bluuebunny/crossref_metadata_2025 huggingface dataset:
Using Non-Human Primate to Investigate the Generation of Liver Immunity in Malaria
Authors: Osii Rowland , Kirtley Payton , Aleshnick Maya , Boggey Gregory Boggey , Marrow David , Mutai Koko , Beebe Roxanne , Tonelli Rachael , Smedley Jeremy , Fisher Miranda , Feltham Shana , Ojha Sohita , Bimber Benjamin , Wilder Brandon
Published: November 2024

Malaria is still a global health issue that afects more than half of the worlds population. While a number of malaria vaccines are currently under development. The most potent and long-lived vaccine have been whole sporozoite vaccines that use live-attenuated sporozoites to generate both antibodies and liver resident memory T cells capable of blocking infection. This vaccine has a higher efficacy in a malaria naïve population (up to 100%), but this efficacy drops in a malaria endemic population (0-30%). It has been hypothesized that the immune profile of people living in malaria endemic regions is altered in a way that negatively affects vaccine induced immunity, but a better understanding of how this occurs is required to generate optimal vaccine strategies. Yet to understand how the parasite alters the immune system, thus causing vaccine hyporesponsive in endemic regions, requires detailed information on previous parasite exposure and access to tissue resident cells that reside in the liver and other lymphoid organs—both limiting our ability to study this phenomenon in humans. To overcome this, we used a non-human primate model of malaria that enables us to access tissues before and after infection and vaccination via minimally invasive biopsies. Here, we present ongoing analysis that shows the longitudinal changes in the immune landscape after infection, vaccination and infection after vaccination using flow cytometry and single cell transcriptomics.
Transcriptomic Analyses of B Cells Infected In Vitro with Transmitted/Founder HIV-1 of R5 Phenotype (HIV-R5) Reveal Downregulation of IGLL1 Gene in Persistent Infection.
Authors: Vyas Girish , Bozzini Randall , Nix Matthew , Sanchez Claudia , Santos Yale , Vashishtha Anshu , Wiita Arun , Chiu Charles , Lowell Clifford
Published: November 2024

HIV-neutralizing antibodies against T cell-dependent antigens of HIVenv permit persistent infection. Conceivably, B cell repertoire has a clone capable of eliciting antibodies to eliminate any HIV. Receptors for T cell-independent conformational antigens (TiCA) of the trimeric envelope glycoprotein subunits (TEGS) of HIV permit persistent infection of that clone. To test this concept, a human B cell line (CA46, ATCC) was chosen for in vitro infection with HIV-X4 or HIV-R5. Both infected cell lines continued to produce infectious virions. Transcriptomic analyses of uninfected and HIV-infected cells was done, and data analyzed by the Edge R and Surpi software. Differential gene expression data for paired analyses of B cells infected with HIV-R5 versus uninfected cells showed that 108 genes were upregulated, while 148 genes were downregulated. Notably, the IGLL1 gene that determines surface expression of Ig-receptors on B cells, was down-regulated in cells infected with HIV-R5 (logFC= 3.30). Such a difference was not seen in B cells infected with HIV-X4. Heatmaps showing RNA-Seq data for pathway analysis revealed distinct patterns of gene expression in B cells infected with HIV-R5 versus HIV-X4. These data are in accord with HIV-R5 initiating all persistent HIV infections worldwide. We conclude 1) HIV-R5 infects B cells to establish persistent infection, 2) Studies with CD19+ normal B cells would enable synthesis of antibodies against TiCA and TEGSs for immune prevention of HIV.
Is AIDS an Actinopathy? HIV Inhibition of ARP2/3 is Reminiscent of Primary Immunodeficiencies in Normal Progressors but Absent in A Subset of HIV Controllers.
Authors: Furler O'Brien Robert , Kovacs Colin
Published: November 2024

HIV-1 infection disrupts the cortical actin network, leading to morphological and migratory changes in CD4+ T cells. We utilized time-lapse and ultrastructural microscopy to observe primary CD4+ T cells infected with wild-type HIV-1 and Nef-deleted (ΔNef) virus to assess cytoskeletal cytopathicity. Furthermore, we investigated the link between HIV-1-mediated actin disruption and primary immunodeficiencies that affect the ARP2/3 complex to cytopathicities found in HIV infection.

Of the five aberrant phenotypes observed, we identified two novel pathological morphologies unique to HIV-1 infected CD4+ T cells. The observed morphologies mirror those seen in migrating cells containing genetic determinants of primary immunodeficiencies affecting ARP2/3 complex. Nef-deleted virus partially ameliorated the cytopathicity in infected cells. In contrast to normal progressors and healthy controls, two-thirds of HIV-1 controllers tested retained cortical actin stability in the presence of WT infection.

