bluuebunny/crossref_metadata_2025 huggingface dataset:
Nanoparticle immunoadjuvant complexes augment antigen-specific germinal center responses to enhance humoral immunity
Authors: Tursi Nicholas , Kannan Toshitha , Londregan Jennifer , Lederer Katlyn , Helble Michaela , Gary Ebony , Bedanova Nicole , Maillard Ivan , Allman David , Escolano Amelia , Kossenkov Andrew , Kulp Daniel , Weiner David
Published: November 2024

An alluring strategy for improving protective humoral immunity against infectious diseases would be to directly target the germinal center (GC). Robust GC responses are associated with high titer and high affinity antibody responses. Despite advances in formulation strategies and vaccine design, there remains a critical need for next generation approaches to elicit potent humoral immunity. Multimeric nanoparticles offer enhanced immunogenicity compared with monomeric antigen partially due to potent B cell activation and increased antigen density on the nanoparticle. Here, we investigated whether GC cytokine IL-21 could be displayed on nanoparticles to further enhance immune responses. We designed a chimeric nanoparticle using the GT8-60mer, a germline targeting HIV immunogen, as a model to scaffold IL-21. GT8 and IL-21 scaffolded nanoparticles, termed GT8 IL-21 nanoparticle immunoadjuvant complexes (GT8 IL-21 NIC), increased antigen-specific GC B cells, functional Tfh cell responses, and serum antibody titers relative to the antigen-only GT8-60mer. Additionally, GT8 IL-21 NICs reorganized antigen-specific GC B cell responses to the light zone (LZ) of the GC, the canonical site of antigen-driven selection. Single-cell RNA sequencing of antigen-specific GC B cells from GT8 IL-21 NIC immunized mice demonstrated upregulation of selection-associated gene signatures and a shift in the clonal repertoire. Thus, GC targeting NICs may provide value to drive improved humoral immunity.
SARS-CoV-2 WA1 induces distinct immune transcriptome profiles in human and deer primary respiratory epithelial cells
Authors: Nelli Rahul , Sarlo Davila Kaitlyn , Phadke Kruttika , Ruden Rachel , Bellaire Bryan , Sang Yongming , Giménez-Lirola Luis , Miller Laura
Published: November 2024

The ability of SARS-CoV-2 to infect animals, especially white-tailed deer (WTD), is a public health and economic concern. Humans experience varied clinical outcomes, while WTD are usually asymptomatic carriers. The immune factors responsible for these differences have yet to be studied. A comparative transcriptomic analysis in primary respiratory epithelial cells of humans (HRECs) and WTD (Deer-RECs) infected with SARS-CoV-2 WA1 was assessed throughout 48 hours post-inoculation (hpi). Both HRECs and Deer-RECs were susceptible to SARS-CoV-2, with significantly (P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine/chemokine response pathways mediated via IL-17 and NF-κB signaling pathways. SARS-CoV-2 activated AP-1/JUN transcription factor in both HRECs and Deer-RECs. Deer-RECs showed delayed upregulation of genes related to wound repair, including CXCL8, MAP4K4, p21, VEGFA, and HBEGF. The early inhibition of the NF-κB signaling in Deer-RECs predicted at 6 hpi may have contributed to the comparatively less proinflammation observed in Deer-RECs than HRECs. These findings may partly explain differences in WTD and human responses to SARS-CoV-2 WA1 infection.
The proteomic landscape of interferon signaling in primary T cells
Authors: Gordon David , Beusch Christian , Monaco Christopher , Swaldi Hailey
Published: November 2024

Interferon signaling has a central role in host defense against pathogens and modulation of inflammation, and it also impacts angiogenesis, carcinogenesis, and response to cancer therapeutics. Despite the physiological importance of interferon, the diverse molecular pathways mediating interferon signaling remain poorly defined, complicating understanding and treatment of interferon-mediated pathology. To address this knowledge gap, we applied mass-spectrometry based proteomics to map the phosphorylation pathways downstream of type-I interferon treatment in primary human T cells. Through time-course analysis of our phosphoproteomic data, we observe profound and rapid changes across over 1,300 phosphosites, revealing a dramatic rewiring of the proteome caused by interferon treatment. Clustering analysis of our phosphoproteomic data highlights numerous immunity-associated proteins, many of which have not previously been associated with interferon signaling, co-clustering with canonical STAT phosphosites (e.g. STAT1-Y701, STAT3-Y705, and STAT4-Y693). This rich dataset highlights the complexity of interferon signaling, and forms the basis for a drastically improved understanding of interferon-regulated pathways. In follow up work, we are also applying experimental genetics paired with phosphoproteomic analysis, as well as proteome-wide interaction studies, to delineate the network architecture of type-I interferon signaling.
Chikungunya-virus-specific CD4+ T cells are associated with chronic chikungunya viral arthritic disease in humans
Authors: Agarwal Rimjhim , Chang James , Cortes Fernanda , Ha Calvin , Villalpando John , Castillo Izabella , Galvez Rosa , Grifoni Alba , Sette Alessandro , Romero-Vivas Claudia , Heise Mark , Premkumar Lakshmanane , Falconar Andrew , Weiskopf Daniela
Published: November 2024

