bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of C3aR induces immune infiltration and inflammatory microbiota in a new spontaneous model of colon cancer
Authors: Guglietta Silvia , Weber Lukas , Fosso Bruno , Hardiman Gary , Marzano Marinella , Robinson Mark , Krieg Carsten
Published: February 2023

Several lines of evidence suggest that inflammation plays a pivotal role in the development and progression of colorectal cancer (CRC) and can be unleashed by the loss of innate immunosurveillance. The complement system is a well characterized first line of defense against pathogens and a central component of the immune response. Emerging evidence suggests that complement anaphylatoxin C3a produced upon complement activation and acting via its receptor (C3aR) may play a role in intestinal homeostasis. However, to date, it is unknown whether and how the C3a/C3aR axis can affect CRC. By mining publicly available datasets, we found that CpG island methylation of c3ar1 occurs in CRC patients and is associated with significant downregulation of C3aR. By reverse-translating this finding we were able to shift in APCMin/+ mice the tumorigenesis from the small intestine to the colon therefore generating a novel mouse model, which more closely mirrors the CRC in humans. Transcriptomic analysis on colorectal polyps from our newly developed genetic mouse model revealed a significant increase in innate and adaptive immune signatures in absence of C3aR. Furthermore, loss of C3aR significantly impacted the fecal and tumor-associated microbiota and supported the blooming of pro-inflammatory bacterial species as confirmed by experiments of fecal microbiota transplantation.

Future studies will elucidate whether loss of C3aR can be exploited as a biomarker for sub-groups of CRC and whether the C3a/C3aR axis may be exploited for the generation of more effective therapeutic interventions.
Regulatory network of miRNAs, lncRNAs and target genes associated with immune response in bovine mastitis
Authors: Morenikeji Olanrewaju , Tucker Ashley , Salazar Nicole , Ayoola Adeola , Thomas Bolaji
Published: February 2023

Bovine mastitis is mainly caused by Escherichia coli and Streptococcus uberis. It is associated with complex multifactorial phenotypes, requiring an integrative approach to elucidate the molecular networks underlying variable disease outcome. The association of lncRNAs, miRNAs and their target as key players in immunity regulation has not been studied in bovine mastitis. We hypothesize that non-coding genes could mediate immune response in bovine mastitis and have potential as disease markers or drug targets. Through bioinformatic analyses, we investigated the networks of lncRNAs, miRNAs and mRNAs, and identified key regulatory elements driving immune response in bovine mastitis. Our analyses reveal 16 highly significant immune response genes including MYD88, IL-10, IL-4, ICAM1, CXCL8, IL-18 and CSF2. Notably, of the 2904 miRNAs reported, six - bta-miR-24-3p, bta-miR-149-5p, bta-miR-223, bta-miR-185, bta-miR-328, and bta-miR-874, were predicted to bind multiple regions of target genes. Likewise, eight out of 22 lncRNAs including NONBTAT001181.2, XR_003030515.1 and XR_003030515.1 bind 13 mRNA targets. Of interest, some of these are conserved in 15 different species, including Homo sapiens. Our functional analyses show that these lncRNAs and miRNAs may regulate pathogenesis of bovine mastitis through lipopolysaccharide-mediated signaling pathway, regulation of chemokine (C-X-C motif) ligand 2 production, regulation of IL-23 production, positive regulation of chemokine production and pattern recognition receptor activity. This interactome lays a foundation for molecular interconnectivity of regulatory elements in bovine mastitis, deserving further elucidation for potential vaccine and therapeutics.
A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions
Authors: Yoder Jeffrey A , Wcisel Dustin J , McConnell Sean , Hernandez Kyle M. , Andrade Jorge , de Jong Jill L. O. , Litman Gary W , Dornburg Alex
Published: February 2023

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide some level of immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby challenging our ability to understand the evolutionary origin and specific function of these genes. In order to better understand the diversity of NILT genes and their possible roles in immune function, we identified 5 NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3 Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternate haplotype for this same genomic region. We then identified transcripts that define 43 different NILT genes and reflect an unprecedented range of sequence diversity and combinatorial diversity of Ig domains for a single family of non-recombining receptors. Zebrafish NILTs include a single putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. Whole genome sequences from different genetic backgrounds reveal at least three alternative haplotypes of the zebrafish NILT gene cluster that display gene content variation. This observation indicates that different individual fish can encode different combinations of NILTs which could impact their immune function. Furthermore, these results reveal a higher level of genetic complexity and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.
Macrophages Harbor Transcriptionally Active Defective HIV Provirus Which Contribute to Immune Activation
Authors: Kuniholm Jeffrey , Armstrong Elise , Coote Carolyn , Henderson Andrew
Published: February 2023

