bluuebunny/crossref_metadata_2025 huggingface dataset:
An image-based real time method to measure antibody-dependent cellular phagocytosis
Authors: Shi Yongchang , Wang Jianyong , Sun Yonglian , Koerber JT , Seki Akiko , Rutz Sascha
Published: November 2024

Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. In vitro quantification of ADCP has been technically challenging. Fluorescence microscopy and flow cytometry based assays were traditionally used to measure ADCP activity, these methods were static measurements at certain time point, thus provide no information about engulfment kinetics. Moreover, the flow cytometry assays require lifting the macrophage cells from the labware which may affect macrophage activities. We have developed a real time ADCP assay using a live cell analysis system (IncuCyte). The target cells labeled with a pH sensitive dye only fluoresce when they are engulfed in acidic phagosomes of macrophages in the presence of specific antibodies. Our assay is a plate-based with a simple mix and read protocol without disturbing the macrophages. The new method is quantitative and robust, can be readily used for high-throughput screening for functional antibodies. By studying the ADCP activities in different subtypes of macrophages, we found M2 microphages showed the strongest ADCP activity compare to M0 and M1 macrophages, due to their different expression levels of FcgRs. The assay is highly sensitive to differentiate the antibody variants with enhanced Fc function. It can also be used to study the kinetics and mechanisms of ADCP.
Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca™ PLX Image Cytometer
Authors: Chan Leo , Pierce Mackenzie , Huang Yongyang , Lin Allen , Lin Bo
Published: November 2024

Apoptosis is a critical pathway for programmed cell death that cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare cancer that can initiate the process of apoptosis, allowing for the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V-APC, Caspase 3/7, and Propidium Iodide (PI) using the Cellaca™ PLX Image Cytometer. First, apoptosis was induced in Jurkat and K562 with staurosporine and monitored over the course of 24 h. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early to late-stage apoptosis, and ultimately necrosis. A clear trend was observed during the first 2 h showing differences of up to 25% between the single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis amongst apoptotic populations, Annexin V+ only populations were higher than Caspase-3+ only populations by up to 30% between 0-2 hours. The proposed image-based detection method may provide an effective and efficient tool that allows researchers to better characterize and screen potential cancer therapeutic drug candidates in a high-throughput manner to determine their treatment efficacy.
Development of an ultra-sensitive multiplex cytokine assay utilizing a novel digital multi-omics detection technology
Authors: Lambert Jeremy , Proctor Angie , Seigfried Nate
Published: November 2024

Demand for rapid, highly sensitive measurement of proteins and nucleic acids has increased dramatically over the past several years to support biomarker research, drug development and diagnostics. In addition, the ability to interrogate both protein and nucleic acids (multi-omics) in a single sample allows critical information to be obtained relating the transcriptome to expressed protein levels. Despite this, current platforms for multiplex measurement of circulating biomarkers can lack sensitivity and specificity required for analytical and clinical validation of low abundance proteins and nucleic acids.

We report the development of an automated microfluidic-based technology capable of multiplexed measurement of protein and nucleic acid biomarkers utilizing a novel approach combining digital and analog detection modalities. Significant improvements in sensitivity and specificity are achieved through use of a bead-based microfluidic array resulting in a spatially-resolved series of single-plex signal amplification reactions. Critically, the isolation of reactions allows multiplex panels to be constructed in a modular manner while eliminating interference that often limits specificity of traditional multiplex biomarker detection technologies. A specific application of this technology we report here is a multiplexed assay measuring low abundance cytokine biomarkers from serum and plasma samples with utility in a broad range of research and clinical studies.
Genetic targeting of the mouse immunoglobulin locus to permit the expression of heavy-chain only antibodies against viral antigens
Authors: Borbet Timothy , Zaldana Kimberly , Morrison Tate , Boehringer Thomas , Gonzalez Sandra , Chan Kun-Wei , Luo Christina , Kirilov Milen , Kong Xiang-Peng , Wang Wei , Sheiffele Paul , Koralov Sergei
Published: November 2024

Conventional antibodies are composed of two identical sets of heavy and light chains, forming a dimer. The CH1 domain of the antibody heavy chain is essential for the pairing of heavy and light chains. However, certain animals, such as camels and sharks, evolved to produce heavy-chain-only antibodies (HCAbs) by losing this crucial CH1 domain in some of their antibody heavy chain constant regions. These HCAbs boast unique physicochemical properties that confer significant advantages: their smaller size allows for improved access to target antigens, increased hydrophilicity, enhanced tissue penetration, and greater structural stability compared to standard antibodies. Our research has successfully established a mouse model that intrinsically produces HCAbs from the mouse's own immunoglobulin heavy chain locus. Our findings demonstrate that heavy-chain-only B cell receptors, once introduced, facilitate normal B cell development, promote germinal center formation, and enhance plasma cell differentiation relative to B cells producing conventional antibodies. These genetically modified mice were vaccinated with viral antigens, resulting in the production of HCAbs that specifically target the SARS-CoV-2 spike protein and the HIV-1 Env trimer. Through single-cell sequencing, we have identified unique nucleotide sequences of clonotypes; the HCAbs are being synthesized to assess their viral neutralization potential and antigen-binding affinity to highlight the efficacy of our model.
Synthetic TCR/BCR RNA spike-in controls for assessing immune receptor profiling assay accuracy
Authors: Chenchik Alex , Liu Tianbing , Makhanov Mikhail , Hu Dongfang , Ghias Khadija , Diehl Paul
Published: November 2024