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes recurrent outbreaks in tropical and subtropical regions of the world. In the first 2-7 days of infection, most individuals experience high fever, skin rash and joint pain and swelling. However, about 30-60% of the infected individuals, primarily middle-age females, develop debilitating chronic arthritis-like symptoms such as joint pain, muscle aches and edema. Despite ongoing treatment and vaccine development efforts, the immune responses to CHIKV in humans remain largely understudied. In this study, we tested peripheral blood mononuclear cells (PBMCs) collected from a cohort of 39 individuals diagnosed with CHIKV disease during the 2014-2015 epidemic in Colombia against pools of overlapping sequentially spanning the entire CHIKV genome. Using high-resolution spectral flow cytometry, we detected robust CHIKV-specific CD4+ T cell responses, but not CD8+ T cell responses in the periphery. Patients experiencing chronic symptoms displayed a significantly higher abundance of CHIKV-specific memory CD4+ T cells, compared to their recovered counterparts. Furthermore, these CD4+ T cells displayed a significantly lower Th1 phenotype but were enriched in the Th17 phenotype, with the predominant production of TNFɑ. Overall, our work demonstrates that CD4+ T cells play a pivotal role in the pathogenesis of CHIKV and provides insight into possible therapeutic options.
RSV-induced Epigenetic regulation of DC via H3K4 demethylase KDM5b
Authors: Mire Mohamed , Cortes Ashley , Morris Susan , Rasky Andrew , Lukacs Nicholas , Ptaschinski Catherine
Published: November 2024

Immune regulation by epigenetic mechanisms has become an intense area of research. The concept that our immune system responses are influenced by environmental factors including microorganisms, pollution, and pathogen exposure is important as a backdrop for understanding disease progression. We previously observed an upregulation of KDM5B, an H3K4 demethylase, following RSV infection of DCs that regulates key innate cytokines, Type I IFN and TNF. As H3K4 tri-methylation is an activating mark, this demethylase should act as a transcriptional repressor to specific gene targets. Our in vitro and transcriptomic data shows genomic ablation of KDM5B in DC leads to greater antiviral response following RSV or TLR stimulation. This includes upregulation of type I IFN and downstream elements such as Cxcl10, Oasl1, and Mx2 genes. While more than 500 genes appear to be regulated in the KDM5b KO DC after RSV infection compared to WT DC, specific transcriptional regulation linked to type I responses were indicated. Utilizing the transcription factor prediction tool BART reveals IRF3, IRF7, and STAT1 as regulatory factors for the differentially expressed genes following TLR re-stimulation of RSV infected KDM5B-KO DC. To further extend these findings to an in vivo setting, KDM5b-CD11c-CRE+ mice were infected by RSV and, after 3 weeks, re-infected with RSV. Compared to KDM5b sufficient mice, there was a reduction in secondary RSV infection induced pathology and altered immune mediators.
Age influences CMV-specific NK and CD8 T cell memory fates
Authors: Jensen Isaac , Redenti Benjamin , Gray Joshua , Caron Daniel , Farber Donna
Published: November 2024

Cytomegalovirus (CMV), a prevalent persistent infection often unnoticed in healthy adults, is the most common congenital infection of neonates/ infants. NK cells and CD8 T cells control CMV via cytolysis of infected cells; yet their coordinate response to CMV remains uncharacterized, particularly in infants. We interrogated how NK and CD8 T cell responses differed between adults and infants in a murine CMV (MCMV) infection model. Infant mice had higher viral burden and delayed viral clearance in multiple tissues. T cell depletion enhanced infant MCMV-induced morbidity; yet, co-adoptive transfer of adult and infant OT-I CD8 T cells followed by MCMV-OVA infection revealed phenotypic and transcriptional limits in infant T cell capacity to form memory precursors and subsequent memory. This impairment was age-dependent, co-transfer of OT-I cells from sequentially aged mice had increasing memory formation, overall infant CD8 T cells are effector biased with limited memory potential. To reconcile how infants bridge this gap in T cell immunity we interrogated NK cell responses. Unlike adult mice, infant Ly49H+ (MCMV-specific) NK cells potently responded to infection, adopting an effector-memory (KLRG1+CD62L+) phenotype. Notably, loss of memory NK cells with age coincided with the increasing OT-I memory potential. Our data suggest discrete roles for NK cells and CD8 T cells in MCMV infection during infancy with NK cells as key responders prior to T cell ability to acquire memory.
Neurotropic viral infections induce priming and maintenance of a novel antigen-specific CD8 T cell population within the bone marrow that dysregulates the stem cell niche upon reactivation
Authors: Reesman Rachael , Wolf Delaney , Tritz Zachariah , Pfaller Christian , Hoyt-Miggelbrink Alexandra , Hansen Michael , Fain Cori , Johnson Aaron , Ayasoufi Katayoun
Published: November 2024

Bone marrow (BM) is the primary immune organ responsible for stem cell maintenance. Neurotropic infections elicit sequestration of T cells within the BM. Changes of the BM niche during brain viral infections are unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) in C57BL/6 mouse is cleared by 30 days due to generation of an antigen-specific CD8 T cell response. We determined that 1-10% of CD8 T cells present in the sternal, femoral, and cranial BM were virus antigen-specific 7 days post intracranial infection. Over 70% of these CD8 T cells were tissue resident. We determined antigen-specific CD8 T cells were generated in the BM within 4 days of infection, a timeline similar to lymph nodes. Continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in any BM compartment. CD8 T cells in the BM established durable memory populations and were reactivated upon antigen reencounter. Both acute infection and reactivation caused an increase in lineage-, Sca-1+, ckit+ (LSK) cells in the BM. This LSK dysregulation was abrogated upon CD8 T cell depletion and was limited to the intracranial routes of infection. We conclude that brain viral infections induce in situ effector and memory T cell responses within the BM and cause stem cell dysregulation. Our data paves the way for crucial studies of BM resident antigen-specific CD8 T cells in health and disease.
Loss of myeloid-KLF2 mediates LPS-induced mortality in neonatal pups by NLRP3 inflammasome signaling and altered neutrophil aging
Authors: Satyavolu Sriram , Mukherjee Devashis , Thomas Asha , Nayak Lalitha
Published: November 2024

