bluuebunny/crossref_metadata_2025 huggingface dataset:
CD3ζ adaptor structure determines functional differences between human and mouse CD16 Fc-gamma receptor signaling in natural killer cells
Authors: Aguilar Oscar A , Fong Lam-Kiu , Ishiyama Kenichi , DeGrado William F , Lanier Lewis L
Published: January 2023

Natural killer (NK) cells are innate lymphocytes capable of distinguishing between healthy and pathogenic cells using a myriad of receptors expressed on their cell surface. The CD16 receptor detects the Fc portion of IgG on antibody-coated cells resulting in NK cell activation. The importance of this receptor on human NK cell biology has long been appreciated; however, how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that key residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function.

Supported by grants from the Cancer Research Institute (CRI), Burroughs Wellcome Fund (BWF), the Parker Institute for Cancer Immunotherapy (PICI) and NIH grants AI068129 and AI146581.
LncRNA U90926 is induced in activated macrophages, encodes a novel secreted protein, and is protective in endotoxic shock
Authors: Krementsov Dimitry N , Sabikunnahar Bristy , Caldwell Sydney , Varnum Stella
Published: January 2023

Thousands of long non-coding RNAs (lncRNAs) are encoded in mammalian genomes, yet the majority of them remain uncharacterized. In previous studies, we identified a mouse lncRNA of unknown function called U90926, expressed in activated macrophages. Here, we functionally characterized this gene in vitro and in vivo. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene is highly induced in myeloid cells by toll-like receptor activation, in a p38 MAP kinase and MYD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency on gene expression in cultured macrophages. Given this lack of a macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel protein (termed U9-ORF). U9-ORF contains an ER signal peptide sequence, traffics to the Golgi, and is secreted from the cell. In vivo, the LPS endotoxemia model showed that U9-KO mice exhibited increased sickness responses and mortality in comparison with WT mice. Mechanistically, serum levels of IL-6 were elevated at in U9-KO mice compared with WT, and IL-6 neutralization improved sepsis outcome in U9-KO mice. Taken together, these results suggest that the protective effect of U90926 expression during endotoxic shock is orchestrated by regulating IL-6 levels, likely mediated by the paracrine and/or endocrine actions of the novel U9-ORF peptide secreted by activated myeloid cells.

Supported by grants from the NIH (R21AI151116 to DNK and P30GM118228 to Dr. Ralph Budd).
Glutamine addiction in NKT cells is regulated by AMPK-mTORC1 axis
Authors: Kumar Ajay , Yarosz Emily Lynn , Andren Anthony , Zhang Li , Lyssiotis Costas , Chang Cheong-Hee
Published: January 2023

Cellular metabolism is essential in dictating conventional T cell development and function, but its role in natural killer T (NKT) cells has not been well studied. We have previously shown that NKT cells operate distinctly different metabolic programming from CD4 T cells, including a strict requirement for glutamine metabolism to regulate NKT cell homeostasis. However, the mechanisms by which NKT cells regulate glutamine metabolism for their homeostasis and effector functions remain unknown. In this study, we report that steady state NKT cells have higher glutamine levels than CD4 T cells and NKT cells increase glutaminolysis upon activation. Among its many metabolic fates, NKT cells use glutamine to fuel the tricarboxylic acid cycle and glutathione synthesis, and glutamine-derived nitrogen enables protein glycosylation via the hexosamine biosynthesis pathway (HBP). Each of these functions of glutamine metabolism was found to be critical for NKT cell survival and proliferation. Furthermore, we demonstrate that glutaminolysis and the HBP differentially regulate IL-4 and IFNg production. Finally, glutamine metabolism appears to be controlled by AMP-activated protein kinase (AMPK)-mTORC1 signaling. These findings highlight a unique metabolic requirement of NKT cells which can be potentially serve as an effective immunotherapeutic agent against certain nutrient restricted diseases.

This work was supported in part by National Institutes of Health Grants R01 AI121156 and R01 AI148289 (to C-H.C.).
Influence of estradiol deficiency on the acute inflammatory response following a traumatic skeletal muscle injury
Authors: McMillin Shawna L , Noel Richard , O’Farrell Laura , Warren Gordon , Lowe Dawn
Published: January 2023

Estradiol (E2) influences the inflammatory response. Research has shown that skeletal muscle chemokine mRNA expression and neutrophil infiltration after a traumatic freeze injury are blunted in ovariectomized mice treated with placebo compared to E2-treated mice. How E2 deficiency alters the acute muscle inflammatory response is unclear with potential mechanisms including alterations in the number of circulating leukocytes, leukocyte chemotaxis, and/or muscle chemokine expression. To investigate these possibilities, female C57BL/6J mice underwent a sham or ovariectomy (OVX) surgery, 21–28 d later tibialis anterior (TA) muscles were freeze injured. TA muscles and blood were collected 24 h post injury. Total white blood cell (149%) and monocytes (427%) were elevated in OVX compared to Sham mice but there was no difference in the neutrophil counts (p≥0.64). To determine if E2 deficiency impacts leukocyte chemotaxis, isolated leukocytes from bone marrow of non-injured Sham or OVX mice were placed in a transwell migration assay. OVX leukocytes had greater chemotaxis towards CXCL1 (37%), CXCL2 (43%), and CXCL5 (27%) than Sham. To examine if E2 deficiency alters muscle CXCL1 and CXCL5 protein content contributing to chemotaxis, ELISA assays were performed. Surprisingly, muscle CXCL1 and CXCL5 contents were similar between OVX and Sham. Collectively, mice lacking E2 have relatively high blood leukocytes and chemotaxis suggesting a greater acute inflammatory response which appears not to be driven by changes in muscle chemokine expression. Additional studies need to consider possible negative regulators of the acute inflammatory response in OVX muscle.

