that IRs play a major role in limiting autoimmunity. Immunotherapies targeting IRs have revolutionized the treatment of many cancers and provided evidence that IR modulating therapeutics can significantly impact patient outcomes. However importantly, analogous IR-targeted autoimmune therapeutics have just begun to be tested.

Despite interest, IR agonist antibodies have proven difficult to generate. Optimal agonist antibodies require IR superclustering, which is not efficiently induced by Fc-null antibodies. Successful agonists will likely rely on simultaneous Fc (constant region) tethering on antigen presenting cells (APC), thereby allowing efficient IR superclustering and downstream signaling. Current agonistic antibodies targeting IRs contain an IgG1 wild-type Fc, which by binding both activating and inhibitory Fc receptors can trigger unwanted production of inflammatory cytokines by APCs. Here we show that antibodies binding selectively FcγRIIb, support superior agonism compared to IgG1 wild-type antibodies and mitigate potential liability of current IR agonists under clinical investigation. Indeed, dual targeting of the APC and T cells shows promising pre-clinical results and will be used to create novel IR agonists.
Large-scale analysis of CoVIC antibodies by a global consortium identifies predictors of in vivo protection
Authors:   Members of the Coronavirus Immunotherapeutic Consortium, Jarjapu Mahita ,   CoVIC-DB Team, Saphire Erica Ollmann , Peters Bjoern
Published: August 2023

The Coronavirus Immunotherapeutic Consortium (CoVIC) was established to develop prevention and therapeutic strategies against SARS-CoV-2 that could be used in low- and middle-income countries. The CoVIC compiled a unique panel comprising nearly 400 candidate therapeutic antibodies contributed by 60 groups in industry, academic, and government settings. All antibodies in the CoVIC panel were first blinded at the CoVIC headquarters at the La Jolla Institute for Immunology through the assignment of a code name. Identical sets of antibodies were then sent under the assigned code names to seven different partner reference labs that carried out side-by-side, standardized analyses of binding affinity, epitope location, high resolution structures, pseudovirus and authentic virus neutralization, in vivo protection, and Fc-mediated effector functions. The reference labs deposited their data, which was then validated and made publicly accessible, in the CoVIC database (CoVIC-DB). Using various statistical analyses of the data, we investigated relationships among functional properties of the antibodies. For example, we could discern that binding affinity to the full-length spike is more predictive of neutralization potency than binding affinity to the RBD, and that binding affinity and epitope location together correlates with neutralization. However, neutralization alone is not sufficient to explain in vivo protection in the K18 mouse model of SARS-CoV-2 infection, as other factors such as antibody pharmacokinetics were also important. Overall, the results of this study revealed important insights into antibody properties that are good predictors of in vivo protection by CoVIC antibodies.

COVIC-19 Therapeutics Accelerator (a partnership of Bill and Melinda Gates Foundation), Bill and Melinda Gates Foundation, Mastercard, Wellcome Trust, National Institute of Allergy and Infectious Diseases of the National Institutes of Health, GHR Foundation, FastGrant from Emergent Ventures
Splicing of ultraconserved poison exon elements in RNA binding proteins reprograms the T effector immune response
Authors: Karginov Timofey , Menoret Antoine , Leclair Nathan , Chandiran Karthik , Suarez-Ramirez Jenny E , Karlinsey Keaton , Murphy Patrick , Adler Adam J , Zhou Beiyan , Cauley Linda S , Anczukow Olga , Vella Anthony T
Published: August 2023

Our current understanding of pathways governing the strength and longevity of T cell mediated immune responses primarily focuses on transcriptional and epigenetic changes. However, our prior work demonstrated that optimal T cell effector function is also dependent on RNA binding proteins (RBPs) that carry out post-transcriptional RNA splicing and polyadenylation. Here, we find that a specific family of RBPs, serine- and arginine-rich (SR) RBPs, are significantly upregulated by T cell activation and required for CD8+ T cell mediated responses. SR transcripts contain ultraconserved poison exon (PE) elements that are alternatively spliced and introduce premature stop codons that trigger nonsense-mediated decay. We show that differential splicing of specific RBP PE regions is both induced by TCR stimulation and required for CD8+ T cell activation in both human and mouse. Further, we demonstrate that PEs are cell-state associated by using single-cell RNA sequencing analysis of splicing changes across mouse thymic T cell subsets, in CD8+ T cells over the course of an acute Listeria infection, as well as in human lymphoblastic leukemia, atherosclerosis, and melanoma patient T cells. CRISPR-Cas9 mediated deletion of the PE element of SR-like RBP Tra2β in mouse T effector cells during influenza infection resulted in a significant increase in proliferating and surviving T cells and altered the splicing pattern of critical cell survival, proliferation, and adhesion genes. In total, our work demonstrates a fundamental role for alternatively spliced ultraconserved elements in defining T cell states, controlling T effector-mediated immunity, and altering the T cell response in pathologic conditions.