Preterm neonates have disrupted myeloid cell activation and aging. KLF2 inhibits myeloid cell activation by repressing NF-kB, which controls NLRP3 transcription. In our myeloid-KLF2 deleted (MKO) murine model, postnatal day (P) 4 pups have higher LPS-induced mortality than controls but 100% survival in both groups after LPS at P12. P4 and P12 reflect preterm and term physiology, respectively. We aimed to to determine the role of neutrophil-NLRP3 and neutrophil aging in LPS-induced mortality in P4 MKO pups. Serum IL-1β was measured 4h post-LPS injection. BM neutrophils were treated with LPS and nigericin for NLRP3 protein expression in lysate and IL-1b levels in the supernatant. Daily MCC950 injections (P1-P4) were used to inhibit NLRP3 before LPS injection on P4. BM neutrophils were treated with LPS ex vivo and flow-sorted to examine CXCR4/CD62L expression. MKO pups have increased serum IL-1β, neutrophil-NLRP3 expression, and IL-1b release, P4>P12. In vivo NLRP3 inhibition rescued survival in P4 MKO and controls. P4 MKO have reduced BM and higher circulating neutrophils. MKO BM neutrophils are more aged (CD62Llo), but CXCR4lo, affecting aged neutrophil homing to the BM. Myeloid-KLF2 loss increases LPS mortality via NLRP3 activation in P4 pups. It accelerates neutrophil aging, but leads to persistence of aged neutrophils in the circulation by reducing CXCR4 expression. Future mechanistic studies will focus on KLF2's regulation of NLRP3 and CXCR4 expression at P4 and P12.
P120 catenin enhances macrophage efferocytosis and facilitates resolution of lung inflammatory injury
Authors: Wang Jianguo , Kanmani Paulraj , Hu Guochang
Published: November 2024

Efficient clearance of apoptotic neutrophils (PMNs) (efferocytosis) after sepsis plays a crucial role in facilitating tissue repair, resolving inflammation, and maintaining immune system balance. Here, we identified p120 catenin (p120) as a pivotal regulator in efferocytosis and subsequent recovery from inflammatory lung injury. We found that specific macrophage p120 deletion had a profound delay in the resolution of PMN infiltration, extracellular protein accumulation, and lung edema and injury following LPS challenge. p120 deletion increased the levels of TNF-α and IL-6, and reduced the levels of TGF-β1 and IL-10. Depletion of p120 also led to a marked decrease in the phagocytosis of apoptotic PMNs by macrophages. The number of p120-depleted alveolar macrophages containing apoptotic PMNs in bronchoalveolar lavage fluid was significantly lower. p120 deficiency caused a reduction in the expression of key efferocytosis-related molecules, namely AXL and CD36. p120-depleted macrophages exhibited a shift in polarization towards the M1 phenotype. Apoptotic cells triggered the association and co-localization of p120 and PPAR, and p120 deletion decreased the activity of PPARgamma in response to apoptotic PMNs. Inhibition of PPAR abolished p120-mediated efferocytosis and the resolution of lung inflammation. These findings underscore the critical role of p120 in facilitating the resolution of lung inflammation through modulation of macrophage polarization and PPAR signaling pathway.
Deciphering the role of Klf2 in CD8 T cell differentiation and exhaustion during chronic infection
Authors: Shen Jian , Brown Ryan , Kharel Arjun , Zhang Yuqi , Brown Ashley , Lin Siying , Cui Weiguo
Published: November 2024

T cell exhaustion, a state of dysfunction occurring during chronic infections and cancer, is marked by a progressive loss of effector functions and increased expression of inhibitory receptors. In chronic infection, CD8 T cells differentiate into TCF1+ progenitor (TPRO), CX3CR1+ effector (TEFF), and terminally exhausted (TEXH) subsets. The differentiation pathways are governed by complex transcription factor (TF) networks. Employing regulon analysis enabled by single-cell RNA+ATAC multiome sequencing, we dissected the TF networks orchestrating the fate of exhausted CD8 T cell populations. Our integrative analysis, complemented by single-cell CRISPR screening, highlighted Krüppel-like factor 2 (KLF2) as a pivotal TF necessary for maintaining TPRO cells and generating TEFF cells. Our research also revealed that KLF2 regulates the distinct distribution patterns of CD8 T cell subsets across organs. More intriguingly, multiomic analysis uncovered a novel function of Klf2 in limiting T cell exhaustion. Klf2-deficient CD8 T cells exhibited a marked increase in exhaustion signature genes. ATAC-seq and CUT&TAG assays further revealed different chromatin accessibility and histone modifications within Klf2 and its enhancers across CD8 T cell subsets. These insights underscore the differential transcriptional and epigenetic regulation of Klf2, presenting a novel mechanism that influences the cell fate decision of CD8 T cells during chronic infections.
Elucidating the Transcriptional Role of the Nuclear Receptor RORα in CD8+ T Cell Memory vs Effector Cell Development
Authors: Chuck Jonathan , Puvvala Avanti , Campbell Sean , Solt Laura
Published: November 2024