Synthetic TCR and BCR RNA spike-in controls can be used as universal standards to account for biases caused by PCR and NGS steps of adaptive immune receptor (AIR) repertoire profiling assays. We designed 48 BCR and 39 TCR mRNA synthetic constructs to cover nearly full-length V(D)JC structures, which represent all seven TCR (TRB, TRA, TRG, TRD) and BCR (IGH, IGK, and IGL) hypervariable chains. To identify cross-sample contamination, we spiked in 2 ul of BCR (16x3) and TCR (13x3) triplex isoform pools and detected the presence of the sequence in the sample. Furthermore, we integrated 48 BCR and 39 TCR premixed controls which are mixed in a 16:4:1 ratio into Cellecta’s DriverMap™ Adaptive Immune Receptor Profiling Assay, which employs a gene-specific, multiplex RT-PCR method with unique molecular indices (UMIs). The spike-in controls coupled with UMI-based PCR amplification can help distinguish between control and background sequences. We observed a consistent linear relationship between the concentration of spike-in controls and their molecular counts, with an average sequencing error rate between 0.4%-0.8% per base, which is in line with Illumina sequencing standards. These findings validate the effectiveness of our synthetic spike-in controls in correcting biases in AIR protocols, offering a reliable estimation of error and mutation rates in the DriverMap AIR assay or similar NGS-based immune receptor profiling methods.
The First Case of Autosomal Recessive CADINS-like Disease Caused by Novel Compound Heterozygous Hypomorphic Variants in CARD11.
Authors: Spratt Alison , Palterer Boaz , Bauman Bradly , Salvati Lorenzo , Mazzoni Alessio , Maggi Laura , Capone Manuela , Vanni Anna , Provenzano Aldesia , Giglio Sabrina , Cosmi Lorenzo , Liotta Francesco , Parronchi Paola , Annunziato Francesco , Snow Andrew
Published: November 2024

CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor ligation to signaling governing lymphocyte proliferation and differentiation. We and others have shown that heterozygous, dominant negative (DN) CARD11 mutations disrupting T cell receptor (TCR)-induced NF-κB signaling cause severe atopy in humans, often with recurrent infections and humoral immune deficits. Here we describe a patient with a history of recurrent upper respiratory infections, recalcitrant atopic dermatitis and common variable immunodeficiency, carrying novel compound heterozygous CARD11 variants (G689E, R920L) in regions of the protein (PDZ and L4, respectively) not normally associated with deleterious mutations. By transfecting variant expression plasmids into CARD11-deficient Jurkat T cells harboring an NF-κB-driven GFP reporter, we found that G689E was mildly hypomorphic, whereas R920L was a complete loss-of-function mutation with negligible DN activity against wild-type CARD11. However, TCR-induced NF-κB activity was markedly reduced upon variant co-expression, strongly suggesting this patient represents the first autosomal recessive case of CARD11-associated Atopy with Dominant Interference of NF-κB Signaling (CADINS) disease. Additional experiments examining different amino acid substitutions at these residues highlight key biochemical interactions between the PDZ and L4 domains, with important implications for future CARD11 structure-function and clinical diagnostic studies.
Deletion of MOF in CD4 T cells results in mitochondrial dysfunction, Th17 polarization, and colonic inflammation.
Authors: Teixeira Lima Felipe , Goddard Yana , Wheeler Matthew , Qu Ganlin , Diodati Nikolas , Sordo-Vieira Luis , V. Lazarte Arantxa , A. Schaller Matthew , Mehrad Borna
Published: November 2024

Introduction: The enzyme, MOF (males absent of the first), is a histone acetyltransferase that regulates the transcription of nuclear and mitochondrial genes. The role of MOF in acquired immunity has not been established.

Hypothesis: Expression of MOF controls CD4 T cell phenotype by regulating cellular metabolism.

Materials, Methods, and Results: We generated mice selectively deficient in MOF in CD4 T cells (Moff/f CD4cre). After ex vivo stimulation with CD3 and CD28, MOF-deficient splenic CD4 T cells displayed lower proliferation rate and viability, associated with altered mitochondrial morphology by electron microscopy, impaired oxidative phosphorylation, and higher glycolytic capacity, lactate production, and higher ATP production. In vivo, Moff/f CD4cre mice developed spontaneous rectal prolapse and colonic cryptitis. Intraepithelial and lamina propria CD4 T cell from these animals were disproportionately activated and displayed a Th17 phenotype. Treating these animals with a mitochondrion-targeted antioxidant, mitotempo, for a month resulted in normalization of T cell ATP production and lactate production.

Conclusion: The histone acetyltransferase MOF moderates T cell activation and polarization by controlling mitochondrial respiration, protecting against the spontaneous development of colitis.
Defining the role of PSTPIP2 in neutrophil inflammatory function
Authors: Momjian Claire , Ratitong Bridget , Fernandes Sheryl , Jorth Peter , Cassel Suzanne , Sutterwala Fayyaz
Published: November 2024