This work was supported in part by NIH grants AI136955 and R21AI139891 awarded to A.T.V, institutional support, and Boehringer Ingelheim endowed Chair in Immunology.
Early Life Antibiotic Exposures Inhibits Postnatal Enteric Nervous System Maturation and Alters Neonatal Neuroimmune Interactions
Authors: Schill Ellen Merrick , Joyce Elisabeth L , Gaddipati Shreya , Floyd Alexandria N , McDonald Keely G , Rusconi Brigida , Newberry Rodney D
Published: August 2023

The enteric nervous system (ENS) regulates intestinal homeostasis via interactions with smooth muscle, epithelium, and immune cells. To assess the impact of early life antibiotics on postnatal ENS development, we performed neuron specific translating ribosome affinity purification to isolate RNA from control and antibiotic exposed enteric neurons for RNA sequencing. Colonic enteric neurons from antibiotic exposed pre-weaning mice expressed significantly less mature neuron markers, and acquired a more inflammatory cytokine signaling signature (e.g. IL18, MCP-1, TLR4). Immunofluorescence staining and ELISA revealed increased enteric neuron density in antibiotic exposed colons, yet reduced expression of neuronal markers including Tuj1 (neuron specific tubulin), synaptophysin (synapse marker) and acetylcholine. Notably, antibiotics did not affect ENS development in germ free mice. By confocal microscopy, antibiotic exposure appeared to reduce phagocytosis of neuronal cell bodies by macrophages, but phagocytosis of synapses by macrophages was increased. Flow cytometry revealed that antibiotic exposure reduced the proportion of macrophages expressing the “M2” marker CD163 compared to controls. This observation is reminiscent of Hirschsprung disease (HSCR), a congenital absence of the ENS from the distal intestine, which causes intestinal macrophages to adopt a pro-inflammatory phenotype and predisposes patients for HSCR-associated enterocolitis secondary to decreased cholinergic signaling. These data suggest that early life antibiotic exposure disrupts how macrophages participate in postnatal ENS development in a microbiota dependent mechanism.

NIH NIAID U01AI131342, NIH NIDDK P30DK052574, T32DK077653, NIH T32HD43010 American Academy of Pediatrics Marshall Klaus 2022
Dietary fiber and Segmented Filamentous Bacteria interaction with intestinal epithelial cells supports the development of CD4+CD8αα+ intraepithelial T cells
Authors: Marino Naomi Claudette Rodriguez , Rivera-Rodriguez Dormarie E , Royer Charlotte J , Yaceczko Madelyn , Cervantes-Barragan Luisa
Published: August 2023

Double positive intraepithelial lymphocytes (DP IELs) are microbiome induced CD4+CD8αα+ T cells that play a role in maintaining homeostasis and reducing inflammation in the small intestine. While the impact of the microbiome on the intestinal immune system has been extensively studied, little is known about how changes in macronutrients, such as dietary fiber, affect the development of important immune cell populations through changes in the microbiome. Here we show that a diet low in fermentable fiber (LFF) impairs development of DP IELs by reducing MHC-II expression on intestinal epithelial cells (IECs). Bulk RNA sequencing of ileal IECs revealed that LFF fed mice had lower expression of genes associated with the antigen presentation machinery. Subsequently, flow cytometry analysis confirmed increased expression of MHC-II on IECs in standard chow fed mice, which is required for DP IEL development. 16s rRNA sequencing of the microbiota of LFF mice showed that SFB is reduced in these animals. We found that colonizing mice with SFB induces MHC-II expression on IECs and restores development of DP IELs in a fiber-dependent manner. Finally, using the offspring of mice that naturally harbor SFB, we show that MHC-II expression on IECs and IFN-γ levels in the ileum increase gradually over time and correlate with SFB colonization. These results suggest that dietary fiber is required for the development of DP IELs through supporting SFB colonization and increasing expression of MHC-II on small intestinal epithelial cells.

Supported by grants from NIH (1R011DK129950) and startup funds from Emory University
Neuronal cells co-ordinate myeloid-epithelial cell interaction to regulate intestinal inflammation
Authors: Sharma Deepika , Malik Ankit , McGrath Shaina , Zabala Sarah , Yang Daping , Chiu Isaac M , Jabri Bana
Published: August 2023

Epithelial response to injury is coordinated through an intricate interaction with neuronal and myeloid cells, however the signaling modules involved are not well understood. In humans, somatic mutations in Tet methylcytosine dioxygenase 2 (TET2), a DNA demethylase, are commonly observed during ageing in myeloid cells and known to modulate inflammatory responses. Using a mouse model that lacks TET2 in myeloid cells (Tet2ΔLysM), we show that myeloid cells and sympathetic neurons form a signaling nexus that controls differentiation of enterochromaffin cells and serotonin production during colonic inflammation. Under physiological conditions, TET2 restricts IL-1β production by myeloid cells which in turn controls the intestinal sympathetic architecture. During inflammation, IL1R signaling limits sympathetic cues that drive differentiation of enterochromaffin cells through α1-adrenergic signaling. As a result, enterochromaffin differentiation and colitis progression in response to mucosal injury is attenuated in Tet2ΔLysM mice. Further, protection from colitis in Tet2ΔLysM mice is mediated by its catalytic activity, and dependent on sympathetic neurons and IL1R signaling. Adrenergic control of epithelial response and pro-colitic serotonin production is also evident under conditions of physiological stress that leads to increased colitis susceptibility. Overall, our study reveals a sympathetic-epithelial axis that controls the severity of colitis and is modulated by myeloid-derived IL-1β and physiological stress. Our findings may also explain why inflammatory bowel disease in the elderly, where TET2 mutations in myeloid cells are common, is less severe and suggests TET2 activity as an attractive target for IBD.

