bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiratory syncytial virus infection alters the gut immune response to commensal bacteria
Authors: Siddikova Anisa , Brown Julia , Zeng Melody
Published: November 2024

Respiratory Syncytial Virus (RSV), characterized by inflammation of the bronchioles, is a major cause of hospitalizations among infants and older adults. Early-life gut dysbiosis, an imbalance of microbiota, has been linked with elevated risk of lung infections later in life. However, our understanding of the impact of respiratory infections on the gut and its long-term consequences is limited. We hypothesize that RSV infection in mice increases gut inflammation. To test this, we intranasally infected mice with RSV, collected blood and stool samples over the course of the infection, and analyzed immune cells in the lung and colon. We saw an increase in immune cells and cytokine production in both lung and colon of infected mice. We further found an increase in antibodies binding to commensal bacteria in the intestinal lumen, which was more prominent in the colon than in the small intestines, suggesting a tissue specific difference in response to RSV. Lastly, we saw gut microbiome changes in response to RSV, such as reduced Muribaculaceae and elevated Alistipes. Together, these data suggest that respiratory infections may disrupt homeostasis beyond the lung, and respiratory virus-induced changes to the gut could influence the risk of subsequent enteric infections or chronic conditions such as inflammatory bowel disease. A better understanding of how RSV influences the gut could help to identify patients at risk for these conditions as well as potential therapeutic targets.
InTraSeqTM: a multimodal single cell technology for simultaneous measurement of RNA and proteins in single cells to uncover new single cell biology
Authors: Ariss Majd
Published: November 2024

InTraSeqTM is a novel technology for single cell analysis that enables unbiased measurement of both RNA and proteins in individual cells. This innovative approach employs validated and functionally tested antibodies at CST, which allows the identification of cell subtypes and Signaling Pathways in single cells by measuring post-translational modifications (PTMs).

One of the significant advantages of using InTraSeqTM is its ability to quantify extracellular, cytoplasmic, and nuclear proteins, including transcription factors, and their PTMs while preserving the RNA and heterogeneity of the sample. This method offers an extensive view of the cellular mechanisms of complex biology, which was previously missing in single cell RNA sequencing datasets. Researchers can use InTraSeqTM to accurately characterize single cell acute and chronic responses, uncover new cell states, and more accurately analyze cell differentiation trajectories. Moreover, InTraSeqTM offers a streamlined protocol that requires only one hour of benchwork and allows for multi-day sample storage, making it a practical and efficient tool for high-throughput applications.
Revolutionary Selection, Activation, and Expansion with Microbubbles for CAR-T Cell Manufacturing
Authors: Badea Dominique , Huang Effie , Snow Tiffany , Chen Jesse , Roussey Jon , Ku Chia , Wegner Casey
Published: November 2024

Despite several chimeric antigen receptor-modified T cells (CAR-T) products being approved and commercialized, CAR-T cell therapy has manufacturing challenges. Conventional CAR-T therapies are manufactured by collecting apheresis material from a patient’s blood, then modifying the isolated T cells from the apheresis material to express a cancer cell-specific CAR, eventually infusing the CAR-T cells into a patient. Challenges include early exhaustion of the therapeutic cells, manual/laborious processes, and complex workflows. Akadeum’s innovative microbubble technology streamlines human T cell selection, activation, and expansion into a single step. Here, we demonstrate over 90% capture efficiency while reducing the selection process to under one hour. Because of the microbubbles’ natural buoyancy, T cell activation occurs at the top of the culture. As the T cells divide and the bound receptors are recycled, T cells fall to the bottom of the media, avoiding CD3/CD28 overstimulation. PD-1 expression is reduced throughout expansion compared to traditional activation workflows. T cell activation was not compromised, as evidenced by the upregulation of CD69, CD25, and HLA-DR+. Additionally, microbubble technology can increase the percentage of desired effector T cell populations. As CAR-T therapy expands, innovations like Akadeum's microbubble technology play a crucial role in overcoming manufacturing challenges and ensuring the development of high-quality, effective therapies.
A Fully Automated, Walkaway Flow Cytometry Workflow for Immune Cell Subsets Identification
Authors: Bowers Rita , Grazia Giulia , Maraman Jason , Lacaille Vashti , Weit Nicole
Published: November 2024

In the fast-paced competitive landscape of life science research, the adoption of an automated workflow offers the advantages of improved data reproducibility and increased throughput with less operator dependency, contributing to robust and efficient research studies.

We present here an automated flow cytometry workflow using a fully integrated system comprising a Biomek i7 Multichannel Workstation, a CytoFLEX LX flow cytometer, DURA Innovations dry reagents and automated data analysis using the Cytobank platform.

In our study, a DURAClone T cell subset dry reagent panel in a 96-well plate format was used to characterize maturation stages in T cells present in PBMC samples from 16 healthy donors. The walkaway workflow leveraged the Biomek i7 Multichannel liquid handler for automated sample preparation, automated sample acquisition on an integrated CytoFLEX LX flow cytometer, automated data upload to the Cytobank platform via the API (application programming interface) with automated data analysis performed using a pre-defined gating model applied to the newly created experiment, achieving auto-generated results ready for review.

Additional features and functionality within the Cytobank platform were leveraged to inspect results, apply dimensionality reduction algorithms, export tables of statistics and graph the final data.

By embracing automated workflows and advanced technologies, scientists can conduct their research more efficiently and reliably.