Our lab and others have found that neutrophils isolated from Pstpip2cmo mice, that are deficient in proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), display a hyperinflammatory phenotype with increased IL-1b and ROS secretion following stimulation. The hyperactive neutrophil phenotype is critical to the spontaneous development of an autoinflammatory disorder called chronic multifocal osteomyelitis. However, there are gaps in our understanding of the underlying mechanism that leads to the observed phenotype in Pstpip2cmo mice which could further our understanding of neutrophil biology. We first infected WT and Pstpip2cmo mice with P. aeruginosa to elucidate the importance of PSTPIP2 in vivo. 24-hours post infection, Pstpip2cmo mice had significantly lower bacterial burden compared to WT mice. We found increased neutrophils and monocytes in the bronchial alveolar fluid (BAL) of infected Pstpip2cmo mice, and these neutrophils are lower in side-scatter measured by flow cytometry. Under homeostatic conditions, we observed higher numbers of neutrophils in the bone marrow, blood, and spleen of Pstpip2cmo mice, and more granulocyte progenitors. In addition to elevated cell numbers, we found increased IL-1β secretion NETosis compared to WT mice. Together these data suggest that PSTPIP2 plays a critical role in restraining neutrophilic inflammatory response to bacterial infection and affects granulopoiesis in naïve mice.
Regulation of B1 cells response to Francisella tularensis LPS, a type-2 T-independent antigen
Authors: Barbosa Carlos Henrique D , Mello Guilherme Afonso , Re Fabio
Published: November 2024

Francisella tularensis (Ft), a Gram-negative bacterium, posesses atypical LPS that does not activate TLR4 and is considered a type 2 T-independent antigen (TI-2 Ag). Our group has recently shown that immunization with Ft-LPS elicits the production of protective Ft-LPS-specific IgM (IgMFt) by B1 cells independently of MyD88, in a mechanism that is dependent of the IL-25-ILC2-IL-5 axis. Immunization, but not infection, generates peritoneum-resident memory B1 cells that differentiates into IgMFt plasmacells upon secondary challenge but does not boost the production of IgMFt.

TI antigens were believed to be unable to generate memory response or long-lived plasma cells. This view is being challenged by several studies. This work aims to elucidate the mechanisms driving the class switch and memory response of B1 cells to Ft-LPS.

Here we show that peritoneal memory B1 cells transferred into Rag-/- mice produce higher amounts of IgM compared to naïve B1 cells. Moreover, the transferred naïve B1 cells did not class switch to IgG3/IgG2b upon immunization, suggesting involvement of FDCs and/or T cells. The presence of IgGFt inhibited the IgMFt response. Complement depletion in WT mice also inhibited class switch to IgGFt but did not affect IgMFt production and resulted in better boost effect. Our results might shed light on new strategies to improve vaccination against TI-2 Ags and on involvement of germinal center and complement proteins for the activation of B1 cells
Dietary and circadian cues regulate gut barrier integrity by synchronizing epithelial cell-macrophage crosstalk
Authors: Dende Chaitanya , Srinivasan Tarun , Propheter Daniel , Quinn Gabriella , Li Yun , Ruhn Kelly , Pendse Mihir , Brooks John , Maples Robert , Raj Prithvi , Pfeiffer Julie , Blander Julie , Lira Sergio , Hooper Lora
Published: November 2024

Small intestinal epithelial cells (sIECs) form a critical barrier essential for nutrient absorption and host defense. sIECs undergo continuous renewal to maintain their integrity. Phagocytes, such as macrophages, facilitate the clearance of apoptotic cells that enter subepithelial tissues. However, little is known about how sIEC-phagocyte crosstalk maintains barrier integrity. Our study reveals synchrony between sIEC death and the accumulation of a specific subset of macrophages engaged in engulfing dying cells. Notably, we observed a diurnal rhythmicity in the death of sIECs during homeostasis. A fraction of these dying cells enter the subepithelial space, where Tim4+ CD4+ macrophages rhythmically eliminate them. Our findings demonstrate that Tim4+ CD4+ macrophage maintenance depends on dietary vitamin A via macrophage-intrinsic retinoic acid receptor (RAR) signaling. We show that rhythmic Tim4+ CD4+ macrophage accumulation relies on light cues and the macrophage circadian clock. Impaired macrophage RAR signaling disrupts the clearance of dying sIECs, leading to compromised gut barrier integrity and increased bacterial translocation to distant organs. Our work reveals an intricate relationship between sIECs and phagocytes and identifies a crucial role for Tim4+ CD4+ macrophages in maintaining intestinal barrier integrity. Understanding these dynamics could suggest new strategies for maintaining intestinal barrier function during infection and inflammatory disease.
Bhlhe40 in Group 3 Innate Lymphoid Cells Shapes TL1A-dependent Commensal T cell Immunity
Authors: Yang Wei , Pires Silvia , Oguntunmibi Seun , Diehl Gretchen , Edelson Brian , Longman Randy
Published: November 2024

A central feature of intestinal mucosal immune system is the ability enable antigen specific tolerance to the gut bacteria. Genetic variants in TNFSF15 (which encodes protein TL1A) are linked to inflammatory bowel disease and have implicated roles for TL1A in regulating both T cell and innate lymphoid cells (ILC), but how these cellular pathways coordinate a response to limit intestinal inflammation is not completely understood. Deletion of TL1A receptor Tnfrsf25 (also called DR3), we demonstrate that TL1A signaling is required for efficient generation of RORγt+Foxp3+ intestinal iTregs at steady state. To investigate if this response resulted from DR3 deletion in T cells or the potential role for DR3 signaling in ILCs to regulate iTregs indirectly, we generated antigen specific transgenic T cells to H. Hepaticus (Hh) with DR3 deletion. Our results reveal that TL1A signaling is dispensable on T cells but required on ILC3s to regulate the balance between intestinal iTreg and inflammatory Th17 cells. RNA sequencing of ILC3s revealed the induction of Bhlhe40 in response to TL1A stimulation. Using conditional deletion models, we defined Bhlhe40 as a transcriptional regulator of ILC3 expression of Tnfsf4 (OX40L) and the subsequent requirement for ILC3-specific Bhlhe40 and Tnfsf4 in promoting Hh-specific Treg induction. These data reveal a novel function for TL1A and downstream transcription factor Bhlhe40 in enabling ILC3 coordination of gut commensal T cell immunity.
Examining the antitumorigenic effects of Ruminococcus gnavus
Authors: Laughlin Colin , McPherson Alex , Pandey Surya , Shapira Jake , Phelps Catherine , Lee Amanda , Randhawa Simran , Gottschalk Rachel , Yang Zhen , Hinterleitner Reinhard , Meisel Marlies
Published: November 2024