Supported by the US National Institutes of Health (DK067180) to B.J, the University of Chicago’s Center for Interdisciplinary Study of Inflammatory Intestinal Disorders (C-IID) Pilot & Feasibility Award (NIDDK P30 DK042086) to A.M. and D.S, Crohn’s and Colitis Foundation Career Development Award #964209 to A.M. and G.I. Research Foundation Associates Board Award to A.M. and D.S.
Intestinal Barrier Repair in Giardiasis
Authors: Kosile Rita Taye , Angelova Vanessa V , Pannkuk Evan , Singer Steven M
Published: August 2023

Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia duodenalis. Acute symptoms can include diarrhea, but infections are often sub-clinical. It is transmitted via ingesting infectious cysts in contaminated food or water. In developing countries, giardiasis is one of the leading causes of growth stunting in children under two years old. Changes in intestinal barrier integrity have been shown to contribute to impaired nutrient absorption, thereby leading to growth stunting. Although intestinal barrier defects have been linked to infection, there is little understanding of barrier repair dynamics post-infection. Previous studies have demonstrated that Giardia infection is associated with dysbiosis in humans and animals. Because Aryl hydrocarbon receptor (AHR) signaling has been shown to promote intestinal repair in other systems, we are exploring the role of AHR in barrier repair following a Giardia infection. We quantified specific microbiome-derived AHR ligands in the plasma of infected C57BL/6 mice and observed a reduction of indole-3-ethanol and indole-3-pyruvic acid 21 days post-infection. Since IL-22 signaling can also promote barrier repair, we quantified IL-22 transcripts by RT-PCR and found significantly increased levels of IL-22 mRNA in infected mice. These data suggest that Giardia can reduce barrier repair through altered production of AHR ligands by the microbiome, but that IL-22 can counter this effect in some situations. This study will give better insight into developing effective dietary interventions to prevent growth stunting in infected children.

Supported by grant from NIH AI166467
Comparison of intrinsic allergenicity potentials of gliadins from durum and soft white wheats in an adjuvant-free mouse model
Authors: Jorgensen Rick Dean , Gao Haoran , Sundar Saanjh , Chandra Shivam , Raghunath Rajsri , Loy Jaden , Ng Perry , Gangur Venugopal
Published: August 2023

Wheat is one of the major allergenic foods capable of eliciting life-threatening anaphylaxis. Discovering potential hypo/hyper-allergenic wheat classes is imperative to advance our knowledge and manage wheat allergy. Durum wheat (genome AABB) and soft white wheat (genome AABBDD) are two commonly consumed wheats. However, whether there are differences in the intrinsic allergenicity potential between them is unknown. Here, we tested the hypothesis that gliadins extracted from durum and soft white wheats will differ in their intrinsic allergenicity in an adjuvant-free mouse model that uses skin sensitization followed by intraperitoneal (IP) allergen challenge. Balb/c female mice were produced and maintained on a plant protein-free diet during this study. Groups of mice (n=5–10/group) were exposed to gliadins or vehicle via the skin once a week for 9 weeks. IgE responses were analyzed by ELISA to determine sensitization. Systemic anaphylaxis upon IP challenge was quantified by hypothermic shock response (HSR); mucosal mast cell response (MMCR) was quantified by measuring MMCP-1 in the blood. Our results suggest that mice exhibited robust, but comparable levels of specific IgE antibody responses after skin exposures to both wheat gliadins elicited. HSRs induced upon IP challenges with gliadins from the two wheats displayed strikingly similar kinetics. Interestingly, soft white wheat gliadin was significantly more potent in eliciting MMCR compared to the durum wheat gliadin. We demonstrate for the first-time the similarities and differences in intrinsic allergenicity potentials of gliadins obtained from the two wheats.