For Research Use Only
Pharmacological activation of LANCL receptors has three-fold benefit in metabolic dysfunction-associated steatohepatitis
Authors: Leber Andrew , Hontecillas Raquel , Tubau-Juni Nuria , Fitch Sarah , Bassaganya-Riera Josep
Published: November 2024

The LANCL family of receptors includes LANCL2, a membrane receptor associated with glycemic control and regulatory immune responses, and LANCL3, a mitochondrial receptor that is significantly downregulated in multiple inflammatory diseases. In metabolic dysfunction-associated steatohepatitis (MASH), LANCL3 expression is reduced more than two-fold in cholangiocytes, hepatic stellate cells and Kupffer cells relative to healthy control. Based on the mechanistic overlap between the LANCL receptors and MASH, we assessed loss of function and pharmacological activation in MASH relevant in vitro and in vivo systems. Loss of LANCL2 or LANCL3 resulted in increased production of IL-17 and TNF from CD4+ T cells. Production of COL1A1 in hepatic stellate cells increased three-fold after TGF-β stimulation in the KO relative to WT. In vivo, liver weight increased and glucose tolerance decreased in KO mice relative WT after high fat diet feeding for 12 weeks. In both carbon tetrachloride and choline deficient L-amino acid defined (CDAA) models of MASH, LANCL activation resulted in lower liver weight, triglycerides and fibrosis through measures of total, perivascular and bridging collagen. Treatment also resulted in a significant reduction of Th17 cells and TNF+ macrophages in the models of MASH. This data suggests that LANCL receptors are promising therapeutic mechanisms for MASH and other metabolic diseases through an ability to directly modulate the three hallmarks of MASH pathogenesis.
Immunaut: Immunophenotype Analysis with Machine Learning for LAIV Vaccine Response Prediction
Authors: Tomic Ivan
Published: November 2024

The Immunaut approach represents a groundbreaking application of machine learning in immunology, focusing on predicting immune responses to Live Attenuated Influenza Vaccines (LAIV). This novel method combines unsupervised machine learning techniques, including t-distributed Stochastic Neighbor Embedding (tSNE) and advanced clustering algorithms, with the Sequential Iterative Modeling OverNight (SIMON) platform for predictive analysis. By identifying and categorizing immunophenotypic groups based on baseline features, Immunaut offers a sophisticated, data-driven means of enhancing our understanding and prediction of vaccine efficacy. This approach promises to pave the way for more personalized vaccine strategies and a deeper insight into immune response mechanisms.
An IL-12 mRNA-LNP adjuvant durably enhances the CD8+ T cell response to mRNA vaccination
Authors: Aunins Emily , Phan Anthony , Cruz-Morales Elisa , Alameh Mohamad-Gabriel , Christian David , Hunter Christopher , Weissman Drew
Published: November 2024

The design of vaccines that can reliably induce protective CD8+ T cell activity has been considered a challenge for the vaccine field, but the use of viral vectors or mRNA encapsulated into lipid nanoparticles (LNPs) has overcome some of these issues. Because the cytokine interleukin-12 (IL-12) supports CD8+ T cell expansion and acquisition of effector functions, studies were performed to assess its contribution to the ability of mRNA vaccines to promote CD8+ T cell responses. mRNA-LNPs do not stimulate macrophage production of IL-12 in vitro nor does in vivo vaccination, and endogenous IL-12 is not required for the CD8+ T cell response to mRNA vaccination. However, the addition of IL-12 mRNA-LNPs into vaccination with LNPs that contain mRNA for the model antigen ovalbumin (OVA) results in enhanced OVA-specific CD8+ T cell expansion, improved acquisition of effector function, diversification of the CD8+ T cell effector pool through generation of a KLRG1hi effector subset and expanded effector and central memory CD8+ T cell populations. In both preventative vaccination against Listeria monocytogenes-OVA and therapeutic vaccination against B16 F0-OVA melanoma, addition of IL-12 mRNA-LNPs enhanced protection. Thus, modification of current mRNA vaccine formulations to induce IL-12 production provides a strategy to modify the CD8+ T cell response, enhance CD8+ T cell mediated protection and illustrates the utility of cytokine mRNA to tailor vaccine-induced immunity.
The ectromelia virulence factor C15 is a potent inhibitor of MHC-mediated T cell activation.
Authors: Carro Stephen , Hedgepeth Emma , Heard Mary , Peauroi Elise , Eisenlohr Laurence
Published: November 2024

Orthopoxviruses (OPXV), home to mammalian pathogens such as variola and Mpox, encode a myriad of immunomodulatory proteins, many of which are understudied, to promote pathogenesis. One protein family, the B22 family, are of interest as potential antiviral targets due to their conservation across pathogenic OPXV. B22 proteins are surface glycoproteins that inhibit T cell activation. In ectromelia virus, we previously found that its B22 protein, C15, is essential for lethality in BALB/c mice and inhibits CD4+ T cell activation both in vitro and ex vivo. However, the mechanism of C15 immune modulation remains unknown. To bridge this gap, here we define the breadth of C15-mediated T cell inhibition and explore its molecular mechanisms. Using T cell hybridomas, we found that C15 inhibits MHCIa/b- and MHCII-mediated T cell activation. This inhibition is not mediated by a secreted factor but is in effect upon expression by MHC-negative cells. This finding, along with the observation that C15 does not impact the surface density of peptide-MHC complexes, suggests that C15 acts directly on the T cell rather than the antigen-presenting cell. In vivo, we demonstrate that C15 significantly dampens the generation of antigen-specific cytotoxic effector CD8+ T cells (Teff), resulting in a significant decrease in KLRG1+CX3CR1+ Teff. Overall, our results offer novel insights into the molecular mechanisms of C15-mediated T cell inhibition and the modulation of adaptive immunity by OPXV.
Antigen-Specific Germinal Center Tfh cells are highly heterogenous in lymph nodes and Tfh1/Tfh17 cells are associated with viral load levels during chronic SHIV infection
Authors: Govindaraj Sakthivel , Styles Tiffany , Ibegbu Chris , Amara Rama , Velu Vijayakumar
Published: November 2024

HIV infection leads to alterations in HIV-specific CD4 T cells including increased expression of inhibitory receptors and skewing toward Tfh cell signature. Tfh cells have been shown to increase during chronic HIV infection. However, the heterogeneous nature of antigen-specific GC-Tfh cells and the relative contribution of specific GC-Tfh subset to viral persistence is not understood in HIV/SIV infection.

