Sun, Jie

Abstract Description During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3). However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer’s patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 pathway as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells. Funding Sources National Institute of Allergy and Infectious Diseases (NIAID) - AI162678 National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) - AR077518 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Abstract BACKGROUND Intraductal papillary mucinous neoplasm (IPMN) is one of the two most common precursor lesions leading to the development of pancreatic ductal adenocarcinoma (PDA). IPMNs comprise a heterogeneous group of tumors with a wide range of grades and histotypes, and the emergence of single-cell RNA sequencing (RNA-seq) and multiplex digital spatial profiling have characterized unique cell populations, including dysplastic epithelial and immune cells, within the heterogeneous tumor microenvironment that carry signature gene expressions, which could be used as markers for disease progression. However, it is not clear on the heterogeneity of the epithelial and immune response of the evolution during progression of IPMN to PDA. OBJECTIVE Combining nuclear GSK-3β with oncogenic KRas mice (referred to as KNGC – KRas, nuclear GSK-3β, Cre) resulted in the development of IPMN. We have found that 4-week-old KNGC mice show progressive desmoplasia and ductal lesion development with increased M2-like macrophage infiltration. Interestingly, CX3CL1 (fractalkine), a chemokine involved in the recruitment of CX3CR1-expressing monocytes and M2-like macrophage conversion in various disease models is expressed by IPMN progenitor ductal cells. We aim to investigate expression and contribution of CX3CL1-CX3CL1 axis in the development and progression of IPMN. METHODS We utilized a newly generated CX3CL1-mCherry and well-established CX3CR1-GFP knockin/knockout mouse for crossbreeding with KNGC and KGC (KRas, GNas, Cre) for lineage tracing and function analysis by immunofluorescent staining, flow cytometry and CyTOF. RESULTS Both KNGC- and KGC-3LmCherry/+ mice harbor a large population of CX3CL1-mCherry/EPCAM+ epithelial cells as compared to 3LmCherry/+ and wildtype (WT) control mice. In KNGC and KGC mice, we show that there was a unique population of macrophages (CD45+ CD11b+ Ly6G- F4/80+ Ly6c- ) with significantly higher expression of CX3CR1 (CX3CR1Hi) that was not seen in WT mice. Significantly, Knockout of CX3CR1 in KNGC-3RGFP/GFP animals had a paucity of M2-like macrophages, reduced T regulatory cells (Tregs), substantially reduced desmoplasia and impaired development of IPMN. CONCLUSIONS The study provides important knowledge regarding the role of CX3CL1-CX3CR1 axis in recruitment and generation of M2-like macrophages, and their impact on the initiation of the immune/stroma microenvironment facilitating IPMN development. Further investigations using these models have the potential to identify new biomarkers for risk stratification and therapeutic intervention. Citation Format: Li Ding, Kaely Roeck, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Esther Rodman, Mark Maynes, Bibo Zhu, Aaron Johnson, Jie Sun, Li Hu, Daniel Billadeau. Uncoupling CX3CL1-CX3CR1 Axis in IPMN Development Through Transgenic Mouse Models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A064.

Abstract Despite intensive characterization of immune responses after COVID-19 infection and vaccination, research examining protective correlates of vertical transmission in pregnancy are limited. Herein, we profiled humoral and cellular characteristics in pregnant women infected or vaccinated at different trimesters and in their corresponding newborns. We noted a significant correlation between spike S1–specific IgG antibody and its RBD-ACE2 blocking activity (receptor-binding domain–human angiotensin-converting enzyme 2) in maternal and cord plasma (P < .001, R > 0.90). Blocking activity of spike S1–specific IgG was significantly higher in pregnant women infected during the third trimester than the first and second trimesters. Elevated levels of 28 cytokines/chemokines, mainly proinflammatory, were noted in maternal plasma with infection at delivery, while cord plasma with maternal infection 2 weeks before delivery exhibited the emergence of anti-inflammatory cytokines. Our data support vertical transmission of protective SARS-CoV-2–specific antibodies. This vertical antibody transmission and the presence of anti-inflammatory cytokines in cord blood may offset adverse outcomes of inflammation in exposed newborns.