Supported by DAL and GW (R01AG031743 & R01AG062899), and SLM (T32AR007612)
Brain resident memory CD8+ T cells trigger CNS immune activation and infiltration
Authors: Musial Shawn C , Searles Tyler G , Kleist Sierra A , Degefu Hanna N , Isaacs Jordan F , Skorput Alexander G , Rosato Pamela C
Published: January 2023

The shifting paradigm regarding immune surveillance of the central nervous system (CNS) has shed light on immune cell networks thought to be absent from healthy brain tissue. Resident memory T cells (TRM) are a unique subset of memory T cells that persist within non-lymphoid tissues to provide rapid onset protection against reinfection. Recently, brain TRM (bTRM) have been identified in humans, with CNS or peripheral infection, or vaccination leading to bTRM establishment in mice. While bTRM are important for protection of the CNS against reinfection, their regulation and involvement in shaping the neuroimmune landscape remains unknown. Here, we take a reductionist approach employing viral-derived peptides to show that CD8+ bTRM reactivation is sufficient to initiate a cascade of innate and adaptive immune activation in the brain. Specifically, bTRM reactivation triggered activation of NK cells, T cells, microglia, and induced dendritic cell (DC) maturation, including upregulation of lymph node homing molecules. Reactivated bTRM also promoted accumulation of DCs in draining lymph nodes, and macrophages, monocyte-derived DCs, T cells and NK cells in the brain. Our preliminary data suggests that PD-1:PD-L1 signaling regulates the magnitude of this response, as PD-L1 blockade led to enhanced bTRM activation and neuroinflammation. We anticipate our results to illuminate roles for PD-1 signaling on bTRM in the context of pathogen clearance, and neurologic toxicities seen in cancer patients following PD-1 inhibitor treatment. This study will also provide insight into the pathologic or protective capacity of bTRM in neurologic diseases where T cells are implicated, such as Alzheimer’s disease, multiple sclerosis, and brain cancer.

Supported by grants from NIH (K22 AI148508-02, T32 AI007363)
Role of NF-kappaB c-Rel O-GlcNAcylation in the regulation of cytotoxic T cell function
Authors: Ramakrishnan Parameswaran , Centore Joshua T
Published: January 2023

Autoimmunity in type 1 diabetes (T1D) is primarily caused by destruction of insulin producing pancreatic beta cells by cytotoxic T cells, resulting in hyperglycemia. Adverse pathological effects of hyperglycemia include the increased O-GlcNAcylation of cellular proteins. We found that the transcription factor nuclear factor kappaB (NF-kB) subunit c-Rel is a target of O-GlcNAcylation at serine residue 350 in T cells. c-Rel is the major regulator of CD4 and CD8 T cell function and T regulatory cell development that controls autoimmunity and immunosuppression, respectively. c-Rel O GlcNAcylation increases the expression of pro-autoimmune cytokines and decreases the expression of the transcription factor forkhead box P3 (FOXP3) in CD4 T cells. However, the role of c-Rel O-GlcNAcylation in cytotoxic T cells remains unknown. The cell death pathways mediated by Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) are major contributors of T cell mediated cytotoxicity. Our preliminary microarray study showed that FasL and TRAIL expressions are dependent on O-GlcNAcylated c-Rel. We also found that hyperglycemia and c-Rel O-GlcNAcylation enhances FasL expression in both CD4 and CD8 T cells. Mechanistically, O-GlcNAcylation enhances and a point mutation at S350 O-GlcNAcylation site prevents c-Rel binding to FasL promoter. Our future studies aim at studying how hyperglycemia-induced c-Rel O-GlcNAcylation enhances cytotoxic ligand expressions in T cells, augmenting their potential to destruct host cells in T1D. This study will define the critical role of c-Rel O-GlcNAcylation in cytotoxic T cell function, revealing a new molecular mechanism that could be targeted to control autoreactive T cell function in T1D.

P.R was supported by National Institute of Allergy and Infectious Diseases at National Institute of Health grants R01 AI116730 and R21 AI144264, and National Cancer Institute at National Institute of Health grant R21 CA246194. JTC was supported by National Institute of Health NIH/NEI T32 pre-doctoral training grant T32EY007157.
A critical role for the RNA binding protein HuR in CD4+ T cell-mediated airway inflammation in mouse and human
Authors: Fattahi Fatemeh , Ellis Jason S , Sylvester Michael , Bahleda Kristin , Lugogo Njira L , Atasoy Ulus
Published: January 2023