Here, we characterized the phenotype and function of antigen-specific GC-Tfh cells based on the expression of chemokine receptors, Th1 (CXCR3), Th2 (CCR4), and Th17 (CCR6) along with activation-induced markers (AIM assay) CD25, OX40 and 41BB in the lymph nodes following chronic SHIV1157ipd3N4 infection in rhesus macaques.

In SHIV naïve RM, only a small fraction of total GC-Tfh expressed CXCR3, CCR4, and CCR6. During chronic SHIV infection, there was an increase in GC-Tfh cells. Interestingly the majority of antigen-specific GC-Tfh cells expressed CXCR3 (Tfh1), while a significant proportion of them also expressed CCR4+ (Tfh2) and CCR6+ (Tfh17) cells. These cells expressed OX40, 41BB, and CD25 at higher levels than non-Tfh cells. Importantly, the expansion of GC-Tfh1 and GC-Tfh-17 cells is associated with high viremia.

These data demonstrate that chronic SHIV infection promotes the expansion of antigen-specific GC-Tfh cells with heterogeneous populations, which are distinct from conventional GC-Tfh, and associated with higher viral RNA levels during chronic SHIV infection.
Tissue-resident memory CD8+ T cell development in the upper respiratory tract
Authors: Kost Kirsten , Michalets Sarah , Mattingly Cameron , Chen Thien Duy , Jimenez Ariana , Williams Mark , Lawrence Laurel , Kohlmeier Jacob
Published: November 2024

CD8+ tissue-resident memory T cells (TRM) in the upper respiratory tract (URT) limit viral replication and can prevent spread to the lower respiratory tract (LRT), which is the primary cause of morbidity and mortality after respiratory virus infection. Utilizing mouse models, our work explores the interesting dichotomy of URT CD8+ TRM development after intranasal (i.n.) priming, in which localized respiratory antigen is present, and intraperitoneal (i.p.) priming, in which URT CD8+ TRM develop without localized antigen. We explore the early kinetics of TRM formation and demonstrate through bulk RNA sequencing that URT CD8+ memory precursor TRM have unique transcriptional signatures in these distinct infection settings. Using immunofluorescent microscopy, we identify localization of URT CD8+ TRM with and without local antigen. We are currently exploring the role of CD4 T cell help and the respiratory microbiome on the development of URT CD8+ TRM in the presence and absence of local antigen. Additionally, utilizing conditional knockout mouse models, we show that cytokine signals in the URT microenvironment, namely TGF-b and IL-15, drive CD8+ TRM formation in combination with localized antigen. Our work outlines the unique requirements for URT CD8+ TRM formation in the absence of localized respiratory antigen, which can inform T cell-based vaccine strategies against respiratory viruses.
Determining the role of T follicular helper cells in a multi-antigen germinal center model
Authors: Bilanovic Jana , Jacobsen Johanne , Nakandakari-Higa Sandra , Barbulescu Alexandru , Victora Gabriel
Published: November 2024

High-affinity antibodies produced by germinal center (GC) B cells are a crucial line of protection from viral infections. Canonically, T follicular helper (Tfh) cells provide essential helper signals to GC B cells through cognate Tfh-B cell interactions, thereby driving the iterative cycles of GC B cell selection. While our understanding of how Tfh cell help influences GC B cell selection in single-antigen GCs has significantly increased over the last few years, less is known in the setting of multi-antigen GCs, such as those induced by Influenza infection.

Notably, during the course of Influenza infection major structural epitopes are accessible to B and T cells to various extents as either external or internal proteins. In theory, GC B cells can therefore receive help from different pools of Tfh populations, containing cells specific for the same (intramolecular) or another (linked help) antigen as the B cells.

Recently, our lab developed LIPSTIC, an enzyme-mediated, proximity-dependent intercellular labeling technique, enabling us to directly record and quantify T cell help provided to B cells in vivo. Using LIPSTIC, we are studying how T cell help varies and is distributed among different viral protein-specific GC B cells and how competition for T cell help between different B cell clones shapes the antibody repertoire against flu. Furthermore, we aim to quantify the occurrence of intramolecular and linked T cell help as well as their contribution to GC selection.
Lack of IFN-g signaling leads to enhanced susceptibility to eschar-associated scrub typhus in mice
Authors: Liang Yuejin , Wang Hui , Gonzales Casey , Sun Keer , Sun Jiaren , Soong Lynn
Published: November 2024