Melanoma is one of the most commonly occurring cancers in the world, with increasing occurrence over the past several decades. Common treatment regimens for melanoma include immune checkpoint inhibitor (ICI) therapy, which enhances interferon-γ production in CD8 T cells (Tc1). Despite showing efficacy in some patients, a majority of melanoma patients are resistant to ICI therapy. Prior studies have demonstrated the ability of the gut microbiota to impact Tc1 antitumor responses and ICI therapy efficacy. In analyzing top gut bacteria enriched in ICI-responders, Ruminococcus gnavus (R. gnavus) is a commonly identified species. Here we demonstrate the tumor restraining properties of R. gnavus in subcutaneous, spontaneous, and metastatic cancer models. Further, gnotobiotic monocolonization experiments demonstrate the ability for this bacterium to restrain tumor growth independent of a complex microbiome. Analyzing the mechanism behind how R. gnavus causes melanoma growth suppression in our model, we have demonstrated requirements of bacterial viability and aryl hydrocarbon receptor (AHR) activation. Colonization with R. gnavus at various tumor timepoints was able to induce a significant Tc1 response locally in the tumor and systemically in lymphoid organs. Finally, we found R. gnavus colonization to facilitate ICI efficacy in our model. This study uncovers a potential mechanism of how a common ICI-response associated bacterium enhances ICI efficacy in melanoma.
Preclinical pharmacology of S-1117, a novel engineered Fc-fused IgG cleaving enzyme, for chronic treatment of autoantibody-mediated diseases
Authors: Sanmarco Liliana , Pellerin Alex , Anderson Jordan , Rollins Nathan , Newton Andita , Green Tobias , Le Nam , Peckner Ryan , Mande Purvi , Xing Xi , Manasson Julia , Higginson-Scott Nathan , Sundy John , Otipoby Kevin , Mascanfroni Ivan
Published: November 2024

Pathogenic autoantibodies are key effectors of inflammation, promoting complement activation and immune cell responses that cause tissue damage in autoantibody-mediated diseases such as myasthenia gravis. IgG-proteases represent a new therapeutic opportunity. Here we present S-1117, a novel Fc-fused pan-IgG protease, engineered using a proprietary machine learning enabled platform to reduce immunogenicity and augment manufacturability and stability while maintaining enzyme activity. S-1117 cleaves soluble IgG and the BCR on memory B cells with comparable potency and significantly reduces antibody-dependent cytotoxicity (ADCC) and immune complex-mediated PBMC activation in vitro. When tested in vivo, a deep reduction of human IgG (>90%) occurred within 30 minutes after dosing. A PK/PD model developed for human projections predicts rapid, deep and sustained reduction of IgG levels. In addition, in an anti-glomerular basement membrane (GBM) model, a prototype Fc-fused pan-IgG protease reduced complement activation, IC deposition, which correlated with reduced proteinuria, blood urea nitrogen and renal pathology. In summary, S-1117 is a novel engineered pan-IgG protease that demonstrates rapid and sustained reduction of IgG levels and reduces multiple pathological manifestations in a murine nephritis model. Given its ability to address multiple pathogenic mechanisms as a single drug, it has the potential to achieve improved clinical outcomes in autoantibody-mediated diseases.
Biochemical and Immunological Characterization of 3HKA; a Novel Anti-inflammatory Biogenic Amine
Authors: Clement Cristina , Buque Aitziber , Mota Ines , Nanaware Padma , Santambrogio Laura
Published: November 2024

Tryptophan (Trp) catabolism generates over fifty metabolites, including kynurenines, powerful mediators of immune regulation. We recently reported that: (i) lymphatic endothelial cells (LEC) and dendritic cells (DCs) secrete a previously unidentified biogenic amine, 3HKA, which derives from a lateral pathway of Trp catabolism (ii) 3HKA exhibits a clear anti-inflammatory profile by inhibiting the STAT1/NF-kB pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines; most notably, IL-6, IL12p70 and TNFa; (iii) in vivo, 3HKA exerts protective effects in the experimental model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring. In a model of nephrotoxic lupus, 3HKA improved proteinuria and serum urea nitrogen, overall ameliorating the immune-mediated glomerulonephritis and renal dysfunction. Additionally, 3HKA and 30 newly generated 3HKA analogs have been proven effective in decreasing ex vivo inflammation in PBMC samples of Lupus patients.
Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice
Authors: Grazda Rachel , Seyfried Allison , MacNamara Katherine
Published: November 2024