The RNA-binding protein HuR (elavl1) positively regulates the expression of its targets by increasing mRNA stability and translation. Th2 and Th17 related genes play critical roles in airway inflammation in both mouse and human. Using in vitro and in vivo studies, we have previously shown that these proinflammatory genes are regulated by HuR. Here, we generated a conditional ablation of HuR in T cells (distal Lck-cre HuRfl/fl). We found significant decreases in Th2 differentiation, cytokine production, and lung inflammation in an alum-emulsified ovalbumin (ova-alum) model of allergic airway inflammation using this novel mouse model. We hypothesized that HuR might similarly regulate lung inflammation in human asthma. Using peripheral CD4+ T cells from 45 asthmatic patients, we found the inhibitory effect of the HuR-specific inhibitor CMLD-2, as well as an AMPK activator (acadesine aka AICAR), on both Th2/Th17 signature cytokines in both protein and mRNA levels. Our asthmatics were classified into type 2 high or non-type 2 high based on their blood eosinophil and FeNO levels. Interestingly, there were significant positive correlations between Th2 cytokine levels in CD4+ cells with levels of eosinophils and FeNO from asthmatics. Surprisingly, IFNγ (protein and mRNA) were also markedly decreased by HuR inhibition. Our bioinformatic studies also revealed proteins with high interaction with HuR including MAPK14/p38α and PTEN, common in the pathways with IFNγ, are affected by HuR inhibitor. Taken together, our mouse and human data suggest that HuR plays a crucial permissive role in both allergen- and nonallergen-driven airway inflammation by regulating key genes and that interfering with its function may be a novel way to treat asthma.

Supported by R01: AI080870, R21: AI079341
QRICH1 is a CARD11 interactor that negatively regulates T cell activation
Authors: Carter Nicole M , Pomerantz Joel L
Published: January 2023

Precise regulation of lymphocyte activation is crucial for fighting off pathogens while preventing autoimmunity and lymphoma. CARD11 is a scaffold protein and signaling hub that links antigen receptor engagement to activation of the NF-κB, JNK, and mTOR pathways. We identified QRICH1 as a previously unrecognized binding partner of CARD11 that binds CARD11 in a signal-dependent manner during T cell receptor (TCR) signaling. To identify the function of QRICH1 in TCR signaling, we used CRISPR/Cas9 genomic editing to disrupt QRICH1 expression in Jurkat T cells. QRICH1 knockout resulted in enhanced TCR-induced NF-κB activation and IL-2 secretion, while QRICH1 overexpression in HEK293T and Jurkat T cells dose-dependently inhibited NF-κB activation by wild-type CARD11 and by constitutively active and oncogenic CARD11 mutants. Domain deletion experiments revealed that the inhibitory activity of QRICH1 resides in the N-terminal half of the protein, while the C-terminus contains an autoinhibitory domain that participates in intramolecular interactions and modulates the ability of QRICH1 to inhibit CARD11 signaling to NF-κB. To study the function of QRICH1 in vivo, we generated mice with a conditional QRICH1 allele. We found that QRICH1-deficient CD4+ and CD8+ T cells produced higher levels of IL-2 and IFN-ɣ, respectively, following stimulation by anti-CD3/CD28. Our findings identify QRICH1 as a negative regulator of T cell activation that modulates the signaling output of CARD11.

This work was supported by NIH grants F31CA254167, RO1AI148143, and T32GM007445.
Malnutrition Disrupts T Cell Migration
Authors: Dadzie Kwesi A , Foster Takesha R , Mister Abigail , Goulmamine Syreen , Gibson David , Adams Olivia , Gubbels-Bupp Melanie R
Published: January 2023

Malnutrition is associated with reductions in the number and function of T lymphocytes. Previous studies in the lab suggest that malnutrition may also impart a “super-quiescent” phenotype to T cells, perhaps affecting the efficiency of their migration within and between lymph nodes. Thus, the purpose of this study is to evaluate the effect of malnutrition on T cell migration in vivo and to characterize malnutrition-induced changes in the expression of proteins known to be important for T cell migration. To determine if malnourishment alters T cell migration in vivo, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. In agreement with other studies, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+ T cells entered the lymph nodes at equivalent rates, suggesting that malnourishment eliminates distinct lymph node entry efficiencies for CD8+ and CD4+ T cells. We also found important differences in the expression of key proteins involved in T cell migration between malnourished and control mice. Overall, we found that malnutrition disrupts T cell migration including the distinct migration efficiencies of CD4+ and CD8+ T cells. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration and shed light on how organisms adapt to malnutrition.

Supported by NSF-MRI [DBI- 1920116] NSF-RUI [IOS-1951881]
IL-4 Signaling Regulates the Fate of B Cell Differentiation and limits BCR repertoire
Authors: Oviedo Juan Marcos , Gibbs Lisa C , James Kimra , Fairfax Keke C
Published: January 2023

Il-4 has a critical role in organizing the peripheral lymph nodes in homeostasis and after vaccination. To further understand the molecular mechanisms and their impact in the immune response, we used single cell RNA-Seq of CD45+ cells in 4get and 4getIL4R−/− mice following tetanus/diphtheria immunization. We found that the lack of IL-4 signaling restrains the expression of key co-stimulatory and differentiation molecules CD83, CD86, and xbp1, while upregulating the jun/junb/fos pathways. These alterations result in reduced B cell proliferation and differentiation into plasma cells. Surprisingly, we found that in the absence of IL-4 signaling the ratio of dark to light zone residing germinal center shifts from the normal ratio to one dominated by the dark zone. The increase in Dark Zone germinal centers lead to an increase in the diversity of the repertoire in vaccinated 4getIL4R−/− mice as confirmed by VDJ-seq. We also observed that the upregulation of the transcription factor T-bet has the capacity of inducing a similar phenotype. Finally, even though the amount of memory B cells was increased in the absence of IL-4 signaling, we observed that these cells are not able to respond to a secondary challenge. These data suggest a pivotal role for IL-4 in governing B-cell differentiation and the development of optimal cellular and humoral immunity to vaccination.