CD8+ T cells play a critical role in controlling viral infections and cancer and have the potential to be harnessed for the development of new therapeutic strategies. Defining the transcriptional regulatory networks that control CD8+ T cell differentiation and function is of great importance to achieve these goals. The retinoic acid receptor-related orphan receptor alpha (RORα), a member of the nuclear receptor superfamily of ligand-regulated transcription factors, has emerged as a potential transcriptional regulator of CD8+ T cell immunity. However, exactly how RORα functions in these processes is poorly understood. To this end we seek to identify the transcriptional role of RORα in regulating the differentiation of memory CD8+ T cells. Utilizing a mouse model with T cell-specific deletion of RORα we identify that RORα deficiency in CD8+ T cells leads to increased IFNγ production and increased cell-specific cytolytic function in vitro. Conversely, RORα overexpression results in decreased IFNγ production and defects in proliferative capacity in vitro. Our in vivo data further reveals that deletion of RORα in CD8+ T cells may affect the trafficking of these cells to non-lymphoid tissue and play a role in the development of tissue resident memory (TRM) cells. Our study provides important insights into the role of RORα in regulating CD8+ T cell function and highlights the potential for targeting nuclear receptors in the development of novel cell-based therapeutics.
Cellular immune responses in the guinea pig model with a 9-color phenotypic flow cytometry panel
Authors: Anthony Scott , Marketon Anthony , Pahar Bapi , Rivera Deja , Barr Bobbi , Bernbaum-Cutler Rebecca , Caravan Peter , de la Torre Juan Carlos , Mani Venkatesh , Carey Brian
Published: November 2024

Guinea pigs serve as a valuable animal model for many aspects of infectious disease research. A primary limitation of the guinea pig model is the lack of a robust toolbox to probe the underlying immune responses in these animals. We developed an optimized 9-color flow cytometric assay for immunophenotyping peripheral and tissue-associated immune cells in the guinea pig. Using this assay, we can delineate CD45+ cells as well as monocyte and macrophage populations, in addition to further differentiating lymphocyte populations of B and T cells. Importantly, we found that this panel is able to detect these populations in both inbred Strain 13 and outbred Hartley guinea pigs in whole blood and from fresh and frozen cells isolated from several peripheral tissues. We utilized this panel to separately interrogate the immune responses in whole blood and spleen tissue samples after exposure of Hartley guinea pigs with Ebola virus (EBOV) and Strain 13 or Hartley guinea pigs with Lassa virus (LASV). We believe this flow cytometry panel will serve as an important tool to interrogate the underlying immune responses to risk group 4 pathogens in addition to numerous other infectious diseases with the guinea pig model.
A Simplified Workflow for Flow Cytometric Analysis of Extracellular Vesicles on the Invitrogen™ Attune™ NxT Flow Cytometer
Authors: Ellis Samantha , Zahner Sonja , Nguyen Chau , Defelippi Kendall , Li Xiao
Published: November 2024

Extracellular vesicles (EV) are miniscule particles composed of a bilipid membrane that are released by all cells. In recent years, it has become clear that EV play an important role in cell-cell communication by shuttling their cargo of proteins, lipids, and nucleic acids. As their content can be indicative of the health of their cell of origin and ability to deliver functional biomolecules, EV have become an increasingly popular topic of research for their diagnostic and therapeutic potential.

EV vary in size, surface markers, and content. Their heterogenous nature introduces challenges for analysis when using technologies that were developed for cells. In flow cytometry, EV have a lower refractive index and paucity of epitopes comparative to cells, which hampers detection. In addition, few commercially available antibodies have been specifically validated for EV flow. This combined with the need for tedious isolation processes and a limited range of instruments capable of small particle detection can make EV flow cytometry a daunting endeavor.

Despite this, flow cytometry holds promise as an accessible approach to analyze EV. Here, we present a streamlined protocol of EV characterization by flow cytometry in which we adapted the Invitrogen™ CELLection™ Pan Mouse IgG Kit for EV capture and staining and optimized the Invitrogen™ Attune™ NxT Flow Cytometer for small particle resolution to validate monoclonal antibodies targeting EV from human plasma. For research use only.
Development of a High-Throughput Image Cytometric Screening Method as a Research Tool for Immunophenotypic Characterization of Patient Samples from Clinical Studies
Authors: Chan Leo , Nitta Carolina , Patel Samir , McDonald James , Mohammed Hamza , Hem Sopaul , Pierce Mackenzie , Parthasarathy Vaish , Hernandez Veronica , Stuckey Tyanna , Lin Bo , Reading Julian
Published: November 2024

Immune cell characterization from patients undergoing therapeutic treatments in clinical research is critical for understanding disease progression and treatment efficacy. Recently, a novel image cytometry system (Cellaca™ PLX Image Cytometer, Revvity, Inc.) was developed to complement flow cytometry immunophenotyping. Initial studies investigated the feasibility of identifying T-cell, B-cell, NK-cell, and monocyte populations in healthy donors in comparison to flow cytometry. Donor PBMCs were stained with three panels of surface markers: B-cell with CD3-KIRAVIA Blue 520 ™(KB) and CD19-PE; NK-cell with CD3-KB and CD56-PE; and monocyte with CD3-KB and CD14-PE. Stained samples were acquired on the image cytometer and the CytoFLEX (Beckman Coulter, Inc.). Results show similar population percentages within 5%. Furthermore, we tested the screening method using patients enrolled in autoimmunity (rheumatoid arthritis) and oncology (multiple myeloma) disease cohorts. 48 primary PBMC samples were analyzed with Panel 1 of CD3-KB, CD56-PE, and CD14-APC, and Panel 2 of CD3-KB, CD56-PE, and CD19-APC. We validated the image cytometry method by comparing the Cellaca PLX to the Aurora (Cytek™). Results demonstrated comparable CD3, CD14, CD19, and CD56 cell populations between both instruments, showing ~ 5 – 10% differences. The proposed image cytometry method provides a novel high-throughput research tool to streamline the immunophenotyping workflow for patient sample characterization.
ThresholdR: An Automated Method for Thresholding CITE-seq-based Antibody Expression Data for Immune Cells
Authors: Oliaeimotlagh Mohammad , Kumar Sunil , Khan Amir , Silas Armstrong Suthahar Sujit , Suryawanshi Vasantika , Makings Jeffrey , Durant Christopher , Drago Fabrizio , Taylor Angela , McNamara Coleen , Lanier Lewis , Hedrick Catherine , Ley Klaus
Published: November 2024