Treatments for severe aplastic anemia (SAA) rely on stem cell transplantation and immunosuppressive therapies. Immune-mediated destruction and inflammation drive SAA, but the mechanisms that lead to persistent inflammation are unknown. Using an established mouse model of SAA, we observed increased apoptotic cells within the bone marrow (BM), correlating with impaired efferocytosis. Single-cell transcriptomics revealed heterogeneity among SAA BM monocytes and enhanced inflammatory signatures. Notably, increased expression of Sirpa and Cd47 were observed on monocytes in murine SAA, as well as human SAA patients. The don’t eat me signal CD47 was increased on apoptotic BM cells, concurrent with markedly increased signal regulatory protein alpha (SIRPα) on monocytes. Functionally, SIRPα blockade improved cell clearance and reduced CD47-positive apoptotic cells. Lipidomics revealed a reduction in the precursors of specialized pro-resolving lipid mediators (SPMs) and increased prostaglandins in the BM during SAA, indicative of impaired inflammation resolution. Specifically, 18-HEPE, a precursor of E-series resolvins, was significantly reduced in SAA mice. Resolvin E1 (RvE1) therapy improved efferocytosis, BM cellularity, platelets, and survival. Our data link impaired efferocytosis with SAA and suggest that failed inflammation resolution contributes to disease progression. We demonstrate that SPMs, such as RvE1, offer novel treatments for SAA that do not rely on immune suppression.
Regulatory T cells as key modulators of breast cancer brain metastasis
Authors: Adam Isam , Longworth Aaron , Lawson Devon
Published: November 2024

Breast cancer brain metastasis (BCBM) is a disease with a dismal prognosis. Current therapeutics often fail due to the distinct immune environment within the brain. Thus, there is a critical need to understand infiltrating immune cells within BCBM. Regulatory T cells (Tregs) are a population of immunosuppressive T cells that have been implicated in the pathogenesis of several tumor types. Their functions are widely explored in these contexts, but an understanding of these functions in the brain is lacking. To elucidate the role of Tregs in BCBM, we intracranially injected E0771 cells into C57BL/6J mice and monitored tumor development and immune infiltration for 14 days. Using Foxp3-DTR mice, we assessed the effects of Treg depletion on tumor burden and the immune microenvironment. We found that Tregs prominently infiltrated BCBM tumors throughout tumor progression. Furthermore, Treg depletion led to a massive accumulation of CD8+ and CD4+ T cells and a near-complete loss of tumor burden. Additionally, we demonstrated that microglia, a population of brain-resident macrophages, displayed a more prominent antigen presentation phenotype in Treg-depleted mice. We hypothesize that BCBM-infiltrating Tregs suppress the infiltration and activation of CD8+ and CD4+ T cells by inhibiting antigen presentation in microglia. The mechanisms by which Tregs exert this function are unclear but can lead to the identification of novel therapeutic targets for the treatment of brain metastases.
Mitigating adverse effects of B cell depletion therapy on acute respiratory virus infections through IFN-β intervention treatment.
Authors: Allushi Bujana , Chlebicz Magdalena , Turner Sean , Kovats Susan , Axtell Robert
Published: November 2024

In autoimmune patients, the use of disease-modifying therapies (DMTs) can compromise immunity to viral infections. The COVID-19 pandemic generated interest in the effects of two DMTs on acute viral infections:B cell depletion therapy (α-CD20) and Interferon-beta (IFN-β) therapy. Severe acute viral infections have been associated with B cell depletion therapy, while IFN-β is believed to provide protection due to its antiviral properties. The goal of our study is to elucidate the individual and combined effects of α-CD20 and IFN-β during acute respiratory viral infections in mice. In our study, B cell depletion was achieved by administering α-CD20 three times every 5 days, starting 7 days before influenza A virus (IAV) infection, while IFN-β was given on days 1 and 2 post-infection. When administered alone, α-CD20 resulted in increased weight loss, reduced viral clearance, heightened lung-infiltrating myeloid cells, and elevated inflammatory cytokines in the blood on day 9 post-infection. T cell responses to IAV were not inhibited by α-CD20. While IFN-β alone did not protect against IAV, co-administration of IFN-β with α-CD20 effectively mitigated adverse effects of B cell depletion, by reducing viral load, mitigating morbidity, and slowing the infiltration of myeloid cells into the lung. These data demonstrate IFN-β’s ability to counteract α-CD20’s negative impact on viral infections, providing insights for potential treatment strategies in autoimmunity during viral outbreaks.
Neuroinflammation in Cryptococcal Post-infectious Inflammatory Response Syndrome (PIIRS) is reduced by Ruxolitinib, a Janus Kinase (JAK) inhibitor.
Authors: Hargarten Jessica , Anjum Seher , Xu Jintao , Ssebambulidde Kenneth , Song Brian , Ganguly Anutosh , Vaughan Malcolm , Scott Terri , Hammoud Dima , Olszewski Michal , Williamson Peter
Published: November 2024

Recently, a PIIRS was described in non-HIV infected cryptococcal meningoencephalitis (CM) as a significant cause of neuropathology and mortality. Inflammatory syndromes are particularly severe in neurological infections due to the skull's rigid structure. In the present study, transcriptional profiling of RNA from brains of PIIRS model mice at peak inflammation identified a predominance of STAT1/STAT3-associated downstream genes. Since JAK is known to activate both pathways, we hypothesized that the JAK/STAT inhibitor ruxolitinib could reduce neuro-damaging inflammation from CM. Treatment with ruxolitinib reduced intrathecal STAT1/STAT3-associated gene expression as well as CD44hiCD62loCD4+ effector CD4+ T-cells and MHC II+ inflammatory myeloid cells with improved neuropathology and weight. Based on these data, the first human treatment of PIIRS with ruxolitinib was initiated in 5 patients with residual inflammation despite corticosteroids (CS). Ruxolitinib reduced cerebrospinal fluid (CSF) activated HLA-DR+ CD4+ and CD8+ cells, NK cells, and inflammatory monocytes as well as soluble markers sIL-2R, and IL-10 with a trend in IL-6 on a constant dose of CS. MRI brain imaging also demonstrated significant improvements and therapy was well tolerated. Together, these findings demonstrate a role for the JAK/STAT pathway in PIIRS and support ruxolitinib as a potential adjunctive therapy for further randomized clinical trials of PIIRS as a steroid-sparing therapeutic.
Host guanylate binding proteins participate in parasite control during Leishmania infection
Authors: Fry Lucy , Kelner Noah , Weinkopff Tiffany
Published: November 2024