supported by grants from NIH R01 AI135045
Lineage specification of killer versus helper Innate lymphoid cells
Authors: Ding Yi , Lavaert Marieke , Harly Christelle , Das Arundhoti , Bhandoola Avinash
Published: January 2023

Helper innate lymphoid cells (ILCs) combat pathogen infection by rapid secretion of cytokines whereas killer ILCs (NK cells) eliminate virus-infected cells or tumor cells during early defense. However, the early development of NK cells and lineage relationships between NK cells and helper ILCs remain elusive. In PLZF lineage tracing model, normal NK cells are traced at a much lower ratio than helper ILCs, which indicates most NK cells develop from progenitors that do not express PLZF. However, it is also possible that most NK cells develop from PLZF-expressing ILC progenitors (ILCPs), but PLZF-labelled NK cells may proliferate less than unlabeled NK cells. To address these possibilities, we transferred ILCPs and its upstream progenitors early innate lymphoid progenitors (EILPs) and all lymphoid progenitors (ALPs), and then assessed PLZF labelling in NK cells derived from these progenitors. We found that ILCP-derived NK cells were traced at a ratio as high as in helper ILCs whereas EILP and ALP-derived NK cells were labelled at a much lower ratio, which is similar to the ratio in normal NK cells. These results indicate that NK cells develop from both ILCP-dependent and ILCP-independent pathways. Our findings establish novel lineage relationships between NK cells and helper ILCs. We are currently assessing whether different developmental pathways of NK cells lead to functionally distinct subsets of NK cells.
Sympathetic nervous system-mediated β-adrenergic signaling maintains the pool of mature natural killer cells
Authors: Geyer Rory , Moussa Marmar , Mandoiu Ion , Srivastava Pramod K , Nevin James
Published: January 2023

The sympathetic nervous system (SNS) innervates all lymphoid tissues and is, thus, well-positioned to influence the activity of immune cells. Accordingly, the coordinated responses of the SNS and immune system are crucial for immune responses to infection and cancer. Natural killer (NK) cells are essential for the immune response to certain viral infections and cancers in which they kill infected or cancerous cells and produce cytokines and chemokines to instruct the activity of other cells contributing to the response. We find here that steady state SNS signaling maintains the balance of mature and immature NK cells in the spleen and bone marrow. Using 6-hydroxydopamine to ablate sympathetic nerve terminals, we show by single cell RNA-sequencing and flow cytometry that SNS-ablated mice have decreased frequencies of mature CD27− CD11b+ NK cells and increased frequencies of immature CD27+ CD11b− and intermediate CD27+ CD11b+ NK cells. An increased ratio of immature NK cells to mature NK cells has been linked to decreased anti-tumor immunity. Along these lines, SNS-ablation also increases the growth rate of RMA-S tumors, the rejection of which depends on NK cells. This accelerated tumor growth was dependent on the absence of both α- and β-adrenergic signaling. Culture of early NK cell precursors in the presence of selective α- or β-adrenergic agonists suggests that signaling through β-adrenergic receptors promotes the maturation of NK cells. These data suggest that signaling through β-adrenergic receptors mediated by norepinephrine produced by the SNS maintains the pool of mature NK cells, allowing them to effectively survey peripheral tissues and participate in anti-tumor immunity.
Exposure to diverse microbial flora accelerates the development of infant immunity
Authors: Schuldt Nathan J , Burger Sarah , Stenger Terran , Pierson Mark , Huggins Matt
Published: January 2023

The newborn immune system is considered underdeveloped and ill-equipped to defend itself against many pathogens. Indeed, infection is the leading cause of hospitalization and death for infants under 6 months. Relative to the adult immune system, little is known about infant immunity or the signals that orchestrate immunologic maturation. The constraints of human research and the use of sterile animal models limit advancements in this area. Commonly used animal models are raised in “specific pathogen free” (SPF) conditions to minimize the confounding effects of diverse microbial exposure. We sought to understand the physiologic effects of exposure to those diverse microbes on infant immune development. We co-housed laboratory mice with pet store mice from conception to expose them to diverse microbial flora. Microbial exposure led to a dramatic shift in immune cell composition with increases in the proportion of T cell memory, helper T cell subsets, macrophages, and neutrophils, as well as general increases in the number of most immune cell subsets as early as 2 weeks of age. The gap between the microbially-exposed mice and SPF mice continued to widen at 3 and 6 weeks of age. We also observed increased TNFα, IL-6, IL-5, IL-17, and RANTES expression at 3 weeks of age. By 6 weeks most of the cytokines/chemokines tested where increased. These observations demonstrate that exposure to diverse microbial flora during early life dramatically alters the trajectory of the developing immune system. A better understanding of how healthy immune systems develop, and the risks and benefits of modulating infant immunity could lead to important advancements in treatments for early life infection and novel infant vaccination approaches.