Supported by grants from USDA/NIFA Hatch Project MICL02486 (Accession Number: 1012322)
A single species bacterial therapy reduces type 2 immune responses in the gut to prevent food allergy
Authors: Hesser Lauren A , Hubbell Jeffrey A , Nagler Cathryn
Published: August 2023

The rising prevalence of food allergy is strongly correlated with dysbiosis of the gut microbiome. Rescuing this dysbiosis by reintroducing specific bacteria is a promising method to prevent or treat food allergy. We have shown that monocolonization with Anaerostipes caccae, a species representative of the healthy infant microbiota, protects against an allergic response to the milk protein b-lactoglobulin (BLG). Recently we developed a synbiotic bacterial therapy (A. caccae plus prebiotic lactulose) which rescues the allergic phenotype of gnotobiotic mice colonized with the fecal bacteria of a dysbiotic, cow’s milk allergic (CMA) infant. Synbiotic treatment minimizes the anaphylactic response to intragastric BLG challenge and reduces circulating BLG-specific IgE and IgG1. To understand how a single bacterium regulates mucosal immunity during the onset of IgE-mediated allergy we first examined the alarmin response. CMA colonized mice express high transcript levels of both IL25 and TLSP in ileal epithelial cells after sensitization with BLG plus cholera toxin. The synbiotic abrogated this alarmin response, potentially via increased mucus production. Synbiotic treatment also reduced the proportion and number of T follicular (Tf) helper cells in the ileal-draining lymph node (LN) and increased Tf-regulatory cells in the colon-draining LN, suggesting a role in T/B cell regulation. Ongoing experiments are examining how the synbiotic affects localization and expansion of IgE producing cells in mucosal tissues including Peyer’s patches and mesenteric LNs. These findings will help to uncover the mechanisms by which specific, protective bacteria prevent food allergy and inform the development of effective live biotherapeutics.
Continued food allergen consumption exacerbates beta-amyloid accumulation in allergen-sensitized AppNL-G-F mice
Authors: Brishti Afrina , Kearney Angela E , Setness Rylan , Combs Colin K , Nagamoto-Combs Kumi
Published: August 2023

Alzheimer’s disease (AD) is the most common neurodegenerative disease, with β-amyloid (Aβ) plaque deposition being one of the hallmark pathologies. However, the etiology of AD remains elusive. While chronic inflammation from recurrent infections or injury seems to contribute to AD development, it is unclear whether atopic diseases, such as allergies, are associated with AD. We previously reported that continuous consumption of a whey protein (WP)-containing diet led to lasting neuroinflammation in C57BL/6J mice that were sensitized but tolerant to a bovine milk allergen, β-lactoglobulin (BLG; Bos d 5). Thus, we hypothesized that the persisting neuroinflammation due to repeated allergen consumption would exacerbate AD pathology over time in genetically predisposed, allergen-tolerant individuals. To address this hypothesis, we sensitized 1-month-old male and female transgenic AppNL-G-F knock-in mice to BLG and subsequently fed them either a whey-free control or a WP diet for 6 months. Despite their lack of overt allergic reactions, BLG-sensitized mice showed elevated plasma levels of BLG-specific IgE and IgG1. Leukocyte infiltration was observed in the hippocampus of sensitized mice, and WP-fed sham mice to a lesser extent. In contrast, mast cell accumulation was apparent in the dura of sensitized mice regardless of the diet. Importantly, Aβ plaque load and Aβ peptide levels were greater in the hippocampus of BLG-sensitized mice on the WP diet compared to the respective sham group. These results indicated that continuous allergen consumption exacerbated AD-like pathology in BLG-sensitized AppNL-G-F mice, suggesting that chronic food allergen exposure may promote the progression of AD in susceptible individuals.

Supported by the National Institute of Health/National Institute on Aging (NIH/NIA) under grant number R21AG070412
Role of CCR2 in Staphylococcus Enterotoxin B-induced Acute Respiratory Distress Syndrome
Authors: Wilson Kiesha Maria , Carter Taylor , Aladhami Ahmed , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: August 2023

Acute Respiratory Distress Syndrome (ARDS) is a fatal condition in which damage to the lungs results in fluid buildup and eventually hypoxia. Elucidating the immunological mechanism of this disorder is vital for reducing fatality. Staphylococcus enterotoxin B (SEB)-induced ARDS mimics the cytokine storm presented in patients with severe COVID-19. In previous studies, we found that SEB induced the infiltration of multiple CCR2+ macrophage subgroups and neutrophils in the lungs. Here, we investigated the role of CCR2 in the regulation of lung inflammation in SEB-induced ARDS, by utilizing CCR2-deficient mice. Mice were administered 50μg i.n. of SEB and evaluated for clinical distress and lung function over 72 hours. We performed flow cytometry and scRNA-seq on cells isolated from the whole lung of CCR2+/+ and CCR2−/−mice. We found a decrease in Ly6c+monocytes, macrophages, and CCR2+ macrophages in CCR2−/−mice, using flow and scRNA-seq analysis. Interestingly, alveolar macrophages were increased in the CCR2−/−mice. The CCR2−/−mice illustrated significant differences in their lung immune cell landscape. Mice exhibited increases in multiple T cell populations not observed in the wild-type mice. CCR2−/−mice expressed novel naïve T cells and a population of T cells which expressed genes associated with chromosome condensation (Hist1h2ap, Hist1h2ae, and Hist1h4d). Overall, top genes expressed in CCR2−/−mice such as Tcf7, Trbc2, and Stmn1, have been associated with T cell differentiation and tolerance induction. These results suggest that CCR2 deficiency prevents the migration of inflammatory monocytes to the lung but in return increase the presence of multiple unique T cell populations which alters the innate response in ARDS.