Cutaneous leishmaniasis (CL) is a debilitating neglected tropical disease causing lesions ranging from self-healing to permanent disfigurations. A predominant Th1 response relying on IFNγ production leads to parasite control during CL. Although IFNγ is mainly responsible for activating macrophages to produce NO leading to parasite control, IFNγ can activate other downstream pathways involved in cell autonomous immunity including the activation of guanylate binding proteins (GBPs), a class of interferon-inducible GTPases. GBPs play a role in host defense against some intracellular pathogens, but minimal work has been done to understand the role of GBPs during Leishmania infection. Utilizing RNA-Seq we found several GBPs are upregulated in lesions during L. major infection in mice. Single-cell RNA-Sequencing revealed macrophages as the main cell type expressing Gbps, including Gbp2, Gbp5, and Gbp7 during infection. Additionally, Gbp expression was not unique to L. major infection; both L. amazonensis and L. mexicana infection in vivo resulted in elevated Gbp expression. Preliminary in vitro experiments show macrophages from GBPChr3 KO mice harbor increased numbers of parasites compared to control macrophages with and without IFNγ treatment. Consistent with in vitro findings, infected GBPChr3 KO mice exhibit increased pathology compared to control mice. Together these data suggest GBPs participate in the defense against Leishmania parasites.
Monocyte-derived macrophages in gut mesentery contribute to the host defense against Salmonella Typhimurium infection.
Authors: Kim Jiseon , Dominguez Joseph R , Kim Seung Hyeon , Chan Carissa , Groisman Eudardo , Kim Ki-Wook
Published: November 2024

Macrophages are essential in clearing microbial pathogens and orchestrating inflammation and resolution of tissues. Recently, two distinct resident macrophage populations (LYVE1hi and LYVE1lo/-) were identified in the mesothelium of serous tissues, including mesentery and peritoneum; however, their pathophysiological functions are poorly understood in inflammatory conditions such as pathogen infection. In the present study, we investigated the heterogeneity and ontogeny of macrophages residing in the mesothelium of gut mesentery, and their function in Salmonella Typhimurium (STm) infection. Fate-mapping approaches using CX3CR1CreER and Ms4a3Cre mice demonstrated that Tim4(+) mesenteric macrophages originate from embryonic precursors and Tim4(-) subsets are replenished from circulating monocytes. The ontogeny of Tim4(-) macrophages was confirmed by CCR2 KO mice. Intragastric infection of STm showed that bacteria first reached the mesenteric lymph node through the gut mesentery and induced immune cell infiltration prior to the dissemination to other tissues. To determine the role of Tim4(-) macrophages, mouse mortality was observed in mice lacking Csf1 in Wt1-expressing stromal cells (WT1Δcsf1 mice) and CCR2 KO. The mouse survival rate was significantly decreased in CCR2 KO or WT1Δcsf1 mice in comparison with their control mice. Our data highlight that monocyte-derived Tim4(-) macrophages might be essential in host defense against enteric bacterial infection.
Establishment of a humanized mouse model for the study of co-infection with Human immunodeficiency virus (HIV) and Mycobacterium tuberculosis
Authors: Bohorquez Jose , John Sahana , Ansari Danish , Florence Jon , Yi Guohua
Published: November 2024

Tuberculosis (TB), caused by Mycobacterium Tuberculosis (Mtb), continues to be one of the most important public health threats worldwide, being the second deadliest infectious disease, after COVID-19. The human immunodeficiency virus (HIV) is another equally important global public health issue. Co-infection with HIV and Mtb has severe effects in the host, with people living with HIV being 15-21 times more likely to develop active TB. The use of an appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans would be a very useful tool for basic and applied research. The present study aimed to develop a humanized mouse model for the study of HIV-Mtb coinfection. Using NSG-SGM3 mice that can engraft human stem cells, our results showed that these mice differentiated a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, the humanized mice showed decreased CD4+ T cell counts overtime with elevated HIV-1 load in sera, similar to the infection pattern in humans. Additionally, Mtb also caused infections in lungs and spleen, and induced the development of lesions in the lungs, detected by CT scan and histopathology. Distinct metabolomic profiles were observed in the different mice groups after infection. Our results suggest the capacity of this mouse model to recapitulate the effects of HIV and Mtb infections/co-infection, in the human host at a pathogenic, immunologic and metabolic level
RUNX2 transcriptional activity is required for CD26high CD4+ T cell development
Authors: Ruffin Ayana , Ware Micheal , Wyatt Megan , Cole Anna , Wittling Megen , Rangel Rivera Guillermo , Lesinski Gregory , Paulos Chrystal
Published: November 2024