Supported by Grants from: Medical School/University of Minnesota Foundation Award Program Masonic Cross-Departmental Grants in Children’s Health Research University of Minnesota Department of Pediatrics “R” Award
Neonatal Immunity to Malaria using a mouse model
Authors: Opata Michael M , Gbedande Komi , Smith Margaret Rose , Onjiko Robert Sifa
Published: January 2023

High mortality rates caused by malaria are seen in children under five years, but there is limited knowledge on neonatal immunity to the Plasmodium infection due to the lack of a reliable neonatal animal model to study the disease in children. By utilizing a newly developed young rodent model in our lab, we have observed that purified CD4+ T cells from day 14 old young mice (pups) proliferate slower than those purified from 8-week adult mice when stimulated in vitro. To investigate neonatal CD4+ T cell response to malaria, we infected 14-day old pups or adult mice with Plasmodium chabaudi and determined the activation status (CD127− CD44+ CD11a+) and differentiation of effector (Teff) subsets, using the expression of CD62L or CD27. Pup CD4+ T cells were activated by downregulating CD127, even though in lower proportions and numbers when compared to adult CD4+ T cells. Surprisingly, there were more proportions of TeffEarly (CD27+ CD62L+) and TeffInt (CD27+ CD62L−) subsets in the pups than adult mice, which had mostly TeffLate (CD27− CD62L−), suggesting that the transition of Teff subsets is slower in the young mice. We next determined cytokine secretion and Tbet expression by the Teff. Apart from IL-2 which was high in the pups, there was no difference in the proportions of protective cytokines including IFNγ, IL-10 and TNFα in both groups. Effector CD4+ T cells from the pups expressed high proportions of the Tbet transcription factor when compared to adult cells. Taken together, these data suggest that pup CD4 T cells may require more Tbet to differentiate into functional effector cells during Plasmodium infection.

Supported by Depart of Biology Appalachian State University
Th1/Tfh hybrid (Ifng+Il21+) T cells contribute to delay of germinal center formation in Plasmodium chabaudi infection
Authors: Clark Lucinda Puebla , Gbedande Komi , Domingo Nadia , Stephens Robin
Published: January 2023

Germinal center (GC) formation in P. chabaudi rodent malaria is delayed for three weeks, which can prolong the infection. T follicular helper (Tfh) cells express CXCR5 for homing to the B cell area, and IL-21 which promotes antibody production. T helper type 1 (Th1) cells express CXCR3 and IFN-γ to promote phagocytosis. Previous data suggests that Th1 responses control peak parasitemia while systemic Th1 cytokines inhibit GC formation. In P. chabaudi, majority of CD4+ effector T cells (Teff) express markers of both Th1 and Tfh (e.g., CXCR5, CXCR3, Th1/Tfh hybrid), including IFN-γ and IL-21. The contribution of Th1/Tfh hybrid T cells in protection and antibody production remains unclear. We tested the impact of Il21+ Tfh-like, Ifng+ Th1-like, and Ifng+ Il21+ Th1/Tfh in protection by transferring these subsets from infected triple cytokine reporters (Il21-rfp Il4-gfp Ifng-Thy1.1) into TCR KO mice that are then infected. We are currently assessing changes in parasitemia and pathology. We found that Th1/Tfh cells do not help in vivo like recent data suggest they do in vitro. Th1/Tfh significantly generated few GC B cells, compared to Il21+ Tfh-like cells. Recipients of Th1-like cells had significantly lower parasite-specific IgG and GCs by immunohistology than Tfh-like at 21 dpi, hybrid Th1/Tfh were intermediate. A cytokine kinetics study of Th subsets in infected cytokine reporter mouse distinguished Il4+ Il21+ GC Tfh from Th1 cells. The inflammation-driven chemokine receptors CXCR3 and CXCR6 are highly expressed on GC Tfh and Th1/Tfh hybrid during the first week of infection, but decline as GCs develop, suggesting a role for T cell migration which could explain the delay in GC formation and parasite-specific antibody production in malaria.

Supported by R01AI 135061
A forward genetic screen in alveolar macrophage-like cells highlights the essential role of peroxisome membrane regulation in hallmark alveolar macrophage phenotypes
Authors: Thomas Sean M , Wierenga Kathryn , Pestka James , Olive Andrew J
Published: January 2023

Alveolar macrophages (AMs) are a tissue resident macrophage population within the lung that are critical to maintain lung homeostasis and respond to inhaled antigens. Mechanistic investigations of AM function employing genetic approaches like CRISPR-Cas9 are hindered by the inability to obtain large numbers or maintain the AM-likeness of these cells ex vivo. We recently developed fetal liver-derived alveolar-like macrophages (FLAMs), a model that maintains the expression profile and immune functions of AMs long-term ex vivo. Importantly, FLAMs are genetically tractable using CRISPR-Cas9, enabling previously impossible interrogation of AM functions. Leveraging this innovative tool, we generated a genome-wide knockout FLAM library and are now completing forward genetic screens to dissect the regulation of AM maintenance and function. One such screen identified genes that regulate the surface expression of the AM-specific protein, Siglec-F. Among the numerous AM-associated pathways we identified as positive regulators of Siglec-F, the most robust signature was for genes related to the peroxisome. Thus, we hypothesize that the peroxisome is an essential signaling platform for AM functions. To test this prediction further we are using chemical and genetic approaches to dissect peroxisome functions in AMs. We are examining how peroxisomes modulate gene expression and metabolic flux in AMs and determining how this alters AM responses to stimuli. Together, these results highlight the utility of our novel genetic screening platform in FLAMs to unlock our understanding of AM functional mechanisms like never before.