Cellular Indexing of Transcriptomes and Epitopes (CITE-Seq) enriches single cell transcriptomic insights by incorporating information about the cell surface phenotype through the application of oligonucleotide-tagged monoclonal antibodies. Similar to observations in flow cytometry, surface protein quantification in CITE-Seq introduces technical noise, potentially obscuring underlying biological processes and giving rise to misleading results. This noise originates from ambient antibodies within the reaction compartment (such as droplets or wells) as well as non-specific binding. Here, we present ThresholdR, an R-based automated tool, to reliably and systematically find the threshold for each antibody under the experimental conditions used in each experiment. We show that ThresholdR helps identification of cell types and enhances the presentation of biological differences across cell populations. Flow cytometry validation confirmed the proportions of cell populations per patient identified by ThresholdR, demonstrating strong and statistically significant correlations. Furthermore, our findings reveal that cell type-specific noise stems from the non-specific binding of antibodies, with larger cells, such as monocytes, exhibiting higher levels of noise compared to other cell types. We systematically assess the performance of ThresholdR across diverse datasets and platforms, underscoring the agreement between individual cell data and well-established surface markers.
Highly efficient non-viral delivery of macromolecules to unactivated human T cells using microfluidic transfection
Authors: Chehal Manreet , Ouellet Eric , Binson Elinor , Nguyen Quan , Liao Tina , Chau Phillip , Chen Kris , Lambertz Ulrike , Yu Jessie , Chen Kevin , Loughhead Scott , Dalton Bob , Eaves Allen , Louis Sharon , Kokaji Andy
Published: November 2024

Traditional methods for transfecting unactivated human T cells are limited in their delivery efficiency while maintaining cell viability, phenotype, and function. To address these challenges, we developed the CellPore™ Transfection System. This microfluidic platform creates transient disruptions in the plasma membrane via pressure-induced mechanical deformation, allowing for cytosolic delivery.

First, we developed a workflow to optimize delivery pressure to T cells using FITC-dextran. This optimized pressure was used to deliver mRNA for expression of eGFP (91.8%) and mCherry (92.9%), and Cas9 RNP complexes targeting B2M and TRAC, with knockout efficiencies of 74.0% MHC-I and 93.9% TCRαβ, respectively. Importantly, while electroporation of T cells resulted in increased expression of the activation marker CD69 (41.2%) and proinflammatory cytokines (77.7 pg/mL IL-2, 8.8 pg/mL IFNγ), CellPore™-transfected T cells maintained an unactivated phenotype (0.8% CD69+, 0.1 pg/ml IL-2 and 0.2 pg/ml IFNγ) and were capable of downstream activation/expansion via CD3, CD28, and CD2 cross-linking. Using this optimized workflow, efficient cytosolic delivery of FITC-dextran to human CD4 (97.4%), CD8 (96.6%) and regulatory (98.3%) T cells was also achieved.

The CellPore™ Transfection System enables robust and efficient delivery of cargo to T cells without impacting cell function in a simple workflow that can easily be integrated into research protocols.
Fast and Convenient Human Immune Profiling of Fresh and Long-Term Stabilized Whole Blood Samples Enabled With CyTOF
Authors: Watson Jessica , Hasan Shakir , Li Stephen , Loh Christina
Published: November 2024

Whole blood (WB) analysis within 24 hours of collection is crucial for monitoring clinical efficacy of therapies. WB collection and cytometric analysis are often performed at different sites, leading to significant delays. Fixation of WB with stabilizer reagents can overcome these challenges. However, not all antibody panels are compatible with these reagents. A high-parameter CyTOF™ panel was created to be compatible with commercial WB stabilizers to enable fast and convenient human immune profiling.

The CyTOF panel contains 20 metal-tagged antibodies to identify over 30 immune cell populations in a single tube of fresh or stabilized WB. There are more than 30 open channels for additional markers. Flexibility of the panel and its workflow was shown using two methods of stabilization, in which WB is either stained followed by freeze storage or WB is first frozen then later stained. To reduce technical variability in staining and acquisition, the CyTOF panel was pooled and frozen as single-use aliquots ahead of time, and samples were barcoded and acquired as a single tube.

The CyTOF panel for broad immune profiling overcomes analysis challenges caused by logistical delays as WB samples can be frozen with various workflows. Furthermore, freezing antibody cocktails is a unique feature of CyTOF flow cytometry, ensuring staining consistency. Thus, CyTOF workflows enable fast and convenient cytometry of WB samples.