Adoptive T cell therapy (ACT) is rapidly growing as a promising treatment option for patients who relapse or fail to respond to immunotherapy. However, in the treatment of solid tumors with ACT, many therapies fail to maintain long-term durability. We posit that CD4+ T cells expressing high levels of CD26 (CD26high T cells) would be a potent ACT product. We previously reported that they express an array of chemokine receptors, dynamic cytokine profile and regressed tumors better than other T helper subsets. Here, we developed novel cytokine priming strategies to enrich CD26high T cells from the blood of healthy donors and cancer patients. We identified a unique suite of transcription factors, including RUNX2, that are elevated on CD26high T cells. We also discovered that CD26high T cells overexpress a distinct patterns of cytokine receptor including IL-2Ra, IL-7R, IL-18R and IL-12Rβ1. Targeting these receptors via recombinant cytokine priming strategies resulted in three-to-five-fold expansion of CD26high T cells from bulk or Naive CD4+ T cells. Inhibiting RUNX2 DNA binding via small molecule inhibitors CADD522 and DIPQUO resulted significant reduction in development of CD26high CD4 T cells and decreased their cytokine production. These data suggest that RUNX2 may regulate the development and function of CD26high CD4 T cells. Taken together, these results provide evidence for the use of CD26high CD4 T cells in the treatment of solid malignancies with ACT.
Engineered IL2 for improved treatment of bladder cancer
Authors: Liao Yiji
Published: November 2024

Bladder cancer (BC) remains a formidable challenge despite its immunogenic nature and responsiveness to FDA-approved immunotherapy. Interleukin-2 (IL2), a potent cytokine with potential for cancer treatment, faces obstacles such as the simultaneous activation of immunosuppressive regulatory T cells (Tregs), high toxicity, and a short half-life. CD122-directed IL2 therapy (IL2c) has shown success in mouse BC models. To address clinical challenges, we engineered two IL2/anti-IL2 antibody (F10) fusion proteins: F10 IC (native IL2), potentially biased toward effector cell activation, and F10 H9T (H9T, an engineered IL2), potentially favoring memory T cells.

In vitro F10 H9T induced stem-like T cells (TCF-1+) and central memory T cells (CD44+CD62L+), while IL2c promoted Teff cell (CD44+CD62L-) differentiation. In vivo, using an orthotopic mouse bladder cancer model (n=10/group), F10 IC was superior in activating anti-tumor Teff and NK cells over F10 H9T and IL2c. Unexpectedly, F10 IC also induced TCF-1+ T cells, suggesting a potentially valuable dual effect, but further validation is required.

We provide a roadmap for designing and translating immunomodulatory cytokine/antibody fusion proteins to overcome challenges associated with IL2-based cancer therapies.
IL-33 downregulates human IL-33 expression in the pulmonary vasculature during allergic airway inflammation
Authors: Hollinger Maile , Howard Chanie , Decker Donna , Blaine Kelly , Hrusch Cara , Grayson Emily , Aneas Ivy , Camacho Daniel , Schoettler Nathan , Ober Carole , Nobrega Marcelo , Sperling Anne
Published: November 2024

IL-33 is abundantly expressed in the human lung, where it is involved in pathogen defense, barrier homeostasis, and development of type 2 allergic responses. We recently identified a 5 kb region within a GWAS-defined segment that acts as an enhancer barrier element in vivo and in vitro. We showed that the asthma-associated SNP rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein in human plasma. To study this human regulatory region in vivo, we engineered a novel IL33 reporter mouse containing the human 5kb regulatory element to reflect human IL33 expression. As expected, human and mouse IL33 reporters were primarily expressed in non-overlapping cell types: human IL33 reporter was mostly expressed in vascular and lymphatic endothelium, while mouse IL33 reporter was expressed in ATII epithelium. Interestingly, we demonstrate negative regulation of human IL33 in lung endothelium during house dust mite (HDM)-induced allergic airway inflammation, which was dependent on the human 5kb regulatory region. Similar downregulation of endothelium IL-33 was induced with only IL-33 treatment in vivo and in vitro. We found that this IL-33-induced negative feedback loop worked through the induction of cytoplasmic IL33 mRNA instability. Together, these data reveal the importance of the asthma-associated human 5kb enhancer element in regulating human IL33 expression in a cell type- and context-dependent manner.
Examining the outcomes of enhanced CD28 signaling in vivo using ‘humanized’ mice
Authors: Brady Alexander , McGeachy Mandy
Published: November 2024

CD28 signaling is stronger in humans than in mice, as demonstrated by the phase one clinical trial of a CD28 superagonist antibody. In mice CD28 superagonist expanded regulatory T cells and reduced autoimmune disease, but in humans it induced life-threatening cytokine storm. The final signaling domain in human CD28 is PYAPP, while in mice it is PYAPA. We used CRISPR/Cas9 to develop a ‘humanized’ mouse with a CD28 A->P substitution (CD28A210P) to examine the outcomes of this variation on T cell-driven disease. In vitro, CD28A210P T cells had increased signaling, cytokine production, and proliferation. In vivo, after injection with CD28 superagonist, CD28A210P mice had increased weight loss, cytokine production, and less Treg expansion, supporting that humanized CD28 signaling could contribute to the excessive inflammatory response. Following LCMV cl13 infection, CD28A210P mice display enhanced early Th1 and CD8 T cell responses with exacerbated weight loss but then underwent increased exhaustion, leading to rapid recovery of weight. Similarly, CD28A210P mice show increased EAE incidence but milder symptoms, coinciding with an increase in CNS-infiltrating CD8 T cells which play a regulatory role in EAE. This study shows enhanced CD28 signaling due to an amino acid variation can alter T cell driven disease and reveals nuances in immunoregulation. This expands our knowledge of CD28, with potential implications for immunotherapeutics that target or utilize CD28 signaling.
Antigen-specific nanoparticle tolerance reduced inflammation at the target tissue and increased target protein expression in recombinant adeno-associated virus (AAV)-mediated gene therapy
Authors: Podojil Joseph , Xu Dan , Chiang Ming-Yi , Kakade Sandeep , McCarthy Derrick , Elhofy Adam , Miller Stephen
Published: November 2024