Abstract Aging has been shown to be one of the major risk factors for host morbidity and mortality following infections with respiratory viruses including influenza virus and SARS-CoV-2. The unique immune properties of the aged hosts not only influence pathogen clearance and tissue damage responses, but also modulate the pathogenesis of the long-term sequalae post acute viral clearance. Currently, the dynamics and features of mucosal immune responses following acute respiratory viral infections in the aged hosts remain to be fully elucidated. Here, we characterized the lung cellular and molecular profiles following influenza virus infection in young (~3-month-old) or aged (~24-month-old) mice with a chronological manner from acute to chronic phase. Using DNA-oligo-conjugated CD45 antibody and i.v. labeling, we were able to distinguish the unique responses of circulating vs lung-resident immune cells to influenza virus infection during aging. These responses were further validated by spectral flow cytometry analysis of the lung innate and adaptive immune cells. Further data analysis and integration are warranted for this unique large data set detailing the single cell molecular profiles of mucosal responses to influenza virus infection comparing young and aged hosts.

Abstract Fetal-derived tissue-resident macrophages exhibit stem cell-like features of self-renewal in adulthood to maintain macrophage population during homeostasis and/or various insults. However, little is known about the cellular and molecular mechanisms modulating proliferative and inflammatory fate decisions of tissue-resident macrophages in vivo. Here, we show that WNT-β-catenin signaling inhibited lung-resident alveolar macrophage (AM) self-renewal, while simultaneously promoted AM inflammatory activities in vitro and in vivo during influenza virus infection. Mechanistically, WNT engagement facilitated the binding of β-catenin with HIF-1α over its conventional binding partner TCF-4. Such a binding choice led to the elevated macrophage inflammation in a glycolysis-dependent manner, while inhibited AM self-renewal by causing mitochondrial damage and impairing oxidative phosphorylation. Thus, AM self-renewal and inflammatory activity are uncoupled by WNT-β-catenin signaling through HIF-1α-mediated cellular metabolic choice. Importantly, we showed that AM with high HIF-1α activity had limited proliferative capacity and produced inflammatory cytokines, while AM with low HIF-1α activity were highly proliferative and expressed genes associated with tissue repair function in vivo during influenza virus infection. In accordance, we demonstrated that AM proliferation and repopulation were needed for optimal lung repair following the clearance of influenza virus in the respiratory tract. Our results have revealed key mechanisms modulating macrophage fate choice between progeny production versus inflammatory effector activity, and subsequent effects on tissue inflammation and repair.

Abstract Alveolar macrophages (AMs) are the most abundant immune cells in the lung during homeostasis. AMs are critical for normal lung development, surfactant metabolism and host defense against various respiratory pathogens. Despite being terminally differentiated cells, AMs are able to proliferate and self-renew to maintain their compartment without the input of the hematopoietic system in the adulthood during homeostasis. However, the molecular and metabolic mechanisms modulating AM proliferative responses are still incompletely understood. Here we have investigated the metabolic regulation of AM proliferation and self-renewal. We found that mitochondrial metabolism is critical for AM proliferation in vitro and in vivo. Interestingly, inhibition of glucose uptake or fatty acid oxidation did not significantly impact AM proliferation. Rather, inhibition of the glutamine metabolic process abolished AM proliferation in vitro and in vivo following their partial depletion during influenza infection. We further demonstrated that glutamine metabolism was required for AM mitochondrial respiration. Our data indicate that glutamine is a critical substrate fueling mitochondrial metabolism required for AM proliferation. Our study is expected to shed light on the metabolic regulation of AM maintenance and repopulation during homeostasis and following acute respiratory viral infection.

Abstract Sialic acids are the terminal modification on glycoproteins and glycolipids. The sialic acid transferase ST8Sia6 generates a2,8-linked disialic acids on O-linked glycoproteins, which bind to the inhibitory receptor Siglec-E on innate immune cells and suppress immune activation. Recently, our lab has shown that ST8Sia6 expression on tumor cells inhibits the immune response through Siglec-E, leading to increased tumor growth and decreased survival. Therefore, the products of ST8Sia6 can modulate the immune response to tumors. Our lab has generated ST8Sia6 knockout (KO) mice, and we seek to understand the role of ST8Sia6 in regulating the immune response to pathogens. We find that ST8Sia6 KO mice, when challenged with influenza virus, clear the infection significantly faster than wildtype controls, suggesting that absence of ST8Sia6 reduces inhibition of the innate compartment, thereby increasing immune recruitment to infection. This data suggests an important role for interactions between Siglec-E and ST8Sia6-generated disialic acids in immune regulation and recruitment to infection sites.