The link between these cellular pathologies and disease progression provides insights into HIV-1 induced immune dysfunction. Additionally, we observed that certain HIV-1 controllers lack these cellular pathologies, possibly indicating a protective mechanism against viral replication and immune response. Restoring ARP2/3 function and cortical actin integrity in HIV-1-infected individuals may present a promising avenue for eradicating infected cells from the body.
Tissue resident memory CD8 T cells limit respiratory virus transmission by IFN-γ production and activation of nasal cavity epithelial cells
Authors: Michalets Sarah , Saha Ananya , Jimenez Ariana , Uddbäck Ida , Williams M Elliott , Mattingly Cameron , Kost Kirsten , Hicks Sakeenah , Lowen Anice , Scharer Christopher , Antia Rustom , Kohlmeier Jacob
Published: November 2024

Many vaccines against respiratory viruses have proven suboptimal in generating long-lasting sterilizing immunity and preventing transmission among populations. It is well established that respiratory tract resident memory CD8 T cells (TRM) induced by vaccination can reduce viral burdens and limit immunopathology upon direct inoculation of respiratory viruses. However, the ability of respiratory tract CD8 TRM to limit viral transmission has remained elusive. We developed a murine model to investigate the role of respiratory tract CD8 TRM in viral transmission using Sendai virus, which naturally transmits among mice. Contact mice were first immunized to generate Sendai virus-specific CD8 TRM and challenged by co-housing with a Sendai virus-infected index mouse. We demonstrated that CD8 TRM can limit viral transmission and decrease viral burdens, even in the absence of B cell responses, CD4 T cells, and circulating effector memory T cells. We evaluated multiple CD8 T cell effector mechanisms for their importance in limiting viral transmission and determined that IFN-γ secretion was critical. Upon IFN-γ signaling, nasal cavity epithelial cells adopted an anti-viral program. Furthermore, CD8 TRM in the nasal cavity alone were sufficient to limit respiratory virus transmission. These findings suggest that respiratory tract CD8 TRM, particularly those within the upper respiratory tract, are a promising vaccine target to prevent respiratory virus transmission among populations.
Quality Assessment of Metabolic Fitness during T Cell and NK Cell Expansion
Authors: Wang Guoxiao , Swain Pamela , McGarrigle Ryan , Romero Natalia
Published: November 2024

CAR-T cells have revolutionized cancer treatment, and other cell types like CAR-NK cells are also showing promising results. Engineered cells generally need to be expanded ex vivo before being transfused into patients. Growth conditions, including expansion platform, cell culture media, and cytokine composition, time of expansion, etc., can have a strong effect on the characteristics of the final products and need to be monitored to establish the appropriate development process.

Recent developments highlight the significance of cell metabolism in influencing immune responses and the anti-tumor efficacy of cell therapies. In the present study, we utilized Seahorse XF technology and assays to characterize T cell and NK cell metabolic fitness during cell expansion. We cultured human T cells and NK cells under different expansion conditions including culture media, culture devices, and cytokine combinations, and sampled cells at different time points for quality assessment of metabolic fitness. Using the new Seahorse XF assay alongside immunophenotypic characterization using Novocyte Flow Cytometry, we obtained robust information on mitochondrial and glycolytic energy production, metabolic poise, and the bioenergetic capacity of the expanded cells, allowing us to identify conditions that resulted in increased metabolic fitness of the cell population that can contribute to defining cell expansion conditions during immunotherapy development.
High throughput Targeted Transcriptome Sequencing of Bone Marrow Mononuclear Cells identifies a Rare Leukemia-Specific T Cell Subset.
Authors: Papalexi Efthymia , Hariadi Bryan , Kim Grace , Suyama Alexa , Roco Charles , Rosenberg Alexander
Published: November 2024

Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery. These applications often do not require surveying the whole transcriptome (WT), but rather require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required.

Here, we demonstrate an approach to combine split-pool combinatorial barcoding with enrichment of 1000 genes (Immune1000 panel) representing immune cell markers and pathways. We use this gene panel to characterize 730,000 human bone marrow mononuclear cells (BMMCs) from four leukemia and four healthy donors. We found that enrichment with the Immune1000 Panel enabled the characterization of all major immune cell types at low sequencing depth and allowed for the detection of known leukemia-associated shifts in cell type proportions. Moreover, our targeted sequencing method enabled the detection of a rare (~0.35%), leukemia-specific T cell population that showed highly proliferative and cytotoxic potential.

Overall, we demonstrate our modular enrichment strategy preserves biological structure and allows for deep characterization of rare disease-relevant cell types and gene signatures with less sequencing.
Decoding mucosal immunity: A unique natural killer cell profile in vaginal tissue.
Authors: Vick Sarah , Domenjo-Vila Eva , Hughes Sean , Kirby Anna , Fialkow Michael , Hladik Florian , Prlic Martin , Lund Jennifer
Published: November 2024

Mucosal immunity in the vaginal tract (VT) remains poorly characterized despite its critical role in protecting from sexually transmitted infections. NK cells, critical for resolving infections, are found in the VT in significant numbers yet it is unknown how the immune system in the VT differs from that in circulation. To address this important question, vaginal tissue and autologous blood were collected from healthy donors and NK cells were profiled using high-parameter flow cytometry (n=10) and scRNA-seq (n=4) to define differences between NK cells from blood and tissue. The scRNA-seq data revealed a unique gene signature in tissue NK cells with a downregulation in genes related to cytotoxicity. We confirmed this data utilizing a 28-color flow cytometry panel. The frequency of markers expressed on blood and tissue NK cells were analyzed using a paired t-test. We found that NK cells in the VT express increased tissue residency markers CD69 and CD103 (p=0.0001, p=0.0009) when compared to NK cells from the blood. The NK cells from the VT displayed a decrease in maturation markers and cytotoxic molecule Granzyme B (p=0.0002). Despite the quiescent nature of NK cells in the VT, these cells produce increased levels of IFNg upon stimulation with cytokines (p=0.0021). This suggests NK cells in the VT may be regulated to limit tissue damage but are poised to robustly respond to inflammatory stimuli. In sum, I demonstrate how NK cells are phenotypically unique in mucosal tissue.
Unraveling the early transcriptional signature of peripheral blood NK cell transition to uterine residency
Authors: Greene Morgan , Asiimwe Rebecca , Bell Markayla , Wright Emma , Knott Brittney , Yates Stefani , Epstein Daniel , Little Shawn , Locke Jayme , Freud Aharon , Richter Holly , Porrett Paige
Published: November 2024