This work was supported by the Rackham Research Endowment Award from Michigan State University.
Cbf-β is required for development and differentiation of murine mucosal-associated invariant T cells
Authors: Toor Jugmohit , Liu Tingting , Subedi Kalpana , Wang Jie , Loveless Ian , Yin Congcong , Zhou Li , Mi Qing-Sheng
Published: January 2023

Mucosal-associated invariant T (MAIT) cells primarily reside in mucosal tissues and tissues exposed to the environment to act as a defense against microbial threats. Additionally, they are also involved in a broad spectrum of diseases, including infection, cancer, allergy, and autoimmunity. Although some transcriptional factors and micro-RNA have been recently identified to regulate MAIT cell development and differentiation, a significant gap in our knowledge of MAIT cell regulation still exists. The core binding factor subunit beta (Cbf-β) is a non-DNA binding protein forming a heterodimer with RUNX family proteins to regulate diverse signaling pathways which maintain homeostasis of a wide range of immune cells. To understand the role of Cbf-β in MAIT cell development and function, we generated T cell lineage specific Cbf-β knockout mice (CD4cre+Cbf-β KO) and inducible Cbf-β knockdown mice (UBCcre+Cbf-β KO). We found that Cbf-β deficiency impaired thymic MAIT cell development and interrupted MAIT1 and MAIT17 differentiation. Additionally, inducible knockdown of Cbf-β reduced activation and cytotoxicity in peripheral MAIT cells. Our work suggests that Cbf-β is required for thymic MAIT cell development and MAIT1 and MAIT17 commitment in addition to playing a role in MAIT cell activation and cytotoxicity. Overall, Cbf-β may serve as a novel target to modulate MAIT cells in MAIT cell-based immunotherapies.

This study was supported by Henry Ford Immunology Program grants (T71016 and T71017) to QSM and LZ, by the National Institutes of Health (NIH) grants R61AR076803, R01AR063611, and R01AR069681 to QSM, RO1AR072046 to LZ.
Modulating expression of a BTB-ZF transcription factor in adipose resident NKT cells alters function and disrupts metabolism regulation.
Authors: Millick Danielle , Vieth Joshua A , Denzin Lisa K , Sant’Angelo Derek B
Published: January 2023

Natural killer T cells (NKTs) are potent modulators of the immune system that can regulate the function of other cell types largely through their rapid and robust production of a wide variety of cytokines. One tissue that relies on the interactions of NKT cells with various cell types is white adipose tissue. Adipose resident NKT cells (arNKTs) maintain tissue homeostasis and metabolism through anti-inflammatory interactions such as modulating macrophage functions and enhancing the proliferation and function of FoxP3-expressing Tregs. However, arNKTs are phenotypically distinct from other populations of NKT cells in their transcription factor expression. We have found that genetically modulating the expression of a BTB-ZF transcription factor in NKT cells alters the frequency and function of arNKTs, thereby altering weight gain and metabolic function. Our data show that sustained expression of the BTB-ZF transcription factor in arNKT cells leads to lower arNKT frequency, altered function, and enhanced weight gain in a model of obesity (60% high fat diet). While deletion of the transcription factor during NKT cell development leads to lower arNKT frequency, altered arNKT phenotype, and impaired weight gain when fed 60% high fat diet. Collectively, our data show that dynamic changes in the expression of a BTB-zinc finger transcription factor during NKT cell development and tissue-homing are required for normal arNKT cell function, and preservation of adipose tissue metabolism. Our findings imply that NKT cell inability to temporally regulate the expression of a single transcription factor may drive adipose tissue dysregulation and metabolic disease.

Supported by NIH/NIAID R21 AI159066-01
Cell-interaction based CRISPR screening using intercellular barcode transfer
Authors: Tay Neil Q , McManus Michael
Published: January 2023

Cell to cell interaction is one of the fundamental paradigms in cell biology and it is central to immune processes. While recent advances have allowed us to monitor cell interactions both in vitro and in vivo, doing so at large scales with complex interactions remain challenging. We have developed Relay, a scalable technology by which cellular interactions can be tracked via molecular barcoding and intercellular transfer of barcodes. We employed our system to perform a novel cell interaction-based CRISPR screen that was not possible with conventional CRISPR screening strategies. In our screen, we investigated factors that are involved in antigen processing, presentation, and transfer in myeloid cells using a myeloid-T cell co-culture system. By utilizing CRISPR/Cas9 together with Relay, we were able to perform genome-wide perturbations in myeloid cells and concurrently track myeloid-T cell interactions, allowing us to directly associate antigen sampling by T cells to genetic perturbations affecting antigen processing in the myeloid cells.

Supported by grant from NIH (U01 CA217882).
The gut microbiota transfers the therapeutic effect of inhibiting glucose metabolism in lupus-prone mice
Authors: Elshikha Ahmed Samir , Brown Josephine , Kanda Nathalie , Ge Yong , Teng Xiangyu , Abboud Georges , Choi Seung-Chul , Terrell Morgan , Garrett Timothy J , Mohamadzadeh Mansour , Morel Laurence
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoantibodies induce tissue damage including the kidney. Gut microbial dysbiosis contributes to SLE pathogenesis. An abnormal metabolism is a characteristic feature of SLE in which the inflammatory functions of CD4+ T cells rely on glycolysis. We have shown that treatment with 2-deoxy-D-glucose (2DG), a glycolysis inhibitor, reduced the expansion of germinal centers and eliminated the production of autoantibodies, ameliorating disease in lupus-prone mice, including (NZB × NZW)F1 and (NZW x BXSB)F1. Here we show that the 2DG treatment also maintained gut bacterial diversity, reduced the changes in bacterial populations that occurred as disease developed in these mice, and that it altered the distribution of fecal metabolites. We investigated the effect of fecal microbiota transplantation (FMT) from 2DG-treated or control mice into pre-autoimmune lupus-prone mice of the same strain. In both strains, FMT from 2DG-treated mice was highly protective, with a reduction or elimination of anti-dsDNA IgG production, immune cell activation, and renal pathology compared to FMT from control mice. Overall, our results demonstrated for the first time that the therapeutic effect of glucose inhibition in lupus is transferable through the gut microbiota. This implicates either a direct effect of glucose on pathogenic gut bacteria, or an indirect effect through the immune system normalized by glucose inhibition.