Scrub typhus is an infectious disease caused by the bacteria Orientia tsutsugamushi (Ot). Severe infection can lead to organ failure and hemorrhage, resulting in fatality without proper treatment. The mechanism of how the host innate immune system responds to this infection remains elusive. By using an intradermal (i.d.) inoculation mouse model, we found that dendritic cells (DCs) and macrophages (MФ) were the major cell subsets responsible for bacterial uptake in dermis and dissemination into draining lymph nodes (dLN), leading to a systemic infection. This process was dependent on the chemokine receptor CCR7. Interferons (IFNs) are known to play a crucial role in microbial infection defense. Our data showed that the levels of IFNs increased significantly in the skin and lymph nodes after infection. To investigate the role of IFNs in Ot infection, we i.d. infected Ifnar1- or Ifngr-deficient mice and found that the lack of IFN-γ, but not IFN-I signaling, resulted in visible skin eschar lesions, higher bacterial burdens in dLN, exacerbated systemic infection, and animal lethality. Our in vitro study demonstrated that IFN-γ restricted bacterial growth in DCs and MФ, suggesting that IFN-γ may inhibit bacterial dissemination in vivo by signaling on bacteria-carrying myeloid cells. Overall, our findings reveal a Trojan horse mechanism of bacterial transmission and highlight the crucial role of IFN-γ signaling in host protection against scrub typhus.
TLR4 dependent generation of a novel bone marrow-derived inflammatory monocyte population required for elicitation of skin contact hypersensitivity responses
Authors: Kish Danielle , Fairchild Robert
Published: November 2024

Contact hypersensitivity (CHS) is a CD8 cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires infiltration of a novel population of Gr-1+ CXCR2+ cells that express FasL and perforin. The goal of the current studies was to further characterize these myeloid-derived cells and to determine mechanisms generating the cells in the bone marrow during elicitation of CHS. The cells were purified from the bone marrow of hapten sensitized mice 2 hrs after hapten challenge by depletion of CD3+, B220+, NK1+, F4/80+, and class II MHC+ cells. Flow cytometry analyses indicate >90% of the remaining bone marrow cells were CD11c+, Ly6C+, CCR2+, expressing FasL+ and perforin. This cell population was at low numbers in the bone marrow of naïve mice but increased to peak numbers 2 hrs. after hapten challenge of sensitized WT and Rag-/- mice as well as T-cell depleted WT. Generation of these cells was absent in hapten sensitized and challenged TLR4-/- mice. Currently, we are using spectral flow cytometry to focus on sensitization induced differences in hematopoietic progenitor cell populations in the bone marrow of TLR4-/- compared to WT mice. These results indicate that generation of bone marrow-derived Ly6C+ CXCR2+ FasL+ cells in response to inflammation requires TLR4 signaling that occurs independently of T cell activation.
The source of TAFA4 and its role in CNS immune cell recruitment during neuroinflammation
Authors: Tso Hwa-Sung , Yeh Xin-Mei , Wu-Hsieh Betty A , Miaw Shi-Chuen
Published: November 2024

TAFA4, also known as Fam19a4, has been reported to be secreted by C-low-threshold mechanoreceptors (C-LTMRs) and alleviate mechanical pain hypersensitivity. However, its immunological function in the central nervous system (CNS) remains incompletely understood. Our study aims to elucidate the wider source of TAFA4 in the CNS and its role in CNS inflammation. We generated lines of TAFA4-knockout (KO)-reporter mice, wherein the TAFA4 gene is deleted and replaced with a membrane-bound green fluorescent protein (GFP) and secretable nano-luciferase. We discovered that TAFA4 expression extended beyond C-LTMRs, encompassing neurons in the habenula-fasciculus retroflexus, hypothalamus, and olfactory bulb. Notably, the kinetics of TAFA4 expression followed a decreased trend during EAE-induced inflammation by nano-luciferase assay. Furthermore, we showed that mice with TAFA4 deficiency exhibited diminished immune cell infiltration and reduced demyelination in the spinal cord after EAE induction. These results underscore the involvement of TAFA4 in pro-inflammatory responses during EAE pathogenesis. Additionally, we showed that TAFA4 acted with chemoattractant properties towards M1 macrophages and neutrophils but not T cells by trans-well analysis. In summary, our study revealed the diverse sources of TAFA4 within the CNS and unveiled its inflammatory role during EAE inflammation. We propose that TAFA4 is potentially involved in CNS immune cell recruitment and inflammation.
Combination Therapy Blocking TNF Superfamily Members 14 and 15 Reverses Pulmonary Fibrosis
Authors: Steele Hope , Cheng Jason , Willicut Ashley , Dell Garrison , Ghastine Andrew , Nguyen Xinh-Xinh , Feghali-Bostwick Carol , Croft Michael , Herro Rana
Published: November 2024

Currently, anti-inflammatory drugs fail to reduce pulmonary fibrosis in the clinic and there is an urgent unmet need to develop novel anti-fibrotic drugs capable of reversing disease. Our lab has identified two novel mediators of pulmonary fibrosis belonging to the tumor necrosis factor superfamily (TNFSF), LIGHT (TNFSF14) and TL1A (TNFSF15). Here, we show LIGHT and TL1A receptor are both significantly elevated in patient pulmonary fibrosis biopsies as compared to healthy control lungs. Using gain of function studies, we found that LIGHT and TL1A can drive airway remodeling independently of one another and that these TNFSF members synergize to maximize airway inflammation and fibrosis. We show that the combinatorial blockade of LIGHT and TL1A limits TGFb-driven profibrotic effects on fibroblasts in vitro. Moreover, LIGHT and TL1A stimulation of human epithelial cells and fibroblasts reveal distinct fibrotic and inflammatory signatures, including redundant, additive, and synergistic profibrotic activities. Importantly, using antagonistic reagents to neutralize both LIGHT and TL1A signaling in combination post-disease onset, we observe a significant decrease in collagen deposition and smooth muscle hypertrophy in a bleomycin model of pulmonary fibrosis as opposed to respective monotherapies. The implication of this work highlights a therapeutic need of concomitantly targeting LIGHT and TL1A to treat pulmonary fibrosis disorders in humans.
Novel loss of function mutations in the death domain of tumor necrosis factor superfamily receptor 1A (TNFRSF1A) in children with systemic juvenile idiopathic arthritis (sJIA)
Authors: Cruz Anthony , Nakano Hiroto , Chou Sophia , Krantz Marisa , Fu Tianmin , Felicelli Christopher , Wu Hao , Ombrello Michael
Published: November 2024

Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory disease characterized by recurring fevers, arthritis, and hyperinflammation, but the etiology is unknown. Using family- and population-based sequencing approaches, we have identified several novel TNFRSF1A mutations. These include 2 mutations in the death domain (DD) of TNFR1 (P367S; E398K) and 1 that leads to truncation of the TNFR1 protein (K203X), fully eliminating the DD and intracellular domains. Protein modeling predicts that E398K will reduce the strength of TNFR1-TNFR1 interactions, while P367S will alter TNFR1-TRADD binding. Using flow cytometric Förster resonance energy transfer (FRET) analysis, we studied TNFR1 DD-mediated interactions. As predicted, interactions between E398K and both WT TNFR1 and TRADD are reduced, as is the interaction of P367S with TRADD. Further, proximal signaling complex formation is reduced in mutant-transfected cells by immunoprecipitation. Consistent with this, cell death is reduced in cells harboring TNFR1 mutants, while overexpression of mutant TNFR1 in NFkB reporter Jurkat cells reveals abrogated TNF responses. Analysis of soluble TNFR1 released into the supernatant of transfected cells shows K203X is released in a greatly enhanced manner unique to this mutation. Overall, we show that these mutations confer muted TNFR1 responses upon stimulation. Future studies will explore the mechanisms through which these loss of function mutations lead to an inflammatory phenotype.
GITRL expressed by regulatory B cells is the key to expanding T-regulatory cells to the level required to ameliorate autoimmunity
Authors: Gurski Cody , Khalil Mohamed , Beltrame Angela , Burns Robert , Dittel Landon , Sommers Kelli , Dittel Bonnie
Published: November 2024

CD4+Foxp3+ T regulatory cells (Treg) are well characterized for their role in controlling autoimmunity. We have previously shown that mice deficient in B cells (μMT) have significantly reduced Treg and cannot recover from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), demonstrating a regulatory role for B cells in immune tolerance. Using B cell depletion strategies and phenotyping, we discovered B cell IgD Low (BDL) regulatory B cells that when adoptively transferred into μMT mice increased Treg numbers and drove EAE recovery. BDL activity was found to be dependent upon glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL), which engages GITR on Treg inducing their proliferation. To investigate whether BDL activity could be enhanced, B cell-specific GITRL transgenic (tg) mice were obtained. These mice had three-fold more Treg than WT mice consistent with attenuated EAE. Adoptive transfer of GITRLtg BDL into μMT mice did not increase Treg numbers nor reduce EAE severity as compared to WT BDL. Here we posit that sustaining elevated Treg numbers is required to ameliorate EAE to clinical scores below that of WT, as seen in GITRLtg mice. This holds translational implications for many autoimmune diseases for which Treg cell therapies have been proposed. New approaches must be examined to not only increase Treg in autoimmune patients, but also to retain these levels over longer periods of time.
Intranasal and Intramuscular Immunization with a Universal Influenza A Virus Vaccine Induces Distinct and Protective Immune Responses in Mice
Authors: Zhang Alicia (Wanyue) , Li Sean
Published: November 2024

Influenza infections cause more than half a million deaths annually. Despite the prevalence of influenza vaccines, the vaccine efficacy varies widely (19-54%) due frequent mutations of the viral genome, a problem exacerbated by cross-species transmission. Although intranasal (IN) administration could enhance effectiveness, most current vaccines are intramuscular (IM) due to limited understanding of the IN mechanism. This study aims to compare the mechanism of protection conferred by an IN or IM delivered universal influenza vaccine. We designed a vector expressing a conserved antigen, the nucleoprotein (NP), fused with CD40 ligand, acting as both a targeting ligand and a molecular adjuvant. Only IN vaccinated mice were protected against H1N1, H3N2, and a highly pathogenic and transmissive H5N1 avian strain, showing high antibody levels and distinct cytokines along the respiratory tract. To further characterize the cell-mediated response, IN or IM vaccinated animals were treated with an agonist for sphingosine-1-phosphate receptors, inhibiting lymphocyte egress. After the treatment, only IN was protective against the viral challenge, indicating that IN induced a potent local cell-mediated defense without circulating lymphocytes. Further flow cytometric analysis revealed that IN induced proliferative tissue-resident T cells in the lungs. These findings suggest that IN vaccination induces superior cross-subtype protection by stimulating robust mucosal immunity.
The role of B cell and microglial BTK signaling in the pathogenesis of experimental autoimmune encephalomyelitis.
Authors: Ogbaslase Abraham
Published: November 2024