Tissue-resident uterine NK cells are essential for successful pregnancy, yet the molecular mechanisms guiding their tissue residency programming early after infiltration of blood-based precursors remain unknown. We examined the transition of CD56+ peripheral blood NK cells to a uterine resident state by performing scRNA-seq on enriched CD56+ cells from a matched peripheral blood (pb) and endometrial (e) biopsy taken during the secretory phase. Our integrated analysis comparing eNK vs pbNK cells identified a tissue adaptation signature. Key components of this signature included NR4A2 (94% vs 0%, p=9.46e-22), FOS (90% vs 9%, p=9.05e-14), and JUNB (77% vs 16%, p=3.15e-07) respectively. This signature also mirrored genes previously identified in activated murine intestinal CD8+ TRM cells early after LCMV infection. This signature was found in all endometrial CD56+ cells, including CD56bright CD16- eNKs, NKTs, and conventional eNKs (CD56dimCD16+). Pathway analysis implicated IL-17 and IL-18 cytokines, along with NFAT and VEGF signals in tissue residency programming. In conclusion, these data expand our understanding of early transcriptional programs in lymphocyte tissue adaptation, revealing the involvement of cytokines beyond TGF-β. Our data further indicate that signals associated with early tissue adaptation are conserved across species and cell types independent of an antigen receptor.
IL-12 induces a B cell-intrinsic IL-12/IFNg feed-forward loop promoting extrafollicular B cell responses
Authors: Elsner Rebecca , Shlomchik Mark , Smita Shuchi
Published: November 2024

While some infections elicit germinal centers (GC), others produce only extrafollicular (EF) responses. The mechanisms controlling these dichotomous fates are poorly understood. We identify IL-12 as a cytokine switch, acting directly on B cells to promote EF and suppress GC responses. IL-12 initiates a B cell intrinsic feed-forward loop between IL-12 and IFNg, amplifying IFNg production, which promotes proliferation and plasmablast (PB) differentiation, in synergy with IL-12. IL-12 sustains the expression of a portion of IFNg-inducible genes. Together, they also induce unique gene changes, reflecting both IFNg-amplification and cooperative effects between both cytokines. In vivo, cells lacking both IL-12 and IFNg receptors are more impaired in PB production than those lacking either receptor alone. Further, B cell-derived IL-12 enhances both PB responses and TH1 commitment. Thus, B cell-derived IL-12, acting on T and B cells, determines the immune response mode, with implications for vaccines, pathogen protection and autoimmunity.
A novel mutation in IRAK2 results in immune dysregulation in systemic juvenile idiopathic arthritis (sJIA)
Authors: Boyd Brooke , Platukus Alana , Schmitz Elizabeth , Nakano Hiroto , Dell'Orso Stefania , Naz Faiza , Deng Zuoming , Cruz Anthony , Ombrello Michael
Published: November 2024

Systemic juvenile idiopathic arthritis (sJIA) is a severe inflammatory disease marked by innate immune activation with potentially fatal complications. Using trio exome sequencing, we identified a novel homozygous mutation in interleukin (IL)-1 Receptor Associated Kinase 2 (IRAK2) in a child with sJIA complicated by lung disease and hyperinflammation. IRAK2 is a serine/threonine kinase involved in IL-1 receptor and toll-like receptor signaling, leading to expression of pro-inflammatory genes. The patient displayed persistent elevation of IL-6, IL-8, and IL-18 and a strong interferon (IFN) stimulated gene (ISG) signature. To determine how the IRAK2 mutation contributes to the patient’s immunopathology, we used gene editing to create THP-1 cells homozygous for the mutation (THP-IRAK). Following 24 hours of lipopolysaccharide (LPS) treatment, RNA sequencing revealed that THP-IRAK cells display higher expression of IL-6, IL-18, and ISGs than wild type THP-1 cells (THP-wt). Gene set enrichment analysis of MSigDB hallmark sets identified LPS-induced upregulation of IFN signaling and cell cycle regulation related sets in THP-IRAK cells relative to THP-wt cells. Finally, LPS treatment led to higher secretion of IL-8 in THP-IRAK cells than in THP-wt cells. Together, these in vitro results recapitulate the pattern of inflammation observed clinically, which suggests that the IRAK2 mutation contributes to the pathophysiology of sJIA and nominates IRAK2 as a therapeutic target in sJIA.
Controlled Reduction of Reactogenicity in Vaccines - Designing New Molecules to Alter Innate Signal Processing
Authors: Esser-Kahn Aaron , Kim Jeremiah , Roman Maria , Snyder Scott
Published: November 2024