Supported b R01 AI143313
Prevention of Type 1 Diabetes via a Lipid Nanoparticle-Based Oral Immunotherapy that Targets Selective Lymphoid Tissues
Authors: Raimondi Giorgio , Zhang Yichuan , Hou Shenda , Wang Jialu , Calderon-Colon Xiomara , Tiburzi Olivia , Lozano Marcos Igleasias , Patrone Julia
Published: January 2023

Type 1 diabetes (T1D) is an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons contribute to T1D, and the inhibition of inflammatory cytokines signaling via JAK inhibitors like Tofacitinib (Tofa) has shown promise in the prevention of the disease. However, maintenance of a therapeutic concentration of these drugs is challenging and their prolonged use leads to significant side effects. Our objective was to design a controlled and localized delivery of Tofa via lipid nanoparticles (LNp) and assess the impact of an oral short-term administration strategy on T1D development.

We identified a LNp formulation with negligible toxicity, readily taken up by multiple immune cells, and with favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have the unique property of accumulating in lymphoid tissues, particularly in pancreatic and mesenteric lymph nodes when administrated via oral gavage. Importantly, short-term administration (5 gavages once every other day) of Tofa-LNp early (3-week-old) or late (10-week-old) in disease prone NOD mice promoted a significant delay of T1D onset. Cell profiling and in vivo challenges indicated that this therapeutic effect is likely due to localized inhibition of APCs maturation and, surprisingly, promotion of anergy in diabetogenic T cells. Ex-vivo stimulation confirmed the hyporeactivity of these cells and adoptive transfer experiments confirmed their ability to act as reservoir of induced regulatory T cells. These results highlight the versatility of this novel LNp-based drug delivery strategy.

This work was supported by grant 2-SRA-2016-310-S-B from the Juvenile Diabetes Research Foundation.
IL-33 upregulated in fibroblastic reticular cells after recipient conditioning acts as a novel costimulatory signal in the generation of alloreactive Type 1 T helper cells
Authors: Dwyer Gaelen K , Mathews Lisa , Lucas Anna , Blazar Bruce R , Poholek Amanda , Shlomchik Warren , Turnquist Heth Roderick
Published: January 2023

In allogeneic hematopoietic stem cell transplantation (alloHCT), recipient conditioning releases Pathogen- and Damage-Associated Molecular Patterns (PAMPs and DAMPs) that generate pro-inflammatory antigen-presenting cells (APC) that secrete IL-12 to initiate donor Type 1 T helper (Th1) responses causing graft-vs-host-disease (GVHD). Yet, other mechanisms exist to initiate alloimmune responses, as recipients with disrupted APC PAMP/DAMP signaling or lacking IL-12 develop GVHD. We used IL-33 receptor, ST2, deficient B6 mice as T cell donors into BALB/c recipients to test the hypothesis that the DAMP IL-33 bypasses APC and acts directly on donor CD4+ T cells to mediate early alloreactive T cells activation and differentiation. In our model, we transferred equal ratios of B6 Cd4-Cre x St2fl/fl (ST2ko) and Cd4-Cre (ST2wt) T cells into irradiated BALB/c recipients with and without IL-12 blockade. Donor T cells in the secondary lymphoid organs (SLOs) were characterized at days 1–3, 5, and 7 post alloHCT. We established that IL-33 DAMP functions involve the direct stimulation of donor CD4+ T cells, which promoted IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. We demonstrated that IL-33 is induced by recipient irradiation in PDPN+ CD31− fibroblastic reticular cells (FRC) of the SLOs as early as 1 day post-radiation to increase alloreactive CD4+ T cell numbers. Mechanistically, IL-33 amplified CD4+ T cell TCR signaling in response to alloantigen to enhance Th1 cell activation and differentiation, while inhibiting regulatory molecule (i. e. IL-10 and Foxp3) expression. Thus, IL-33 is an unappreciated costimulatory signal in Th1 generation and a promising early target to prevent acute GVHD after alloHCT.

Supported by NIH F30AI147437 and T32 CA082084 (GKD), NIH R01HL122489, R01AR073527, R56AI13927 (HRT)
Deficiency of antibody-suppressor CXCR5+CD8+ T cells (not CD4+ Tregs) drives high alloantibody production in CCR5 KO kidney transplant recipients
Authors: Han Jing Light , Zimmerer Jason , Zeng Qiang , Chaudhari Sachi , Breuer Christopher , Bumgardner Ginny L
Published: January 2023