Abstract Alveolar Macrophages (AMs) are unique innate immune cells that reside in the alveolar space and accommodate the ever-changing need of the lungs against internal and external challenges. During homeostasis, AMs maintained themselves through self-renewal without the need for input from adult hematopoietic stem cells. Currently, little is known about how aging influences AM dynamics, heterogeneity and self-renewal. To this end, we have performed single-cell RNA sequencing (scRNA-seq) on murine lung AMs (CD11+ Siglec F+) isolated from young (~8w) and aged (~2y) mice. We discovered that despite similar transcriptome in the proliferating cells, AMs from aged mice had reduced proliferation and self-renew ability than those from the young mice. AMs from aged mice also displayed elevated senescence markers such as p16, p21 and p53. Consistently, AMs from aged mice had reduced DNA repair ability, which could contribute to their impaired capacity to pass through the cell cycle checkpoints. Interestingly, AMs from aged mice also possessed reduced pro-apoptotic gene profile. Thus, we hypothesize that the reduced proliferation- and apoptosis-associated gene profiles in aged AMs may lead to the enhanced senescence-like phenotype in those cells. Further validation of the senescence-like phenotype of aged AMs and investigation of how they contribute to lung “inflammaging” are warranted.

Abstract Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. Defining research models to alleviate lung damage without compromising viral control is critical. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor in regulating lung inflammation and antiviral innate immune responses. Nmp4-deficient mice are protected from H1N1 influenza infection, losing less body weight compared to the wild-type mice. Nmp4-deficiency does not affect viral clearance or adaptive immune responses, but significantly reduces the accumulation of neutrophils and monocytes in the lungs and the early inflammation. Consistent with fewer innate cells in the airways, infected Nmp4-deficient mice show decreased expression of chemokine genes Ccl2 and Cxcl1 as well as pro-inflammatory cytokine genes Il1b and Il6. We further reveal that NMP4 binds to the promoters and/or conserved non-coding sequences (CNS) of Ccl2 and Cxcl1 in mouse lung epithelial cells. Besides, the knockdown of NMP4 by CRISPR/Cas9 or siRNA significantly lessens the luciferase activities of Ccl2 and Cxcl1 promoters and/or CNS. Taken together, our data suggest that NMP4 regulates monocyte- and neutrophil-attracting chemokine expression in airway epithelial cells upon influenza infection, resulting in exaggerated innate inflammation and lung tissue damage.

Abstract CD8+ tissue-resident memory T (TRM) cells provide frontline immunity in mucosal tissues. The mechanisms regulating CD8+ TRM maintenance, heterogeneity, and protective and pathological functions are largely elusive. Here, we identify a population of CD8+ TRM cells that is maintained by major histocompatibility complex class I (MHC-I) signaling, and CD80 and CD86 co-stimulation after acute influenza infection. These TRM cells have both “exhausted-like” phenotypes and memory features, and provide heterologous immunity against secondary infection. Using recombinant influenza virus, we demonstrate that the development of these PD-1hi “exhausted-like” TRM cells is dependent on the doses of the antigen during primary infection. Furthermore, we showed that lung CD11c+ antigen presenting cells are required for sustaining these TRM cells at the memory stage. Our data suggest that continuous antigen presentation in situ is required for the development and maintenance of these “exhausted-like” TRM cells in the lung. Strikingly, PD-L1 blockade after the resolution of primary infection promotes the rejuvenation of these “exhausted-like” TRM cells, restoring protective immunity at the cost of promoting post-infection inflammatory and fibrotic sequelae. Thus, PD-1 serves to limit the pathogenic capacity of “exhausted-like” TRM cells at the memory phase under specific tissue environment with chronic low-levels of antigen presentation following viral clearance. Our data indicate that TRM cell “exhaustion” is the result of a tissue-specific cellular adaptation that balances fibrotic sequelae with protective immunity.