Vaccines are a critical tool in preventing and managing many diseases, but their efficacy can be improved by enhancing their immunogenicity and lowering their reactogenicity. We have developed a series of molecules that directly reduce correlates of reactogenicity while also improving protection. We present data on the application of these immunomodulator towards a CpG adjuvanted subunit SARS- CoV-2 vaccine in both mice and Syrian hamsters. Immunomodulator-containing vaccines increased neutralizing antibody and T-cell responses in mice. In a challenge model in hamsters, these vaccines reduced weight loss, viral load, and tissue damage. These findings demonstrate the possible improvement of current and future vaccines using immunomodulators. We also present a series of modulators that reduce and direct inflammatory responses in a controlled fashion for use in influenza, hepatitis, typhoid, and many other vaccine applications.
Production of a covalent conjugate vaccine using cell-free protein expression (XpressCF™) and click chemistry
Authors: Rozzelle James , Poston Taylor , Arroyo Daisy , Bruno Antonio , Wu Manhong , Esser-Kahn Aaron , Darville Toni , Fairman Jeff
Published: November 2024

Chlamydia trachomatis is the most common bacterial sexually transmitted infection globally and a vaccine is urgently needed. To address this, we determined that Chlamydia protease-like activity factor (CPAF) is the most immunoprevalent and immunodominant antigen for CD4 T cells and B cells in Chlamydia exposed women and mice. CPAF is a 70-kDa serine protease that is secreted into the host cell cytoplasm and subsequently released into the extracellular milieu where it paralyzes neutrophils to evade phagocytosis. CPAF contains a fold similar to other known serine proteases and is processed into C- and N-terminal fragments that heterodimerize. To improve and facilitate vaccine scale-up and immunogenicity, we developed CPAF variants incorporating non-native amino acids at specific sites using the proprietary cell free expression platform XpressCF™. These CPAF variants were covalently conjugated to DBCO-derivatized adjuvants using click chemistry to make novel antigen-adjuvant conjugate vaccines. Site-specific conjugation enables important T- and B-cell epitopes to be avoided for better immune recognition and provides a method to incorporate a specific number of conjugated adjuvants. These conjugated vaccines provide a means to simultaneously deliver antigen and adjuvant and potentially reduce the amount of adjuvant required for vaccine efficacy leading to fewer side effects and dose sparing.
Repeated allergen consumption impairs intestinal barriers and promotes neurobehavioral pathologies in mice with subclinical cow’s milk allergy
Authors: Ekanayake Dilini , Setness Rylan , Nagamoto-Combs Kumi
Published: November 2024

Cow’s milk and other food allergens are often implicated in behavioral problems in sensitized but tolerant individuals. While the mechanisms are still unclear, we previously showed that male C57BL/6J mice with subclinical cow’s milk allergy (CMA) exhibited depression-like behavior associated with brain neuroinflammation and cortical demyelination after consuming a whey protein-containing diet (WP diet) for 2 weeks. Recently, we also found that adoptively transferred peripheral leukocytes from CMA mice migrated to the dura mater of naïve recipient mice during the duration of the 2-week WP diet. Hypothesizing that consumption of food allergens by subclinically sensitized individuals would cause intestinal damage and promote the trafficking of activated peripheral leukocytes to the dura to adversely affect the brain, we assessed neurobehavioral and intestinal pathologies CMA mice that sensitized to β-lactoglobulin (Bos d 5) and exposed to WP diet for 2 weeks. Behavioral tests indicated that the exploratory activity and short-term memory of CMA mice were significantly lower than controls. Cortical demyelination, microgliosis, and axonal damage were detected in the brain as previously reported. Notably, immunoreactivity for a tight junction protein, claudin 5, was visibly lower in the intestines of CMA mice. Our results suggested that repeated allergen consumption could trigger immune responses by increasing gut permeability in susceptible individuals.
The calpain/ABCA1 pathway drives mast cell- initiated vascular leakage and inflammation in the skin.
Authors: Kohl Lisa Marie , Hay Ahnika , Gupta Vinay , Williams Li'an , Bharadwaj Shreya , Donahue McKenna , Omar Lea , Cattley Richard , Sumpter Tina
Published: November 2024

Cutaneous mast cells (MCs) initiate local and systemic immune responses. To reach this end, activated MCs immediately release an arsenal of pro-inflammatory proteases. In this study, we evaluated the role of the cysteine protease, calpain in MC function. Calpain activity and protein were confirmed in the supernatants of MCs activated via either the FceR or the MrgprB2. In vivo, extracellular calpain induces vascular leakage in a MC- dependent manner. We then investigated the mechanism of calpain release. In vitro and in vivo studies suggest that the cholesterol transporter, the ATP- binding cassette A1 transporter (ABCA1) externalizes calpain. To investigate the role of the ABCA1 in MC biology, we generated mice lacking the Abca1 specifically on connective tissue MCs (Mcpt5-Cre+Abca1fl/fl mice). In vitro, Abca1-deficient MCs accumulate intracellular cholesterol, have reduced calpain release and reduced pro-inflammatory cytokines levels. In vivo, in the MC903-induced atopic dermatitis model, Mcpt5-Cre+Abca1fl/fl mice have reduced inflammation and a fewer MCs in affected skin compared to littermate controls. Corroborating this finding, global ABCA1 blockade with FDA-approved glyburide similarly reduced inflammation induced by MC903. Collectively, these data suggest that targeting the calpain/ABCA1 pathway may open new therapeutic avenues for MC -mediated inflammation in the skin.
GEMINI pipeline: Fecal microbiota and Immune fitness evaluation in healthy, elderly and centenarians populations
Authors: Levast Benoit , Beitz Benoît , Mouharib Manal , Ania Maryne , Bellais Samuel , Ben Abdallah Bouthaina , Koenig Marie , Welsh Jérémy , Beloeil Laurent , Louis Karen , Guyard Cyril
Published: November 2024

The symbiosis between microbiome and its host creates a homeostasic crosstalk regulating the host immune system. Host microbiome richness and diversity are linked with a wide range of diseases and treatment efficacies such as vaccination or immunotherapies.