Kidney transplant (KTx) into CCR5 KO mice is an excellent model to study AMR immunobiology due to a heightened humoral response posited to be secondary to impaired CD4+ Treg cell trafficking to the allograft. We have observed that adoptive cell therapy (ACT) with alloprimed CXCR5+ CD8+ T cells mediates significant reduction of alloAb titer in CCR5 KO KTx recipients. We hypothesized that CXCR5+ CD8+ T cells are more potent inhibitors of alloAb production than CD4+ Tregs. We found that CCR5 KO recipients have significantly fewer CXCR5+ CD8+ T cells (782±183 vs 2058±167 cells/mm3) and graft-infiltrating CD4+ Treg cells (CD25+ FoxP3+; 12.9±8.8 vs 56.2±8.8 cells/mm3) compared to WT recipients, as well as significantly greater quantity of splenic germinal center (GC; GL7+ Fas+ B220+) B cells (5814±436 vs 671±342 cells/mm3; all p<0.003). CCR5 KO recipient alloAb titer is significantly reduced following CXCR5+ CD8+ T cell ACT (1259±436 vs 5951±404 in untreated controls; p<0.0001) but not following CD4+ Treg ACT (5688±436). CXCR5+ CD8+ T cells reduced splenic GC B cell (5813±589 to 4087±537; p=0.002) quantity, while CD4+ Treg had no impact (6916±538/mm3). Compared to untreated controls, CD8-depletion in WT recipients led to significant increase in alloAb titer (1508±338 vs 6078±436) and GC B cells (671±436 vs 7761±430; p<0.0001 for both). CD4+ Treg depletion led to increased alloAb titer (3180±436) and GC B cell quantity (4911±429; p<0.004 for both), though less pronounced than in the CD8-depleted group (both p<0.001). This suggests that CXCR5+ CD8+ T cells are more potent regulators of alloAb production than CD4+ Tregs and their deficiency in CCR5 KO recipients is the major driver of heightened humoral immunity and AMR following KTx.

Supported by NIH R01 AI083456 (to GLB), T32 AI106704-07 and F32 AI161844 (to JLH), CA016058, UL1TR002733, the OSU Division of Transplant Surgery, and the OSU College of Medicine.
CMV drives phenotypic changes in CD4 T cells prior to but not during the first year after solid organ transplantation
Authors: Higdon Lauren E , Ahmad Ayah A , Maltzman Jonathan S
Published: January 2023

Cytomegalovirus (CMV) infection is a major complication after solid organ transplantation. The virus establishes latency after immune clearance of the primary infection. Immunosuppression impairs immune control in transplant recipients. We have previously shown that CMV drives accelerated immune aging of CD8 T cells in the first year after transplant. CMV-specific CD4 T cells also contribute to resolution of post-transplant viremia and reduce severity of disease at least in part by promoting maintenance of anti-viral CD8 T cell populations. Thus, we investigated the impact of CMV on CD4 T cell differentiation in the first year after transplant. Compared to CMV seronegative (CMV−) recipients, CMV+ recipients had an elevated frequency of aged (CD57+) CD4 T cells, with trends towards elevated PD-1+ and skewing toward memory rather than naïve phenotype. Unlike CMV-specific CD8 T cells, these phenotypes were stable over the first year after transplant, indicating these differences demonstrate a pre-established differentiation state. To correlate these findings to T cell receptor repertoire at a single cell level, we conducted single cell sequencing of TCR coupled to gene expression in CMV-responsive CD4 T cells from 6 transplant recipients from pre- to one-year post-transplant. Cell phenotypes were stable over time, and clonally expanded cells predominantly had an aged cytotoxic phenotype. Thus, CMV-responsive CD4 T cells in transplant recipients display evidence of immune aging and cytotoxicity. This is consistent with similar phenotypes in CD8 T cells, but striking in stability. Further study of this population will be important to determine whether this pre-existing phenotype is protective or pathogenic after transplant.

This work was supported by funding from the NIH to LEH (NIDDK 5K01DK123196) and AAA (Stanford PreRenal Initiative, NIDDK KUH 1R25DK122957) and the Department of Veterans Affairs to JSM (CSR&D 1I01CX001971).
Lipoxin A4 mitigates ferroptosis via FPR2 signaling during lung ischemia-reperfusion injury
Authors: Sharma Ashish Kumar , Cai Jun , Leroy Victoria , Tu Zhenxiao , Gonzalez Alejandro , Hartman Joseph , Mulligan Jennifer , Atkinson Carl , Upchurch Gilbert
Published: January 2023

Ischemia-reperfusion injury (IRI) after lung transplantation entails dysregulation of inflammation-resolution pathways leading to primary graft dysfunction. We investigated the role of ω-3-derived specialized pro-resolving lipid mediators, i.e. Lipoxin A4 (LxA4), and formyl peptide receptor (FPR2) signaling in the resolution of lung IRI. We used an established murine model of lung hilar ligation for IRI using C57BL/6 wild-type (WT) and FPR2−/− mice that underwent sham surgery or IRI (1hr left lung ischemia followed by 6- or 24hrs reperfusion). Lung function was measured using an isolated, buffer-perfused apparatus. Cytokine levels were measured in bronchoalveolar lavage (BAL) fluid, and neutrophil infiltration was assessed by immunohistochemistry. Post-lung transplant BAL from human patients was analyzed by liquid chromatography-mass spectrometry that demonstrated a significant increase in LxA4 on day 7 compared to days 0 and 1. In the murine model, we observed a significant increase in hallmarks of ferroptosis i.e. induction of lipid peroxidation (malonyldialdehyde), inhibition of glutathione peroxidase 4 (GPX4) as well as nuclear factor erythroid 2 (Nrf2) after IRI. Treatment of WT mice with recombinant LxA4 significantly attenuated lung dysfunction (decreased airway resistance and pulmonary artery pressure, and increased pulmonary compliance), inflammation (IL-17, TNF-a, CXCL1, HMGB1), injury (neutrophil infiltration) and ferroptosis (decreased MDA as well as increased Nrf2 and GPX4 expressions) compared to IRI alone, that was abolished in LxA4 treated-FPR2−/− mice. Collectively, our results indicate that lung IRI is regulated by ferroptosis which can be prevented by LxA4/FPR2-mediated signaling.