NIH P01AT003961, P20GM103641, R01AI129788, R01ES030144, R01AI123947, and K99GM147910
Discovery of a molecular clock that controls CD8+ T cell function and exhaustion
Authors: Mann Thomas Holladay , Ma Shixin , Furgiuele Jesse , Globig Anna-Maria , Chung Hokyung K , McDonald Bryan , Kaech Susan
Published: August 2023

During cancer and chronic viral infections, the persistence of antigen progressively causes CD8+ T cells to differentiate into a dysfunctional PD1+ “exhausted” state, with reduced production of inflammatory cytokines relative to effector cells that form during acute infections. Antigen and costimulation signals activate kinase cascades to induce distinct T cell transcription programs, but how T cells distinguish acute and chronic signals to program the exhausted or effector transcriptional states remains poorly understood. We found that members of the protein kinase C (PKC) family function together as a “molecular clock,” sensing acute or chronic agonism to drive distinct transcriptional programs. Continuous stimulation of PKC induces many features of T cell exhaustion, including a loss of production of the cytokines IFNγ and TNF, upregulation of inhibitory receptors and TOX, and altered expression of the proteins in the AP-1 family. Mechanistically, CD8+ T cells express several different PKC proteins, and chronic agonism of PKC leads to degradation of multiple family members and selective maintenance of only one PKC protein, PKC-η. This “PKC switch” alters downstream signaling to support the transcriptional reprogramming of T cells into a terminally exhausted state. In summary, continuous signaling through PKCs causes changes in the output from these kinases initially at the protein level, driving transcriptional changes downstream of PKC targets in the AP-1 transcription factor family and thus allowing further widespread transcriptional and functional changes that characterize T cell exhaustion.

Supported by a Damon Runyon Cancer Research Fellowship (DRG-2358-19) and an NIH training grant, T32-CA009370.
M. tuberculosis induced alterations in Siglecs’ expression on neutrophils predicts susceptibility to infection
Authors: Sankar Poornima , Saqib Mohd , Nafiz Tanvir Noor , Mishra Bibhuti B
Published: August 2023

The Presence of neutrophils in tuberculosis (TB) lung lesions is associated with bacterial growth. Despite being potent microbicidal cells, why neutrophils in TB lesions fail to control bacterial replication is poorly understood. By infecting mice with a hypervirulent replication reporter Mycobacterium tuberculosis (Mtb) W-Beijing Strain HN878, we found elevated neutrophils containing bacteria, associated with host susceptibility to infection. Phenotypic characterization of neutrophils from infected lungs revealed a downregulation of canonical neutrophil marker Siglec E (SigE) with concomitant upregulation of Siglec H (SigH), a marker generally used to define plasmacytoid dendritic cells. SigH expression on lung neutrophils increased with disease progression and predicts susceptibility to TB. Importantly, these non-conventional Ly6G+SigH+ neutrophils harbored more replicating bacteria compared to Ly6G+SigH-SigE+ lung neutrophils. In-vitro infection of naïve bone marrow neutrophils showed enhanced SigH expression with increased infectivity, and this was dependent on glycolysis and fatty acid metabolism. Treatment of immunocompetent mice with neutralizing antibodies for SigH reduced live cell bacterial burden, neutrophil infiltration, restricted bacterial replication and increased neutrophil expression of SigE. Further, SigH neutralized mice also showed elevated T cell numbers, indicating protective immunity. Collectively, our data suggests a model where Mtb induces SigH expression on neutrophils to survive within them, as a potential immune evasion strategy. Our future studies will determine the functional significance of this alteration in Siglecs on neutrophils and its role in TB pathogenesis.

R56 AI148239-01A1 and R21AI16359 (sub-award) from NIH/NIAID toDr. Bibhuti B Mishra
Allergen inhalation transforms the lungs into a permissive environment for local IgE responses
Authors: Nelson Alexander J , Wu Yee Ling
Published: August 2023

Immunoglobulin E (IgE) is the primary cause of allergic diseases and must be tightly regulated. Production of IgE occurs when activated B cells undergo class switch recombination (CSR) to IgE. These key events are rare, thus questions on which B cell subsets undergo IgE CSR, where IgE CSR predominantly occurs, and which molecular and cellular signals promote IgE CSR remain unresolved.