The project includes three different cohorts of volunteers: healthy volunteers (18 and 55 years old) – considered as a reference group (n=51); elderly population between 50 and 75 years old - considered as the population of interest for seasonal vaccinations (n=60); and centenarians’ population (> 95 years old) – that may present a gut microbiota and immune system with specific properties partly supporting their exceptional longevity in relatively good health.

By integrating high throughput anoxic culturomics, host/microbiota interaction functional analyses, with metabolism and host immune response analytical capacities, BIOASTER has established a multidisciplinary platform for the development of microbiome derived products.

Here we report the characterization of human donors “immune fitness” data through the analyses of plasma cytokines profiles in whole blood stimulation assays using TruCulture, paired to feces analyses. Our results will guide our strategy for gut microbiome bacterial isolations.

The screening of specific microbiome strains will lead to their genetic identification, their functional characterization and the development of next-generation probiotics with clinical related immuno-modulatory properties.
Deciphering TCR-pMHC Interactions in Human Memory T Cells: Biophysical Insights into SARS-CoV-2 Antigen Recognition
Authors: Chen Xiangcheng
Published: November 2024

Precise antigen recognition by T cell receptors (TCRs) is key for initiating cellular immune responses. This study investigates the biophysical characteristics of TCR-antigen interactions, emphasizing 2-dimentioanl (2D) TCR affinity and catch bond dynamics in human memory T cell subsets, particularly in their response to specific SARS-CoV-2 epitopes.

Utilizing DNA-barcoded pMHC tetramers, our research profiles TCRs interacting with selected SARS-CoV-2 epitopes, identifying antigen-specific memory T cells in lung tissue, peripheral blood mononuclear cells (PBMCs), and antigen-specific precursor T cells from the spleen. Through micropipette adhesion assays and biomembrane force probe, we quantified 2D TCR affinity and examined catch bond formation dynamics at the single-cell level.

Our findings indicate a broader range of 2D TCR affinities in spleen precursor T cells compared to memory T cell subsets in tissue and blood. Notably, tissue-resident and circulating memory T cells exhibit distinct 2D TCR affinity and catch bond properties.

The differential biophysical properties, particularly in 2D affinity and catch bond force lifespan, underscore the unique role of tissue-resident memory T cells, influencing memory formation and tissue-specific immune responses. This detailed characterization of TCR-pMHC interactions in human tissue-resident memory T cells contributes significantly to our understanding of memory generation during viral infections.
Sialic acids from the Group B Streptococcus (GBS) capsule dampen mast cell activation through Siglec-9
Authors: Miralda Molina Irina , Orvis Austyn , Coleman Michelle , Brokaw Alyssa , Lewis Amanda , Rajagopal Lakshmi , Piliponsky Adrian
Published: November 2024

GBS is a leading cause of bacterial infection in newborns, adult diabetics, pregnant women, and the elderly. A key virulence factor of GBS is its surface capsular polysaccharide, which displays terminal sialic acids that suppress host innate immune responses by engaging host inhibitory receptors like Sialic acid-binding immunoglobin-like lectin- 9 (Siglec-9). We reported that human mast cells (MC) express Siglec-9 and that engaging it with native ligands or antibodies results in reduced mast cell activation in vitro. We hypothesized that capsular sialic acids engage Siglec-9 to impair MC ability to initiate the host response. Mice expressing human Siglec -7 and -9 were infected with WT GBS or a sialic acid deficient strain, ΔcpsK, and the ΔcpsK infected mice showed reduced bacterial burden and less dissemination. Using bone marrow-cultured MC from a transgenic mouse expressing Siglec-9, we confirmed that GBS interacts with MC Siglec-9, which inhibited the release of the pro-inflammatory cytokine IL-6. Challenge of primary human MC with WT, ΔcpsK, or ΔneuA, another sialic acid deficient GBS strain, showed that MC interact more with ΔcpsK and ΔneuA GBS, and that MC kill sialic acid deficient GBS. MC challenged with ΔcpsK or ΔneuA GBS also produce more IL-8 and undergo cell death more than WT infected MC. Together, this data shows that sialylated GBS capsule engages MC Siglec-9 to dampen activation, which reduces the immune response and benefits the pathogen.
A humanized ACE2 mouse model recapitulating age- and sex-dependent immunopathogenesis of COVID-19
Authors: Park Uni , Lee Jae Hoon , Kim Uijin , Kim Yuri , Jeon Kyeongseok , Kang Ju-il , Kim Hyeran , Oh Song Hyuk , Kang Jun-Gu , Han Seung Ro , Yoon Sang Ho , Jin Liyuan , Zhang Yinhua , Kim Myoung-Hwan , Lee Myung-Shin , Cho Hyun-Soo , Lee Han-Woong
Published: November 2024