For Research Use Only. Not for use in diagnostic procedures.
Systemic impact of melanoma on bone marrow-derived immune cells
Authors: Carey Alexis , Palm Cheyenne , Chhabra Yash , Weeraratna Ashani
Published: November 2024

Melanoma accounts for the most skin cancer-related deaths in the United States. As patients age, antitumor responses decline, making age a negative independent prognostic factor of overall survival in melanoma. Delving into age-associated changes in lymphoid organs, such as increased bone marrow (BM) adiposity, could improve the understanding of the aged antitumor response. We hypothesize that the age-related increase in BM fat drives the functional decline of immune cells, ultimately hindering the systemic antitumor response to melanoma. We utilized flow cytometry to analyze melanoma and age-related alterations to the BM, blood, and tumor immune cell composition. Our preliminary data suggests that mice challenged with melanoma systemically increased granulocytes. However, only aged tumors had an increase in granulocyte infiltration. Meanwhile, T cells significantly decreased in the BM of tumor-bearing mice. Additionally, there was a significant increase in lipids in the BM T cells from non-tumor-bearing aged mice and a reduction in these lipids when mice were challenged with tumor. Thus, there appears to be an age-dependent systemic immune response when the mice are burdened with melanoma. As a future direction, we plan to investigate the role of the aged BM niche on the effectiveness of immunotherapies. These insights could lead to improved outcomes for elderly patients with melanoma.
Exploring the Biomedical Potential of MXenes in Macrophage Phenotype Identification and Tracking
Authors: Longo Giorgia , Giro Linda , Fusco Laura , Resto-Trujillo Natalya , Chacon Benjamin , Shuck Christopher , Pigazzi Martina , Gogotsi Yuri , Ley Klaus , Orecchioni Marco , Delogu Lucia
Published: November 2024

Macrophages are innate immune cells with varying phenotypes and functions that are influenced by their microenvironment. The distinction between the classical M1 and M2 phenotypes has been crucial in understanding their involvement in inflammation. Recently single-cell technologies have revealed a complex range of macrophage activation states, each contributing differently to maintaining physiological balance and affecting the development and progression of diseases. Identifying the origins and function of these macrophage subsets remains a complex challenge. MXenes, a category of two-dimensional transition metal carbides and nitrides, show promise in the biomedical field owing to their notable features, such as biocompatibility. MXenes' unique chemical properties allow for their detection at the single-cell and tissue level using mass cytometry (CyTOF), which shows great promise for biomedical applications. We found that MXenes can be internalized by myeloid cell precursors, monocytes, and macrophages without affecting their survival or differentiation. Early evaluations of MXenes uptake by immune cells have shown their detectability even after 30 minutes of incubation, attributable to their chemistry and impressive signal-to-noise ratio at CyTOF. These characteristics position MXenes as effective tools for precise single-cell labeling and in vivo monitoring of monocyte-derived macrophage phenotypes across various tissues and in response to specific stimuli or diseases.
Assessment of the Fetal Gut Immunome after Decidua-Placental Inflammation
Authors: Vazquez Jessica , Keding Logan , Schotzko Michele , Antony Kathleen , Racine Jenna , Simmons Heather , Shah Dinesh , Golos Thaddeus
Published: November 2024

Exposure to maternal inflammation in utero has been shown to impact the long-term immunological health of the fetus.

Tisseel (n = 5), a surgical glue, or saline (n = 3) was injected directly into the placenta of pregnant (~GD90) rhesus macaques. C-sections were performed at ~GD150. Fetal colon and jejunum were collected at necropsy and mononuclear cell (MCs) were isolated. Amniotic fluid was collected and cytokine levels assessed by cytometric bead analysis. Colon and jejunum MCs were labeled with fluorochrome-conjugated antibodies against markers for B cells, Innate Lymphoid Cells (ILCs), and T cells. Data was acquired using the Cytek Aurora spectral cytometer and analyzed on SpectroFlo (v3.0) and pre-gated to Live CD45+ cells in FlowJo (v10.8.1). Pre-gated FCS files were then clustered and visualized within R package Specter (v1.0.0). Post-analysis was done within Specter, with appropriate statistical test, p < 0.05 considered significant.

Tisseel fetuses had a unique population of activated ILC3s in the colon. In the fetal jejunum, we found that the Tisseel fetuses, similarly had an activated Tbet+ T cell population. In addition, Tisseel fetuses also had a unique, activated ILC population. Interestingly, we did not find any differences in cytokine levels in the amniotic fluid of these pregnancies.

Overall, decidual/placental inflammation led to disturbance of the fetal gut immunome. The mechanism by which these changes are occurring are still under investigation.
Altered mitochondrial homeostasis in SLE neutrophils promotes aberrant NET release but blunts NETosis during infection.
Authors: Reber Callista , Wise Ashley , TenBarge Eden , Mendonca Jessica , McDaniel Sarah , Mennen Ellie , Holder Bailey , Belevsca Eva , Ferris Jennifer , Burcham Lindsey , Bieber Jeffry , Monteith Andrew
Published: November 2024

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a lack of immune tolerance to nuclear antigens leading to systemic inflammation. Patients with SLE are more susceptible to bacterial infections, which is thought to be the result of immunosuppressants used to treat their disease. However, we showed that SLE neutrophils are impaired in their response to bacterial infection. While the unique cell death process resulting in the formation of neutrophil extracellular traps (NET; NETosis) has been attributed to antagonizing SLE pathology, we demonstrated that SLE neutrophils fail to undergo NETosis in response to the bacterial pathogens. Herein, we identified that production of lactate by phylogenetically distinct bacterial pathogens caused increased generation of mitochondrial superoxide and triggered NET release. In contrast, SLE neutrophils displayed an altered metabolic state that rendered them incapable of sensing bacterial lactate. As such, SLE neutrophils failed to undergo NETosis in response to the bacterial pathogen Staphylococcus aureus and underwent aberrant NET release upon stimulation with apoptotic debris. These results indicate that NET formation in response to bacteria may be distinct from pathways promoting heightened NETosis in SLE. Furthermore, deciphering the metabolic disruptions driving inappropriate NET formation in SLE may lead to clinically actionable biomarkers that limit accumulation of nuclear antigens and restores host defense.
Metabolic modulation unleashes immune vigor in murine pancreas tumors
Authors: Poimenidou Maria , Le Janet , Darnell Chad , AbuEid Mahmoud , McAllister Donna , Dwinell Michael
Published: November 2024