We modeled allergic asthma by intranasal administration of ragweed pollen and ovalbumin (OVA) in mice, and found that allergen inhalation induced the formation of lymphoid aggregates and production of OVA-specific IgE in the lungs. Using fluorescent mice reporting IgE switching, we revealed that allergen-sensitized lungs were a major site of IgE CSR with IgG1+ memory B cells (MBCs) being the dominant IgE-switching cells. Single-cell transcriptomics and flow cytometry analyses revealed a tight correlation between CCR6 expression and lung IgG1+ MBCs, potentially explaining their recruitment/retention in the respiratory mucosa. Furthermore, lung MBCs underwent IgE CSR more frequently than MBCs in the draining lymph nodes or spleen. Since IgE CSR requires interleukin 4 (IL-4), we used IL-4 reporter mice and found that IL-4-producing TH2 cells were enriched in the lungs compared with lymphoid organs. Co-culture experiments using purified lung MBCs from IgE reporter mice with lung or splenic effector CD4+ T cells revealed that IgE CSR was elevated in the presence of lung T cells, suggesting lung-infiltrating TH2 cells drive elevated IgE CSR in the lungs.

This study clarifies that the respiratory mucosa is a key site of IgE CSR – in allergic asthma the lung environment supports IgE CSR with IgG1+ MBCs and CD4+ T cells collaborating to promote local IgE responses.

Supported by grants from NIH (F31 HL156459, T32 AI007508, R01 HL165120, R21 AI159456)
Modulation of mucosal immunity and tissue resident memory formation by NS1-deficient influenza A virus
Authors: Tam Rachel C Y , Liao Yingyin , Mok Bobo Wing-yee , Chen Honglin
Published: August 2023

Recurrent influenza A virus (IAV) epidemics and occasional pandemics as a result of viral genome reassortment and human adaptation pose significant health burdens globally. The non-structural protein 1 (NS1) mediates attenuation of host response and facilitates viral replication in the nucleus. While intranasal vaccination using NS1-deleted live attenuated virus has been shown to induce pulmonary mucosal immunity in animal models, the immunological details have not been fully studied. In vivo mouse infection using wild type (WSN/WT) and NS1-deleted WSN (WSN/Del) showed WSN/Del induced massive type I interferon response in murine lungs, whereas pulmonary inflammation was minimal. Skewed migration of CD103+ dendritic cells (DC) to mediastinal lymph nodes upon WSN/Del infection was observed with enhanced formation and persistence of tissue resident influenza-specific memory CD8 T cells (TRM). After rechallenge, while influenza-specific TRM in both groups expanded comparably, more polyfunctionality was seen in TRM from WSN/Del immunized mice than WSN/WT recovered mice. Single cell RNA-seq of migratory DC in initial immunization showed differential gene programs associated with WSN/Del infection. Ongoing work is undertaken to identify the signals provided by DC that may lead to preferential priming of T cells to tissue resident lineage. Since TRM have been illustrated to confer heterosubtypic protection, knowledge on the specific physiological cues that facilitate TRM formation and retention will be useful to vaccine development.
CD8+ TRM-derived IFNγ is a critical activator of human lung epithelial cells
Authors: Mattingly Cameron , Elliott Jennifer , Thomas Jeronay , Lobby Jenna , Michalets Sarah , Kost Kirsten N , Kohlmeier Jacob E
Published: August 2023

Due to their position in the pulmonary mucosa, CD8+ tissue resident memory T cells (TRM) act as sentinels that rapidly respond to, and mediate protection against, respiratory viruses. In mice, TRM have been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. However, in humans, less is known about the effector functions of virus-specific lung CD8+ TRM. Using cells from healthy human lung donors, we first identified and quantified the frequency of antigen-specific cells against common respiratory viruses by performing intracellular cytokine staining. We next investigated the polyfunctionality and residency profiles of responding antigen-specific CD8+ T cells and found that that CD69+ CD103+ TRM comprise a larger portion of the antigen-specific CD8+ T cells for viruses largely restricted to the respiratory epithelium when compared to cells specific for viruses with a broader tissue tropism. Next, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated how antigen-specific CD8+ T cells impact local innate and epithelial cells. When stimulated with their cognate antigen, lung CD8+TRM-derived IFNγ strongly correlated with epithelial cell activation. Finally, we stimulated lung CD8+ TRMwith cognate antigen +/− anti-IFNγ and performed RNAseq on responding antigen-specific CD8+ TRM (CD25+CD137+) and epithelial cells to determine exactly how TRM-derived IFNγ alters the cellular program of these cells. Results of this study suggest that human lung CD8+ TRM act to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8+ TRM fit into the overall immune response to respiratory viruses.

This project is supported by R35 HL150803 and the Emory Center of Excellence for Influenza Research and Response (CEIRR) 75N93019R0028.
γδ T cells mediate protective immune responses following an insect virus-based Chikungunya virus chimera vaccine
Authors: Rodriguez Leslie , Adam Awadalkareem , Luo Huanle , Osman Samantha R , Weaver Scott C , Wang Tian
Published: August 2023

Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes acute chikungunya fever (CHIKF) which often is accompanied by severe and persistent arthralgia. There are currently no licensed human vaccines against CHIKV. Eilat virus (EILV), a mosquito-specific alphavirus, replicates well in mosquito cells, but not in vertebrate cells. EILV/CHIKV is a chimeric CHIKV vaccine candidate that contains the nonstructural proteins of EILV and the structural proteins of CHIKV. This chimera vaccine candidate has been shown to protect against wild-type (WT) CHIKV challenge in mice and non-human primates (NHPs), yet the immune mechanism of the vaccine-induced host protection remains unknown. γδ T cells are known to react to WT CHIKV infection by controlling CHIKV inflammation, cell infiltration, and tissue damage. We found that γδ T cells are expanded following vaccination with EILV/CHIKV. TCRδ−/− mice, which are deficient of γδ T cells, exhibited a reduced CD8+ T cell effector function response at different timepoints compared to WT mice. This reduced CD8+ T cell response was also observed 7 days post WT CHIKV infection in the vaccinated mice. A significant reduction of CHIKV-specific IgM titers was also observed 7 days post WT CHIKV challenge in the vaccinated mice. Lastly, we noted that the vaccinated TCRδ−/− mice had higher levels of viremia than vaccinate WT mice following challenge with WT CHIKV. Overall, our results suggest that γδ T cells contribute to the host protection by promoting vaccine induced CD8+ T cell and antibody responses against CHIKV infection.
B cells shape naive CD8 T cell programming and fate decisions
Authors: Klarquist Jared , Guerrero Miguel Moreno , Manes Cameron , Cimons Jennifer , Getahun Andrew , Kedl Ross
Published: August 2023

Renewed interest in the relationship between B cells and CD8 T cells has revealed direct interactions between these cells, despite long-prevailing views that they function independently from one another. In fact, an IL-21-producing “helper” CD8 T cell subset has been described; yet, whether B cells have the reciprocal capacity to support CD8 T cells is less well-studied. Here, we find B cells influence the phenotype of naive, effector, and memory CD8 T cells. In MD4 mice, which have a fixed BCR repertoire, naive CD8 T cells exhibit an activated/effector phenotype, including relatively high pAkt, p70-s6K, and 4e-bp1, and low Foxo1 and Eomes. Primary CD8 T cell responses to subunit vaccination in MD4 or B cell-deficient mice are numerically reduced and effector-skewed. Importantly, the magnitude and function of memory CD8 T cells 3 months after vaccination are impaired in MD4 mice, leading to increased susceptibility to bacterial challenge. Adoptive transfer of CD8 T cells from WT into MD4 mice results in a similar effector-skewing upon vaccination, demonstrating that these phenotypic changes result from T cell-extrinsic factors. Furthermore, CAR T cell-mediated B cell depletion in 6-week-old WT mice also recapitulated the reductions in primary CD8 T cell numbers and their effector-skewing after vaccination. In agreement with prior literature, peak T cell responses after Listeria m. infection are of equivalent magnitude in WT and B cell-deficient mice, yet here we also observe reductions in their CD127+ memory phenotype. Together, these data indicate that B cells perform an important, broad role in the establishment of the naive CD8 T cell pool and subsequent regulation of CD8 T cell programming following subunit vaccination or infection.
Canine osteosarcoma as a solid tumor model for evaluation of dual valent B7H3-CXCR2 CAR T cell therapy
Authors: Cao Jennifer Wang , Lake Jessica , Chow Lyndah , Ammons Dylan , Perez Luisanny , Impastato Renata , Kurihara Jade , Verneris Michael , Dow Steven
Published: August 2023

To develop more effective solid tumor CAR T cell therapies, we utilized the spontaneous canine metastatic osteosarcoma (OS) animal model. Our studies evaluated the relative activity of single B7H3 and dual-valent B7H3-CXCR2 CAR constructs. Studies were also designed to determine if modifying the tumor microenvironment (TME) with oral immunotherapy improved CAR activity, based on recent studies of losartan and propranolol in dogs with OS and glioma. We evaluated B7H3 expression on canine OS cell lines using flow cytometry and OS biopsies using IHC. High levels of B7-H3 were observed on OS cell lines and OS biopsies, whereas normal canine tissues were negative or low. To assess the utility of the dual CAR we determined that canine OS cell lines secreted high IL8 spontaneously. To assess the functionality of canine B7-H3 CAR T cells, we generated canine CAR T cells from whole blood from dogs with cancer. We next determined that canine dual CAR T cells exerted specific killing and cytokine activity against B7H3+ canine OS cell lines. To further establish functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the dual CAR construct was significantly more active than the single CAR construct in slowing tumor growth. We found that losartan and propranolol blocked migration of mouse inflammatory monocytes to xenografted canine OS tumors. Future steps will be to evaluate the effectiveness of dual CAR T cells with TME modification in dogs with metastatic OS, to investigate the translational relevance in human patients. Results from these studies in dogs will likely give more clinically relevant results for evaluating new CAR T cell therapies and bridging results between mouse models and human clinical trials.