The COVID-19 pandemic underscores the critical requirement for robust animal models to elucidate the immunological and pathological repercussions of emerging pathogens. In this study, we introduce a novel humanized ACE2 mouse model (hACE2-E1/2/8) developed through precise CRISPR-based substitution of nine human ACE2 amino acids (N24Q, N30D, N31K, Q34H, T79L, S82M, F83Y, S84P, and H353K). This model maintains mouse ACE2 physiological function, exhibiting heightened susceptibility to SARS-CoV-2 without inducing brain damage, thus faithfully representing human COVID-19 pathology. Following infection with the mouse-adapted SARS-CoV-2 strain (MAp30), middle-aged hACE2-E1/2/8 mice exhibit more severe symptoms compared to their younger counterparts, with male mice showing more pronounced symptoms than females. Notably, middle-aged male mice develop long COVID-like symptoms, including persistent weight loss and sustained lung pathology post-acute infection recovery. Furthermore, our model replicates intricate immune responses observed in human COVID-19, such as heightened type I interferon levels and enhanced inflammation associated with neutrophils and eosinophils in plasma. Additionally, mice subjected to eosinophil or neutrophil depletion during early infection stages display milder disease symptoms. This model demonstrates substantial potential to facilitate in-depth exploration of COVID-19 and influence implications for therapeutic and preventative strategies.
Peptide-pulsed MHC class II allogeneic dendritic cell vaccine has superior efficacy providing allogeneic help in a murine cancer model.
Authors: Seishima Noriko , Becker William , Olkhanud Purevdorj , Maeng Hoyoung , Lopez-Lago Miguel , Wiseman Charles , Williams William , Berzofsky Jay
Published: November 2024

Allogeneic dendritic cell (DC) cancer vaccines present a promising alternative to autologous counterparts, offering an “off-the-shelf” solution for multiple patients and providing additional allogeneic help. We assessed the efficacy of a semi-allogeneic DC vaccine in comparison to a syngeneic one for tumor suppression. C57BL/6 mice were inoculated subcutaneously with human papillomavirus E6 and E7-expressing TC-1 cells. Syngeneic bone marrow DCs (BMDCs) were generated from C57BL/6 and semi-allogeneic BMDCs with a point mutation on either MHC class I or II were generated from B6.C-H2-Kbm1/By and B6(C)-H2-Ab1bm12/KhEg, respectively. The TC-1-bearing mice were injected with syngeneic or semi-allogeneic BMDCs pulsed with H-2Db-restricted E743-77 peptide. Compared with saline control, the MHC-I mutant BMDC vaccine reduced tumor growth no better than the syngeneic one. However, the MHC-II mutant BMDC vaccine was significantly better at delaying tumor growth. CD4+ or CD8+ T cell depletion showed that the syngeneic BMDC vaccine worked independently of CD4+ T cells, but the enhanced activity of the MHC-II mutant BMDC vaccine was dependent on CD4+ T cells at an early stage. Surprisingly, later depletion of CD4+ T cells improved vaccine efficacy, and this was confirmed to be due to Treg depletion. Thus, MHC class II allogeneic BMDCs proved more effective by inducing early allogeneic CD4+ T cell help, and this effect can by further enhanced by Treg depletion at a later stage.
PD1 blockade improves survival and CD8+ cytotoxic capacity, without increasing inflammation, during normal microbial experience in old mice
Authors: Dahlquist Korbyn , Huggins Matthew , Yousefzadeh Matthew , Soto-Palma Carolina , Cholensky Stephanie , Pierson Mark , Smith Declan , Hamilton Sara , Camell Christina
Published: November 2024

The elderly and those with chronic diseases face a 2 to 10-fold higher risk of hospitalization and mortality post-infection compared with healthy adults. Disease severity in the elderly is often attributed to the aging of the immune system, including an expansion of exhausted T cells that contribute to inflammation and have impaired activation and function. It is unclear how exhausted T cells from aged individuals respond to infection and the impact of acute restoration of T cell activation produces functional cells that contribute to excessive inflammation. In a mouse model of normal microbial exposure (NME), old specific pathogen-free (SPF) mice exhibit 100% mortality when exposed to microbes ordinarily found in pet store mice. We show that old mice exposed to NME show increased expression of inflammatory pathways and elevated frequencies of a heterogenous exhausted CD8+ T cell pool that express different levels of PD1, TOX, and CXCR5. Treatment with an anti-PD1 monoclonal blocking antibody in old mice during NME significantly improves survival without altering the acute inflammatory response despite the potential for adverse events following checkpoint blockade. Anti-PD1-mediated survival depends on CD8+ but not CD4+ T cells. CD8+ PD1+ T cells from old mice given anti-PD1 have increased GzmB production. These data suggest a novel approach for reducing infection vulnerability in the aged by targeting CD8+ T cell exhaustion through checkpoint blockade immunotherapy.
Towards optimizing cancer immunotherapeutic index: anti-CD3 Mono-Fab, a novel T cell-based therapeutic approach with less auto-Immunogenic profile.
Authors: Okpasuo Onyekachi , Mahadevappa Chandrashekara , Huynh Hien , Cheatam Christa , Radhakrishnan Vijay , Zaghouani Habib , Franklin Craig , Schrum Adam , Gil Diana
Published: November 2024