Our study investigates the transformation of the pancreatic ductal adenocarcinoma tumor microenvironment from an immune-excluded to an immune-inflamed state using metabolic inhibitors metformin and mitochondria-selective conjugates, MMe and pCF3-MMe. In murine models of pancreas cancer, MMe and pCF3-MMe exhibited potent inhibition of tumor growth. Flow cytometry indicated increased levels of total T cells, activated CD8+, and NK T cells in MMe and pCF3-MMe treated mice. The anti-tumor effects of these metabolic inhibitors were reversed upon CD8+ T cell immunodepletion. In vitro experiments demonstrated elevated levels of pro-inflammatory chemokines in MMe-treated tumor cells, aligned with increased CD8+ T cell infiltration. Furthermore, metabolic inhibitor treatment activated both helper CD4+ and cytolytic CD8+ T cells, with the latter demonstrating increased glycolysis and acidification rates indicative of enhanced activation. Flow cytometry corroborated these findings, revealing heightened proliferation and activation markers on treated T cells. In addition to the direct effects on T cells, immunofluorescence microscopy suggested the activation of the mitochondrial antiviral-signaling protein (MAVS) pathway in tumor cells and an increase in apoptosis as indicated by the significant increase in caspase 3/7. Our findings highlight the potential of metabolic inhibitors to activate anti-tumor immune responses directly and indirectly in murine pancreas tumors.
Single cell RNA sequencing analysis of tumor infiltrating γδ T cells in murine and human glioblastoma.
Authors: LA JEONGWOO , Kang In , Lee Heung Kyu
Published: November 2024

Glioblastoma multiforme (GBM) is the most commonly occurring malignant brain tumor. The anatomical location and the immunosuppressive TME have brought disappointing results for multiple therapeutics approaches, including immune checkpoint inhibitors (ICIs). γδ T cells are an attractive target for adoptive cell therapies (ACTs) due to their MHCI-independent tumor killing mechanism and GVHD-Free nature. Thus, investigating GBM infiltrating γδ T cells can provide insight into the pro-tumoral and anti-tumoral roles of γδ T cells and provide potential targets for gene engineering prior to ACT. Our Single-cell RNA sequencing (scRNA-seq) and flow cytometric data demonstrate that GBM harbors subtypes of γδ T cells of different origins and functions. γδ T cells from the meninges produce IL-17A and are enriched for genes related to Rorγt programme and immune checkpoint molecules. On the contrary, blood-borne γδ T cells produce IFN-γ and express genes related to cytotoxicity, co-stimulation and activating NK receptors. Functional dichotomy of γδ T cell is also observed in human GBM, with Areg producing tumor-promoting subset and Gzmb producing cytotoxic subset. Interaction analysis reveals that these subtypes of γδ T cell strongly interact with myeloid cells through unique receptor-ligand interaction. These finding reveal the comprehensive role of γδ T cells during cancer progression and provide insights into potential gene-editing targets for effective ACTs.
Type I IFN regulates neutrophils responses to murine beta-coronavirus infection
Authors: Worley Matthew , Gong Heng , Carver Kyle , Deng Jane
Published: November 2024

Introduction: Type I IFNs play a critical role in the immune response to viral infections. Suppressed type I IFN levels are predictive of severe SARS-CoV-2 infections. Given the critical role of neutrophils in viral-mediated lung injury, we investigated how type I IFNs regulated neutrophil responses during pulmonary coronaviral infections.

Methods: IFNAR-/- and C57BL/6 (WT) mice were infected with 2,500PFU of murine beta-coronavirus (MHV-1) and survival determined. At day 3 post infection, cellular recruitment, cytokine profiles and markers of lung injury were evaluated. Neutrophils were evaluated for activation markers, ROS generation and NET formation.

Results: All IFNAR-/- mice succumbed within 5 days versus 100% survival for WT mice. IFNAR-/- mice had higher pulmonary levels of GM-CSF (p=0.0079) and 3-fold more recruited neutrophils (p=0.0159) and monocytes (p=0.016). IFNAR-/- lung neutrophils had significantly lower TLR3, TLR4, TLR7, TLR9, CD88, CXCR2, CD69 (p<0.05 for all) but increased levels of PDL-1. IFNAR-/- neutrophils isolated during infection showed significantly decreased ROS and NETs compared to WT.

Conclusion:IFNAR-/- mice are highly susceptible to MHV-1, displaying elevated neutrophil recruitment with lower frequency of activation markers, reduced ROS and NETs, and increased expression of PDL-1. Our findings demonstrate mechanisms by which inadequate type I IFN levels subvert critical aspects of neutrophil responses during severe viral respiratory infections.
CRISPR Screens Identify Key Regulators of NK Cell Cytotoxicity in Cancer Therapy
Authors: Rethacker Louise , Romero Hugo , Dulong Joshua , Sasseville Marc , Lalonde Marie-Eve , Gadoury Christine , Beland Kathie , Beausejour Christian , Marcotte Richard , Haddad Elie
Published: November 2024

Natural Killer (NK) cells are innate lymphocytes that exhibit cytotoxic activity against cancer cells and are associated with a good prognosis when present in solid tumors. In the past, NK cells have been used in adoptive cell transfer to treat various cancers with modest relative success. More recently, NK cells modified with chimeric antigen receptor (CAR-NK cells) are being explored as a potential off-the-shelf therapy. However, mechanisms preventing the full cytotoxic anti-tumor activity of NK cells remain understudied.