The V Foundation, The Shipley Family Foundation, and NIH T32 training grant
TLR2 dependent enhancement of bacterial clearance in macrophages via TNFR2
Authors: Luu Abby , Hatton Alexis , Shepardson Kelly , Benson Evelyn , Hall Monica , Rynda-Apple Agnieszka
Published: August 2023

The public health crisis of antimicrobial resistance is especially challenging due to the rapid pace of bacterial adaptation to antibiotics. Thus, development of alternatives to antibiotic-based therapies are crucial. We found that stimulation of murine macrophage TLR2/6 heterodimer with Pam2CSK4 (PAM2), a synthetic bacterial lipoprotein, enhanced clearance of subsequent Staphylococcus aureus infection. Whole lung RNA sequencing identified tumor necrosis factor receptor superfamily member 1B (TNFRSF1B, TNFR2) and its cognate cytokine, TNFα, to be upregulated in response to PAM2. ELISA and qPCR data showed both bioactive forms of TNFα, transmembrane (tmTNFα) and soluble (sTNFα), to be upregulated. To mechanistically address the role that TNFR2 could play in PAM2 induced enhanced bacterial clearance (EBC) ability, antibody blocking of TNFR2 led to reduced macrophage S. aureus clearance. In contrast, sTNFα reduction by small molecule inhibition of ADAM17, the enzyme that cleaves tmTNFα to sTNFα, did not reduce macrophage EBC ability. These data suggest tmTNFα signaling via TNFR2 may have a protective effect which enhances S. aureus clearance. Our work aims to elucidate TLR signaling events through which enhancement of existing host bacterial resolution mechanisms can be achieved. As such, we are investigating whether/how tmTNFα:TNFR2 initiates intracellular signaling pathways that may contribute to upregulation of macrophage mechanisms to enhance pathogen clearance. An in-depth understanding of these signaling events could facilitate development of novel treatment strategies designed to modulate and enhance host immune responses against pathogens, which could be leveraged to circumvent antimicrobial resistance.
Metabolic changes induced by complete Freund’s adjuvant resemble the features of trained immunity
Authors: Mone Kiruthiga , Seravalli Javier , Reddy Jay
Published: August 2023

Using the mouse model of viral myocarditis, we recently reported that animals immunized with complete Freund’s adjuvant (CFA) conferred complete protection against Coxsackievirus B3 infection. Because that Bacillus Calmette-Guérin (BCG) is a well-known inducer of trained immunity and both CFA and BCG possess similar mycobacterial components, we sought to test the hypothesis that the trained immunity might be the underlying mechanism of CFA-mediated protection against CVB3. By using incomplete Freund’s adjuvant (IFA) as a negative control, we investigated the metabolic profiles by mass spectrometry in bone marrow-derived monocytes and CD11b+ cells sorted from splenocytes using LPS as a secondary stimulus. The analysis revealed elevated levels of lactate, α-ketoglutarate, fumarate, malate, and oxaloacetate in the CFA group as compared with the control or IFA groups. In contrast, S-adenosyl methionine and homoserine levels were high in the IFA group. Overall, our data suggest that the metabolites produced in response to CFA were uniquely different from those of IFA. Whether these profiles correlate with the epigenetic changes are currently being investigated.

Supported by grants from NIH (R21, A116673)
TLR4-Independent Mechanisms of Complement Activation During Endotoxemia
Authors: Falguni Mehnaz Mustari , Hunat Junela Cecile , Nunez Kathryn Elizabeth , Ngo Vy , Harberts Erin M
Published: August 2023

Almost two million American adults are diagnosed with sepsis annually of which 350,000 succumb to the infection. Toll-like-receptor 4 (TLR4) canonically recognizes lipopolysaccharide (LPS), an outer membrane structure of Gram-negative bacteria. This recognition leads to pro-inflammatory cytokine release and propagation of other immune compartments. Complement is an acellular innate immune recognition system and has three general categories for activation: classical, lectin and alternative pathway. Previous research has found that complement proteins are dysregulated independent of TLR4 in serum during murine endotoxemia. This study investigates mechanisms of complement activation by describing location of complement gene upregulation and protein distribution in WT and TLR4−/− mice. mRNA was extracted from murine organ samples collected 0, 6, 12, and 18 hours post-induction of endotoxemia. RT-qPCR is then performed to measure expression fold changes of complement genes F5, F13a1, C1qb, Cfd, Masp1, and Mbl1. Genes involved in the lectin pathway were observed to be upregulated at the 6-hour timepoint and then downregulated throughout the progression of disease. C3 is a complement protein that is cleaved to create C3a, an anaphylatoxin, contributing to activation of acute phase inflammatory responses. Levels of C3a are analyzed using Western blots from WT and TLR4−/− mice as a marker of activation. The effects of LPS chemical structure on the efficiency of C3 cleavage is also analyzed. Identification of TLR4-independent pathways will allow for more targeted immune therapies for adverse outcomes related to sepsis.

Supported by National Institute of Health and Towson University Bridges to Doctorate Program NIH (1T32GM146694-01), Fisher College of Science and Math at Towson University, and an FY2022 grant from the Towson University Faculty Development and Research Committee (FDRC) awarded to Erin Harberts titled “Septic Shock: Defining Alternative Pathways of Activation”.