Recombinant Adeno-associated virus (AAV)-mediated gene therapy is an attractive approach for treating diseases where expression of an endogenous protein is defective or absent. While AAV is considered to be non-integrating and weakly immunogenic, AAV delivery elicits antigen (Ag)-specific T cells targeting transduced cells, and neutralizing antibodies inhibiting secondary administration. Effector T cell responses and vector dilution lead to decreased protein expression within target tissue, thereby requiring periodic AAV re-dosing with AAV to sustain the therapeutic benefit. Treatment with Ag-containing biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles (denoted as CNPs) represents a putative co-treatment allowing for decreased therapeutic AAV dose and frequency of AAV re-dosing to maintain therapeutic benefit. In the present studies, AAV8-eGFP was administered intramuscularly or intravenously in the presence or absence of B cell depletion. Prophylactic or therapeutic treatment with CNP(VP1 + eGFP) allowed for higher levels of eGFP expression within the target tissues following primary and secondary AAV8-eGFP dosing. Additionally, Ag-specific CNP(VP1 + eGFP) treatment inhibited the increase in effector CD8+ T cells within the target tissue following secondary AAV8-eGFP administration. Taken together, co-treatment of gene therapy patients with Ag-specific CNPs may allow for enhanced transgene expression over time, while allowing for AAV re-dosing.
Exploring cGAS-STING Pathway in Sjögren’s Disease: Driver of IFN production and Potential Therapeutic Target
Authors: Yamada Eiko , Abed Mehdi , Perez Paola , Jang Shyh-Ing , Warner Blake
Published: November 2024

Sjögren's Disease (SjD) is a prevalent autoimmune disorder characterized by dysfunctional lacrimal and salivary glands (SG) and lacks an effective therapy. Despite unknown pathogenic mechanisms, elevated interferon (IFN) expression is observed in SjD patients. Recent findings indicate activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in systemic lupus, prompting investigation into its role in SjD. We hypothesize cGAS-STING pathway activation in SjD contributing IFN dysregulation and symptoms. Bulk and single-cell RNA sequencing of SjD SG shows elevated IFN-related genes in epithelial and inflammatory cells. Investigation of the affected human SG by flow cytometry confirms upregulated phosphorylation status of cGAS-STING proteins in epithelial and immune cells; and epithelial cGAS-STING pathway activation directly correlates with the level of immune cell infiltration. Treatment of patient’s SG and peripheral blood mononuclear cells (PBMC) with a STING antagonist ex vivo reduces phosphorylated protein levels without cytotoxicity. These findings support our hypothesis that targeting the cGAS-STING pathway holds promise for mitigating salivary gland inflammation in SjD, preventing chronic inflammation's secondary effects and resulting tissue destruction.
Peripheral Natural Killer Cell Cytotoxicity is Suppressed in Amyotrophic Lateral Sclerosis
Authors: Murdock Benjamin , Teener Samuel , Webber-Davis Ian , Monteagudo-Caballero Alina , Famie Joshua , Piecuch Caroline , Goutman Stephen , Feldman Eva
Published: November 2024

Amyotrophic lateral sclerosis (ALS) is a deadly neurodegenerative disease where the immune system contributes to progression, though the underlying immune mechanisms are unclear. Natural killer (NK) cells likely play a critical role in disease progression: they are responsible for dispatching damaged cells, and during ALS they accumulate in the CNS and increase the rate of neuronal death. Thus, we hypothesize that NK cells are more cytotoxic in ALS patients than in healthy controls. To test this, primary NK cells were isolated from the blood of control and ALS patients and co-cultured for four hours at a 1:1 ratio with K-562 cancer cells. K-562 cell death was measured via e500 viability dye staining and used to quantify NK cell cytotoxicity. Surprisingly, we found that NK cell cytotoxicity was significantly reduced in ALS NK cells compared to healthy controls. Next, using flow cytometry, we measured a battery of peripheral inflammatory markers and correlated these measures with NK cell cytotoxicity to identify immune pathways impacting NK cell cytotoxicity. Unsurprisingly, in healthy control patients there was significant correlation between NK cell markers and NK cell cytotoxicity. However, these correlations were absent in ALS patients. Together, these data suggest that NK cell cytotoxicity is downregulated in ALS, likely as a mechanism to slow NK cell-mediated damage to motor neurons, though further studies will be required to elucidate the underlying mechanisms.
Isolation and characterization of epidermal Langerhans cells from human skin
Authors: Yamada Minami , Hasegawa Tatsuya
Published: November 2024

The skin is the largest organ that acts as the protective interface between the body and the environment. The immune system of the skin provides the first line of defense against continuous exposure of various external insults and pathogens. In the outermost layer of the skin, epidermal Langerhans cells (LCs) play a sentinel role as tissue-resident antigen presenting cells to confront the environmental insults. Ontogeny and functional studies of murine LCs have provided fundamental insights into LC biology, however, the nature of LCs and the immunological microenvironment surrounding LCs are different between mice and human skin. Therefore, it is important to study the LC biology in human skin. Here, we optimized a protocol for the isolation of epidermal LCs from sun-protected normal human skin tissues. Flow cytometry and immunocytochemistry staining analysis demonstrated that CD1a and CD207, which are human LC markers, were expressed in isolated epidermal LCs. In addition, we observed that epidermal LCs isolated from human skin highly expressed CD1a and CD207 in comparison with LCs derived from CD34+ hemopoietic stem cells. Our optimized protocol for isolating human epidermal LCs may help to fully capture the nature and role of human LCs in healthy and diseased condition.