To unveil the key genes that govern the activation and inhibition of the cytotoxic activity of NK cells towards cancer cells, we performed whole-genome CRISPR screens in NK cells. Primary activated NK cells from the NK-cell Activation and Expansion System (NKAES) were transduced with the Yusa CRISPR library and then electroporated with Cas9 protein, inducing the knockout (KO) of 18,010 genes. These NK cells were exposed to the colorectal cancer cell line HT-29 for 4 hours. After co-culture, NK cells were sorted based on the expression of the degranulation marker CD107a and the production of IFNg, both associated with NK cell activation. To identify genes involved in the inhibition of NK cell activity, we will compare sgRNA enriched in CD107a+ and IFNg+ cells to unactivated negative cells.

In turn, these genes can be therapeutically targeted or genetically modified in future iPSC-derived NK and CAR-NK cells to improve the outcome of cancer patients.
Natural Killer T cell subsets exhibit tissue-specific transcript factor expression following bacterial challenge
Authors: Rodriguez Luisa , Torres Cameron , Spencer Charles
Published: November 2024

Natural Killer T cells are a population of lymphocyte immune cells that have regulatory effects that influence the inflammatory response. There are two subsets of NKT cells, type I and II which can be experimentally differentiated based on their antigen recognition of CD1d-tetramers loaded with α-galactosylceramide (αGalCer-tetramer). NKTTK cells become signaled upon infection of pathogens and have been observed to respond to pathogenic challenges. Francisella Tularensis (Ft) is a bacterial zoonotic bacterial pathogen that, when transmitted to human hosts, can cause severe clinical disease. Infection with Ft leads to an over-response of the inflammatory immune reaction, causing dysregulation of cytokines, known as the cytokine storm. Type I NKT cells have been observed to express exhibited transcription factor profiles similar to T helper cells such as T-bet, GATA3, and RORγT, that represent Th1, Th2, or Th17 phenotypes. In this study, we utilize flow cytometry to characterize both type I and type II NKT transcription factor profiling in response to Ft infection. We identify Th17 responses by type II in respiratory tularemia and Th1 responses by type I in ulceroglandular tularemia to be key factors contributing to disease outcome.
Blood immune receptor repertoire and transcription profiling to characterize disease progression in a murine tuberculosis model
Authors: Kobzik Lester , Chenchik Alex , Hu Dongfang , Makhanov Mikhail , Paraiso Kitt , Ghias Khadija , Diehl Paul , Yabaji Shivraj , Kramnik Igor
Published: November 2024

Murine models of tuberculosis (TB) can reveal mechanisms and allow testing of drug/vaccine interventions. Current approaches often rely on the evaluation of lung lesions after euthanasia, with a large number of animals needed for time course and dose-response studies. Measuring blood biomarkers in small volumes obtained serially could provide useful insights and reduce the number of animals needed. We evaluated the utility of small-volume blood samples for profiling T- and B-cell receptor repertoires and transcriptomes in mice (n=16) at different stages of TB progression (early, middle, and advanced—defined by lung histopathology and mycobacterial loads). We isolated RNA from 100 uL of whole blood for TCR/BCR profiling using the Cellecta DriverMap adaptive immune receptor (AIR) assay as well as transcriptome quantitation of ~4700 key mouse genes using a targeted sequencing panel. The results showed substantial changes in clonotype usage and diversity over the course of TB disease, most prominent in the BCR IgH (e.g, top 10 clonotype abundance count in control, early, advanced disease, respectively: 62 + 47, 1792 + 589, 3559 + 286, p < .01, mean + SD, n=3-4). Blood transcriptome profiling showed abundant up-regulation of immune and inflammatory genes (e.g, up-regulated genes vs control: 182 (early), 382 (advanced), q.05 FC1.5). The results support direct testing of non-terminal serial blood samples to characterize disease progression & therapeutic or vaccine interventions.
CXCR6+ polyfunctional CD4 T cells are essential for protective immunity against Chlamydia in the female reproductive tract
Authors: Mercado Miguel Antonio , Li Qiang , Kim Yejin , Li Lin-Xi
Published: November 2024

The sexually transmitted Chlamydia infection has been increasing in the last few decades in the United States and worldwide. A better understanding of CD4 T cell effector function during infection is essential to develop a protective vaccine. Recently, we reported that mice lacking the transcription factor basic helix-loop-helix family member e40 (BHLHE40) had a significant defect in their ability to clear infection from the female reproductive tract (FRT). An increase in IL-10-producing T regulatory type 1 (Tr1) cells and a decrease in polyfunctional CD4 T cells co-producing IFN-γ, IL-17A, and GM-CSF in Bhlhe40-/- mice correlated with greater bacterial burden and reduced protective immunity in these mice. Using scRNA-seq, we found that Cxcr6, which encodes the chemokine receptor CXCR6, was enriched in the polyfunctional CD4 T cell clusters in WT mice but not in the stem-like clusters in Bhlhe40-/- mice. Here, we found that both CXCR6- and CXCR6+ CD4 T cells were detected in the FRT following infection, but CXCR6+ CD4 T cells contained most of the polyfunctional T cells. Notably, anti-CXCR6-treated mice exhibited a marked decrease in polyfunctional CD4 T cells, particularly, those capable of producing IL-17A. This corresponded to increased bacterial burden and delayed clearance following intravaginal infection. Overall, our results demonstrate that CXCR6 marks a polyfunctional CD4 T population required for protective immunity against Chlamydia in the FRT.