Bruton’s tyrosine kinase (BTK) is a key signaling molecule expressed in B cells and microglia. While the contribution of BTK signaling in B cells to the pathogenesis of multiple sclerosis has been explored to some degree, the involvement of microglial BTK signaling to disease remains largely uncharacterized. We hypothesize that BTK signaling regulates features of microglia and B cells that drive disease progression in experimental autoimmune encephalomyelitis (EAE). Using a global-BTK KO model we have found that BTK deletion confers a protective effect against the development of EAE. Additionally, in our CD79-cre conditional KO system, deletion of BTK reduces the ability of B cells to serve as antigen presenting cells and dampens T cell responses following EAE induction. Within the central nervous system of BTK KO mice there is a reduction in B cell expression of MHC II and co-stimulatory molecules as well as a reduction in the frequency of overall CD4 (p = 0.0079, n ≥ 13) and effector CD4 (p = 0.0857, n ≥ 13) T cells compared with WT. Additionally, BTK KO mice produce less anti-rodent MOG antibody in comparison to BTK-replete controls. We have also observed that the protective effect of BTK deletion is only partially driven by BTK signaling in B cells, leading us to explore microglial contribution to disease pathogenesis. Induction of EAE in a CX3CR1-cre-ert2 conditional knock-out system will determine the contribution of microglial BTK signaling to disease pathogenesis.
BCLAF1 is a novel regulator of fetal and adult hematopoietic stem cell function
Authors: Crowley Stephanie , White Lynn , Yang Wei , Li Yanan , Wu Jun , Schmidt Haley , Magee Jeffrey , Bednarski Jeffrey
Published: November 2024

Hematopoietic stem cells (HSCs) adapt to blood production and maintenance needs of a developing organism. Mouse fetal liver HSCs undergo rapid perinatal proliferation and possess high repopulation capacity. Adult HSCs establish residence in the bone marrow and lose repopulation capacity with age. This shift is characterized by epigenetic and transcriptional cues. The objective of our study is to determine the developmental context-dependent role for BCLAF1 in HSC function. Conditional loss of BCLAF1 results in a reduction of fetal HSCs that persists into adulthood, while induced deletion of BCLAF1 in adult mice does not affect HSC numbers. These results suggest the context of fetal liver HSC development may exacerbate effects of BCLAF1 loss. In competitive transplant models, we have found that both fetal and adult BCLAF1-deficient HSCs have significantly reduced repopulation capacity. CITEseq of fetal Vav-Cre:Bclaf1fl/fl hematopoietic cells reveals a reduction in long-term repopulating HSCs and elevated AP-1 gene expression. Consistent with these results, in vitro HSC differentiation culture assays demonstrate that BCLAF1-deficient HSCs have increased myeloid differentiation. Current BCLAF1 CUT&RUN sequencing experiments on fetal and adult HSCs will define specific changes in DNA-binding patterns of BCLAF1 over development. Collectively, these results suggest that BCLAF1 acts in fetal and adult HSCs to transcriptionally restrain AP-1 activity and promote HSC function.
Conditionally immortalized cell lines with mast cell potential
Authors: Salehi Faraz , Redecke Vanessa , Häcker Hans
Published: November 2024

Prior to the establishment of definitive hematopoiesis, mast cells (MCs) derived from the yolk sac and fetal liver colonize diverse tissues, maintaining a presence throughout life. Following the establishment of definitive hematopoiesis, hematopoietic stem cells from the bone marrow (BM) also contribute to the MC pool. The diverse origins and tissue-resident nature of MCs complicate their study. Therefore, a system facilitating the expansion of MC progenitors and their controlled differentiation becomes crucial. This report outlines the development of conditionally immortalized MC progenitor cell lines based on two tissue sources, i.e. mouse fetal liver and adult bone marrow. To obtain cell lines, retroviral vectors encoding an estrogen-regulated Hox gene were introduced into progenitor cells derived from both tissues. These transduced cells can be expanded in their non-differentiated state for extended periods and upon estrogen removal, these cells differentiate into MCs in the presence of IL-3 and SCF. Resulting mast cells are functionally comparable to BM-derived MCs, initiating degranulation upon IgE cross-linking. Likewise, these MCs express MC-specific proteases such as chymase 1 and MCPT4. In conclusion, our findings highlight the efficacy of estrogen-regulated Hox genes in targeting MC progenitors in both fetal liver and adult BM. The established cell lines serve as reliable tools for studying MC biology, ontogeny and, possibly, disease conditions, such as allergies.
Chaotic hematopoiesis in homeostasis
Authors: Jia Dongya , Mukherjee Ratnadeep , Altan-Bonnet Grégoire
Published: November 2024

Hematopoiesis is a highly dynamical and stochastic process, challenging classical models of homeostasis. By number, there are around 330 billion cells turning over every day in a human body and 86% of which being blood cells. Clinical studies of leukemia and neutropenia patients suggested that multiple blood cell types are vibrantly fluctuating with large yet controlled variation. Yet the establishment and dynamics underlying homeostasis in the hematopoietic system remains elusive.