Despite advancement of cancer therapeutic approaches with immune checkpoint inhibitors (ICIs) against several tumor types, its anti-tumor benefit is limited by the emergence of immune related adverse events (irAEs). irAEs with an autoimmune phenotype, show a wide spectrum of toxicities that affect almost all organ systems. Several studies suggest a positive correlation between irAEs and anti-tumor response, with higher levels of toxicities seen with combination therapies. Finding strategies that optimize efficacy with less toxicity has been in the frontline of immunotherapy. We have shown a novel therapeutic strategy: anti-CD3e Monovalent Fab fragment (Mono-Fab) that targets the TCR/CD3 complex, strengthening signaling response to moderately weak antigens. Anti-CD3e Mono-Fab as a single therapy and in synergy with combination ICIs is effective against metastatic melanomas. To determine its optimal therapeutic index, we utilized a cell mediated experimental autoimmune encephalomyelitis mouse model as a platform to gauge potential to cause irAEs in the clinic. In this model, anti-CTLA-4 mAb administration to mice increases severity of their EAE symptoms over those observed in mice treated with appropriate IgG control, while anti-CD3e mono-Fab treatment does not. Thus, Anti-CD3e Mono-Fab contrast as an efficient potentially non-toxic cancer immunotherapy when compared with anti-CTLA-4 antibodies that are known to cause irAEs in patients.
Recombinant Fasciola hepatica fatty acid binding protein (Fh15) ameliorates dextran sulfate sodium (DSS)-induced ulcerative colitis in mice.
Authors: Figueroa-Gispert María Del Mar , Ramos-Lugo Claudia , Malavé-Ocasio Carlimar , Espino Ana
Published: November 2024

Ulcerative colitis (UC) is the most common inflammatory bowel disease affecting 5 million individuals worldwide. The Fasciola hepatica-derived molecule termed Fh15 is a TLR4 antagonist that has shown to exhibit powerful anti-inflammatory properties in murine and non-human primate sepsis models. Since TLR4 is involved in UC-pathological mechanisms, we hypothesize that Fh15 will suppress the pathological presentations of UC in mice. To test this hypothesis, we induced UC in C57BL/6 mice by providing animals with 4% DSS drinking water for 7 days. Mice were allotted into three experimental groups and four control groups containing eight mice each (4 male and 4 female). The experimental groups received i.p. injections with 50ug, 100ug or 150ug Fh15 on days 1, 3 and 5 of DSS-drinking. Animals that only drank DSS-water or that only received injections with 50ug, 100ug, or 150ug Fh15 were used as positive and negative controls, respectively. Results demonstrated that Fh15-treated animals had significantly less disease activity index, lower histopathological score, and longer colon-length than DSS-control animals; being 50ug the optimal Fh15 dose to reduce the extension of colon inflammation in male mice. Fh15 also suppressed other inflammatory markers such as MPO, CHI3L1, S100, T cells and macrophages. Preliminary results indicate that Fh15 could be an excellent therapeutic alternative to ameliorate the pathological signs of UC in the mouse model.
Catalytic activity and autoprocessing of murine caspase-11 mediate noncanonical inflammasome assembly in response to cytosolic LPS
Authors: Wodzanowski Kimberly , Akuma Daniel , Schwartz Wertman Ronit , Exconde Patrick , Vazquez Marrero Victor , Odunze Chukwuma , Grubaugh Daniel , Shin Sunny , Taabazuing Cornelius , Brodsky Igor
Published: November 2024

Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammasomes. Inflammasomes recruit caspases to undergo proximity-induced autoprocessing into an enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high molecular weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Expression of fluorescently labeled Casp11 in macrophages revealed that cytosolic LPS induced Casp11 speck formation. Unexpectedly, catalytic activity and autoprocessing were required for Casp11 to form LPS-induced specks in macrophages. Furthermore, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and processing of Casp11 via ectopically expressed TEV protease was sufficient to induce Casp11 speck formation. These data reveal a previously undescribed role for Casp11 catalytic activity and autoprocessing in non-canonical inflammasome assembly, and shed new light on the molecular requirements for noncanonical inflammasome assembly in response to cytosolic LPS.
Airway epithelial cell-specific deletion of EGFR in Scnn1b transgenic mice modulates inflammatory responses but doesn’t affect muco-obstructive features of lung disease
Authors: Choudhary Ishita , Paudel Kshitiz , Kumar Rahul , Patial Sonika , Saini Yogesh
Published: November 2024

Epidermal growth factor receptor (EGFR) is known to regulate airway mucous cell metaplasia, but the roles of EGFR-mediated pathway in the manifestation of cystic fibrosis (CF)-relevant lung phenotypes remain unclear. We hypothesized that airway epithelial cell-specific deficiency of EGFR mitigates muco-obstructive and inflammatory responses in sodium channel, non-voltage gated 1, beta subunit (Scnn1b) overexpressing -transgenic (Tg+) mice, a mouse model of human CF-like lung disease. To test our hypothesis, we generated airway epithelial cell-specific EGFR-deficient mice and performed various endpoint analyses. The deletion of EGFR in airway epithelial cells in Tg+ mice resulted in increased neutrophilic and macrophage infiltration into lung airspaces, which was accompanied by significantly increased levels of KC, G-CSF, MIP-2, MIP-1α, and MIP-1β. Additionally, as compared to EGFR-sufficient Tg+ mice, airway epithelial cell-specific EGFR-deficient Tg+ mice exhibited significantly increased postnatal mortality, compromised bacterial clearance and elevated markers of Th2 inflammation, including Il13, Slc26a4, and Retnla. Counterintuitively, the deletion of EGFR in airway epithelial cells of Tg+ mice did not alter mucous cell metaplasia, MUC5AC/5B production, and degree of mucus obstruction. Our data shows that, unlike inflammation, muco-obstruction-relevant responses in Tg+ mice are not dependent on airway epithelial cell-specific EGFR signaling.
Macrophage expressed TRIM47 is required to control RNA virus infection by targeting MAVS
Authors: Lu Wenting , xing Junji , Wang Junying , Gonzalez Anastasia , Wang Ling , Zhang Zhiqiang
Published: November 2024

In the past half century, there have been multiple RNA virus pandemics (influenza 1957, 1968 and 2009; SARS-CoV and COVID-19), inhabiting 44% of all emerging contagious diseases. Innate immunity is the first line of defense against viral infection. Here we found that TRIM47, a member of the TRIM family E3 ligase, plays a pivotal role in positively regulating host innate immunity in response to Influenza A virus (IAV). Among immune cells, TRIM47 is highly expressed in macrophages. Compared with wild type, TRIM47 deficient bone marrow derived macrophages exhibited a significantly lower level of type I interferon (IFN-I) following RNA mimics infection, encompassing IAV and poly I: C. After in vivo IAV infection, TRIM47 knockout mice showed an extremely lower survival rate, along with a lower level of IFN-I, higher viral load and more inflammatory cell infiltration in lungs compared to wild-type mice. Mechanistically, TRIM47 induces K68-linked ubiquitination of mitochondrial antiviral-signaling protein (MAVS), a key adaptor in RNA-sensing pathway, leading to robust IFN-I production. This K68-linked ubiquitination by TRIM47 is essential for MAVS aggregation and activation in macrophage. These findings provide compelling evidence that TRIM47 interacts with MAVS, enhancing IFN-I production to effectively restrict IAV infection in macrophages. This research not only advances our understanding of influenza pathogenesis but also opens avenues for potential therapeutic interventions.
Neuregulin-1 reduces necroptosis and protects mice from death caused by respiratory viral infection
Authors: Hussain Syed-Rehan , Rohlfing Michelle , Santoro Jennifer , Muralidharan Kaushik , Peeples Mark , Grayson Mitchell
Published: November 2024

Respiratory syncytial virus (RSV) and influenza type A virus (IAV) cause significant morbidity and mortality even with available vaccines. Pre-existing atopy provides protection against death in mice infected with Sendai virus (SeV), a rodent virus related to RSV. We found neuregulin-1 (NRG1) expressed at high levels in atopic mice, and when given exogenously (500 ng/day i.n for five days) to non-atopic mice provides significant protection against mortality from lethal doses of both SeV (2x106 pfu) and IAV (3x103 pfu PR8-mouse adapted). Further, using well-differentiated human airway epithelial cells, NRG-1 was found to improve epithelial barrier function following RSV infection. RSV has been reported to cause necroptosis, which can induce inflammation and alter epithelial membrane permeability. We hypothesized the survival advantage of NRG1 involves inhibition of necroptosis. Treating cells from the murine airway epithelial cell line LET-1 with NRG1 before inoculation with SeV significantly inhibited viral induced necroptosis as measured by a real-time non-lytic apoptosis/necroptosis assay. Furthermore, NRG1 reduced expression of genes promoting necroptosis (RIPK3 and MLKL) in well-differentiated human bronchial epithelial cells 72h post RSV infection. Together, these data suggest NRG1 modulates viral induced cell death through inhibition of necroptosis of epithelial cells and may explain the reduced airway leak and increased survival seen in our model.
Unfolding micro-case studies: a strategy for better student engagement/understanding/recall of difficult concepts in didactic sessions
Authors: Alard Pascale
Published: November 2024

The most common teaching format in professional school is lecturing, i.e., Powerpoint presentation, which minimally engages students. However, engagement of students in the classroom has been shown to improve understanding and retention of material via assessment of previous knowledge, prediction and retrieval of information. Learning from real-world scenarios is a method that medical/dental students favor over lectures covering basic science as it helps them make connections among various taught topics.

We have included very short case studies (micro-case studies) that unfold over time in 50-minute lecture sessions covering basic immunology. Basic concepts were first presented and a micro-case study developed over time to illustrate each taught concepts/details in a clinical setting. Students were asked prediction/retrieval questions about different portions of the session using a patient example. Students were provided with the micro-case study in its entirety at the end of each session, so they could use it to plug in the information learned during the session. The outcome of our study suggested that utilizing unfolding micro-case studies to illustrate basic immunology concepts is beneficial for the students in term of understanding and recalling the material better as well as enhancing student engagement as it can make challenging subjects more palatable. This format can be applied to any field and at any education level, including Graduate, Medical or Dental school.
Contribution of Helios in Ets1-Deficient NK Cells
Authors: Moos Ezra , Melanson Randy , Kee Barbara
Published: November 2024

Natural killer (NK) cells mediate anti-viral and anti-cancer immune responses. However, the gene networks that regulate NK cell development and function remain poorly understood. One transcription factor critical for proper NK cell development is Ets1. Previously we showed that loss of Ets1 at the germline level resulted in a developmental block, a functional defect, as well as a phenotype reminiscent of chronic cytokine stimulation in the NK cell compartment. Ets1-deficient NK cells also overexpress Helios, an Ikaros family transcription factor encoded by Ikzf2. Ikaros family transcription factors regulate development in many hematopoietic cell types; therefore, we hypothesized that Helios may be acting downstream of Ets1 to mediate the observed developmental block or functional defects. To investigate this, we generated NK cell-driven conditional deletion of Ets1 only (Ets1 cKO) or Ets1 and Ikzf2 (2xKO). The data presented here confirmed that Helios is highly expressed at the protein level in Ets1 cKO NK cells. Further, overexpression of Helios in Ets1-deficient NK cells does not contribute to the block in maturation nor the cytokine production defect. However, activated NK cells show high Helios expression, suggesting that Helios expression in Ets1-/- NK cells may be a consequence of the chronic cytokine stimulation phenotype.