We integrated high-dimensional cytometry (mass and spectral cytometry) with mathematical modeling, and performed both cross-sectional study (n=39) and longitudinal study (n=10) of healthy mice. We found that all cell types exhibit large variations of cell numbers (10-100-fold-change in general) in bone marrow, blood and spleen. We showed that the time-series of leukocyte population size (B cells, neutrophils etc.) all exhibit chaotic behaviors i.e. the leukocyte population size demonstrated a strong sensitivity to initial conditions, and a broad spectrum of characteristic timescales of fluctuations. Using mathematical modeling, we accounted for the emergence of chaotic dynamics and predicted that accumulation of B cells (akin to lymphoma development) would transition the blood cell dynamics from chaos to oscillations (as observed for leukemia patients). Such model challenges our textbook understanding of homeostatic hematopoiesis, opening new avenues of research for immunotherapeutic manipulations.
LMAN1 promotes enhanced Alternaria alternata-induced airway hyperresponsiveness through alteration of alveolar macrophage and ILC2 populations
Authors: Sauber Faith , Swaby Lindsay , Zhang Bin , Tigno-Aranjuez Justine
Published: November 2024

Current biologics often fail to effectively manage symptoms in severe allergic asthmatic patients, necessitating the exploration of novel therapeutic approaches. Our laboratory has recently identified LMAN1 as a novel regulatory cell surface receptor for house dust mite (HDM). Binding of LMAN1 to HDM allergens was found to depend on mannosylation, suggesting a potential broader role of this receptor in the recognition of other highly mannosylated allergens such as molds. Alternaria alternata is a ubiquitous mold often associated with severe asthma. Whether LMAN1 can also act to regulate responses to Alternaria, remains to be explored. We demonstrate that the immunodominant Alternaria allergen Alt a 1 can also bind to purified Fc-LMAN1, indicating potential involvement of this receptor. Thus, we subjected both WT and LMAN1 knockout mice to an Alternaria alternata-induced asthma model. The absence of LMAN1 resulted in a substantial increase in airway hyperresponsiveness (AHR) compared to WT animals. Interestingly, changes in AHR did not correlate with enhanced eosinophilia but, instead, went hand in hand with a reduction in alveolar macrophages and an increase in type-2 innate lymphoid cells. Work is currently ongoing to further investigate the cellular and molecular mechanisms underlying these findings. This discovery highlights LMAN1 as a promising target for innovative therapeutic interventions.
Epigenetic reprogramming of CD8+ T cell populations drives exhaustion in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)
Authors: Iu David , Maya Jessica , Vu Luyen , Fogarty Elizabeth , Munn Paul , Grenier Jennifer , Hanson Maureen , Grimson Andrew
Published: November 2024

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a severe, debilitating disease with striking similarities to long-COVID/PASC, but its etiology remains unclear. Mounting evidence implicates immune dysfunction as a potential cause for ME/CFS. We previously reported metabolic changes in ME/CFS CD8+ T cells, including increased fatty acid oxidation, decreased glycolysis, and reduced mitochondrial membrane potential - features commonly found in exhausted T cells. To comprehensively characterize the gene regulatory state underlying potential transcriptional dysregulation, we first re-analyzed a single cell transcriptomic atlas of ME/CFS by reclassifying CD8+ T cells, where we found that various clusters upregulated exhaustion-associated genes. We then generated high resolution profiles of gene expression and chromatin accessibility in effector memory (TEM) and naïve CD8+ T cells by RNA-seq and ATAC-seq at baseline and further analyzed different CD8+ subsets following symptom provocation. We observed in ME/CFS TEM upregulation of key transcription factors associated with exhaustion and downregulation of genes controlling key metabolic activities, as well as an altered chromatin landscape consistent with exhaustion programs, while ME/CFS TN displayed a potential loss of naïve identity. Our dataset serves as an important resource for understanding ME/CFS pathology and supports a novel state of immune dysregulation in ME/CFS which may offer new treatment options.
Investigating biomarkers and therapeutic approaches in mouse models of SARS-CoV2 induced Long COVID with neuroinflammation and cognitive deficits.
Authors: Lu Peiwen , Fernández-Castañeda Anthony , Geraghty Anna C , Song Eric , Monje Michelle , Iwasaki Akiko
Published: November 2024

Survivors of COVID-19 frequently experience lingering neurological symptoms including cognitive impairment. However, the absence of suitable animal models presents a significant challenge in comprehensively understanding the post-acute sequelae of SARS-CoV-2 (PASC) or Long COVID. Utilizing a mild SARS-CoV2 infection mouse model, we explored neuroinflammation caused by respiratory SARS-CoV2 infection in mouse with AAV-hACE2 delivery in lower respiratory system only and found white matter selective microglial reactivity. After subjecting mice to mild SARS-CoV-2 infection for 7 weeks, we observed persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss along with increased CCL11 in cerebrospinal fluid (CSF). Notably, individuals experiencing lasting cognitive symptoms post-COVID exhibited elevated CCL11 levels, suggesting its potential as a biomarker for Long COVID. Concurrently, we developed a model of moderate mortality SARS-CoV-2 infection in K18-hACE2 transgenic mice by modifying the infection route, allowing us to study the development of neuropathology and simulate Long COVID patients with a history of hospitalization at the acute SARS-CoV-2 infection stage. To address neuroinflammation in the mild respiratory COVID and moderate mortality COVID mouse model, we are targeting reactive microglia for therapeutic intervention, evaluating various approaches.
Role of mTOR and HIF-1α in LMP2A-mediated increase in ATP production
Authors: Swanson-Mungerson Michelle , Devitt Grace , Braich Kamiljit , Salimian Melody , Monroy Del Toro Rosalinda , Incrocci Ryan
Published: November 2024

Numerous studies indicate that latency proteins from Epstein-Barr virus contribute not only to the development, but also the survival of B cell lymphomas. Our laboratory has demonstrated that LMP2A promotes B cell lymphoma survival by inducing the activation of STAT3. More recently, pathways that link STAT3 activation with increased cellular metabolism suggested that LMP2A may increase B cell lymphoma ATP production via the mTOR-dependent activation of STAT3. Using LMP2A-negative and -positive B cell lymphoma cell lines, our data suggest that LMP2A increases ATP production in an mTOR-dependent pathway. However, the addition of the STAT3 inhibitor, STATTIC, did not impact the LMP2A-mediated increase in ATP production. Further investigation indicated that LMP2A-mediated activation of mTOR results in increases in HIF-1α protein, but not RNA, that are required for the enhancements in ATP production. Finally, our data demonstrate that LMP2A requires the activation of phosphorylated p70 ribosomal s6 kinase and 4E-BP1 to increase protein levels of HIF-1α that are required to increase ATP levels in LMP2A-expressing cell lines. Taken together, our data suggest that LMP2A gives lymphoma cells a competitive advantage to produce more ATP through increases in HIF-1α that are mTOR and p70s6K/4E-BP-1 dependent.
Amelioration of lung inflammation induced with viral infectious stimulation by a high-molecular weight sulfated polysaccharide compound, Fucoidan mix-AG
Authors: Miyazaki Yoshiyuki , Nakano Hayato , Takeuchi Shugo , Takeuchi Hideaki , Tachikawa Daisuke
Published: November 2024

Fucoidan, viscous components in brown algae, has been reported to possess the effect enhancing cellular immune responses against cancer and viral infections. In contrast, it is also known to contribute to the alleviation of pathology in some inflammatory diseases, and almost certainly be effective in maintaining mucosal immune homeostasis. In this study, we evaluated the mitigating effect of fucoidan on pneumonia in aggravated upper airway infection. Consequently, C57BL/6J mice were orally administered 30 mg of Fucoidan mix-AG (Fm-AG) daily for 2 weeks, and then a solution of TLR ligands (TLR3-L: 62 µg Poly(I:C), TLR4-L: 25 µg rSARS-CoV-2 spike protein, TLR7-L: 5 µg Gardiquimod) were injected via oropharyngeal aspiration to simulate lung inflammation in viral infections. As a result, enhanced cellular infiltration into bronchoalveolar lavage (BAL) fluid were observed after the treatment with TLR ligands, but the expression of class II MHC molecules on the leukocytes was reduced in the Fm-AG-administrated mice as compared with the control mice. In addition, the markedly augmented production of inflammatory cytokine IL-6 in the model mice were significantly lowered by administration of Fm-AG both in BAL fluid and plasma. However, no reduction in the level of anti-viral IFNs were observed in the mice administrated Fm-AG. These results suggested that continuous intake of Fm-AG would prevent the severe complications (pneumonia and cytokine storm) during upper airway infections.
Autism-related CHD8 is required for maintaining Treg cell fitness
Authors: Yang Junqi , Zheng Yi , Guo Fukun
Published: November 2024

CHD8 is an adenosine triphosphate (ATP)-dependent chromatin remodeler, the mutations of which define a subtype of autism spectrum disorders. CHD8 has recently been found to be important for hematopoietic stem cell survival and erythroblast cytokinesis. It remains unclear whether CHD8 plays a role in regulatory T (Treg) cells that suppress effector T cells such as CD4+ Th1/Th2/Th17 cells and CD8+ cytotoxic T cells to maintain immune tolerance. In this study we have generated Treg cell-specific CHD8-deficient mice. We found that CHD8 deletion led to early, fatal systemic inflammatory disorders. The autoimmune responses came from an increase in activated CD4+ and CD8+ T cells and in effector T cells including Th1, Th2 and Th17 cells. The immune activation was due to increased Treg cell plasticity as evidenced by increased effector T cell cytokine expression in Treg cells. Paradoxically, CHD8 deletion upregulated Treg cell functional markers such as CTLA-4, PD-1, GITR and ICOS, indicative of functional exhaustion. RNA-seq revealed that CHD8 deletion upregulated a number of hallmark pathways including inflammatory response, p53 pathway, apoptosis, G2M checkpoint, mTORC1 signaling, glycolysis, oxidative phosphorylation, and reactive oxygen species pathway. Collectively, our findings suggest that CHD8 is required for maintaining Treg cell fitness and function by modulating p53-mediated cell survival and cell cycle progression, and mTOR-mediated mitochondrial metabolism.
Mechanisms of pTreg development from anergic precursors
Authors: Ristin Svetlana , Nivelo Luis , Stelekati Erietta , Villarino Alejandro
Published: November 2024

Regulatory T cells (Tregs) are generated either in the thymus or periphery. While factors governing ‘natural’ thymus-derived Treg (nTreg) development are well described, there is limited understanding of those directing peripheral regulatory T cell (pTreg) differentiation. Using a mouse model of oral tolerance, we demonstrate that T cells must first enter a state of anergy prior to becoming pTreg, suggesting a two-stage process. Stage 1 is characterized by T cell anergy with prominent induction of ‘checkpoint’ inhibitory receptors, most notably PD-1 and CD200. Stage 2 then follows with pTreg conversion or, alternatively, adoption of a ‘deep’ anergic state. Given this induction of inhibitory receptors, we assessed the impact of PD-1 deficiency and found that T cells proliferate more and convert less to pTreg. Intriguingly, we also find that PD-1 expression distinguishes pTreg derived from anergic precursors from those derived from non-anergic precursors. Based on scRNAseq data showing that IL-2-STAT5 signaling is highly active in pTreg, we also assessed the role of IL-2Ra and STAT5 and found that both are strictly required for induction of pTreg, suggesting that this pathway drives transition from Stage 1 to Stage 2 tolerance. Taken together, our data shows that transition from Treg-independent (Stage 1) to Treg-dependent (Stage 2) tolerance is driven by cell extrinsic signals provided by inhibitory receptors and IL-2-STAT5 signaling which combine to induce pTreg development.