People living with HIV suffer from a variety of co-morbidities including neurological deficits and cardiovascular disease even while on antiretroviral therapy (ART). Chronic inflammation has been implicated in these co-morbidities and has long been described in HIV patients. Characterization of the HIV DNA (provirus) reservoir in CD4 T cells from HIV patients on ART has revealed that more than 90% are defective and unable to complete the viral life cycle. Macrophages are susceptible to HIV infection and are positioned at the nexus of pathogen detection, inflammation, and priming of adaptive immune responses. We hypothesized that HIV-infected macrophages harbor defective HIV proviruses which remain transcriptionally competent and affect adaptive immune responses. To test this, we examined the status of HIV proviruses in primary human monocyte-derive macrophages (MDMs) and their expression of HIV transcripts. MDMs were infected with HIV in vitro and using an intact provirus detection assay we observed that roughly half of HIV proviruses were defective. Reverse transcription droplet-digital PCR (RT-ddPCR) detected a distribution of HIV transcripts, many of which lack the full 5′ LTR sequence consistent with transcription starting from an intragenic site. When CD8 T cells were co-cultured with autologous HIV-infected MDMs, they increased their expression of CD107a, a degranulation marker, indicating that infected macrophages could trigger CD8+ activation. Together, these results support that alternative transcriptional mechanisms, which persist in the absence of a fully functional LTR, are active during HIV infection in macrophages, contributing to macrophage dysfunction and persistent HIV-associated diseases.
Profound phenotype alteration and delayed regeneration of circulating antigen presenting cells in COVID-19
Authors: Winheim Elena , Rinke Linus , Lutz Konstantin , Reischer Anna , Münchhoff Maximilian , Hellmuth Johannes , Scherer Clemens , Subklewe Marion , Krug Anne B
Published: February 2023

Disease manifestations in COVID-19 range from mild symptoms to severe/critical illness with lung inflammation, hypercoagulation and cardiovascular complications, which may be caused by a dysregulated innate immune response. Antigen presenting cells may contribute to the inflammatory response and shape the adaptive immune response in COVID-19 patients. We therefore investigated the frequency and phenotype of circulating dendritic cells (DCs) and monocytes in hospitalized COVID-19 patients with mild/moderate or severe disease at different time points (n=65). We found an overall reduction of DCs and a shift towards the CD14+ DC3 population within the DCs in patients vs. controls.

In patients with more severe disease, we observed upregulation of CXCR3, CCR2, CD40 and PD-L1, but reduced expression of CD86 and HLADR in monocytes and conventional DCs (cDCs) within the first 3 weeks after diagnosis (n=26). Increased proliferation and expansion of precursor cells in the blood indicated an enhanced turnover that was observed even in convalescent patients. Proliferating cDC2 and DC3 showed a phenotype similar to that found in healthy controls suggesting that the phenotype alterations are induced in differentiated DCs by external factors. The observed changes were accompanied by increased inflammatory cytokine levels in the plasma and lymphopenia.

Thus, in more severe COVID-19 circulating cDCs and monocytes exhibited a profoundly altered expression of costimulatory molecules and were poised to exit the blood in response to inflammatory chemokines. Increased proliferation even after recovery indicated delayed regeneration of the mononuclear phagocyte system.
In Silico Assessment of SARS-CoV-2 Mutations with Relevancy to Prophylactic and Monoclonal-Based Treatment Strategies
Authors: Benoit Lora , Mikbel Deena , Vilela Mario , Alsalihi Yusur , Shakran Dina , Merino Edward , Mohseni Ali
Published: February 2023

The ongoing pandemic caused by SARS-CoV-2 has inflicted significant disease burden worldwide. Several vaccines are now in use that are based on the virus’s highly immunogenic spike “S” protein, which mediates infection of host cells. However, since the emergence of the reference strain in 2019, the virus has underdone >40,000 nucleotide mutations, many of which have resulted in deletions or amino acid substitutions in the S protein. Most mutations appear to be random, while others deviate from expected outcomes. Indeed, some non-random mutations appear to confer resistance to either innate or adaptive immune functions while other enhance transmissibility. Although the consequences of these mutations are largely unknown, there is concern for the ability of select mutations to detract from the protection conferred by prophylaxis strategies and interventional therapies. We have therefore selected >50 whole genome-length mutant strain from 3 relatively closed populations to compare against widely circulating strains of concern and the reference strain so that we could assess differences in mutation events within the context of distinct population immunogenetics. For each strain we have biochemically characterized potentially critical mutations, assessed the randomness of each mutation, and assessed key mutations occurring in the S, M, E and N proteins for their ability to influence innate or adaptive immune recognition using bioinformatic platforms. The results of this study further our understanding for how these mutations influence population-based immunity to the virus, including biomedical interventions, as well as help explain the selection pressures and external events that are driving this genetic diversification.
T-B lymphocyte interactions mediated by SLAM-associated protein (SAP) are essential for diabetes in transgenic anti-insulin VH125SD.NOD mice
Authors: McNitt Dudley H , Bonami Rachel H , Hulbert Chrys , Thomas James W
Published: February 2023

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing beta cells. An essential role for B lymphocytes in the disease process and molecular signatures for T follicular helper cells (Tfhs) in T1D point to the importance of T-B lymphocyte interactions in the pathological process. To understand mechanisms that underpin these T-B interactions, we introduced deficiency of the signaling lymphocyte activation molecular (SLAM)-associated protein (SAP) into VH125SD.NOD mice. In VH125SD.NOD mice a targeted anti-insulin VH gene generates a small population of anti-insulin B lymphocytes that are highly diabetogenic yet are functionally silent for autoantibody production. We find that SAPko dramatically eliminates germinal centers in spleen, pancreatic draining lymph nodes and pancreas. However, insulitis was similar between SAP-sufficient and SAPko VH125SD.NOD mice early in T1D development (8–12 weeks old) but differed later in disease onset (13–17 weeks old). Strikingly, SAP is essential for T1D development, as SAPko VH125SD.NOD mice did not develop T1D. Despite elimination of germinal centers, the numbers of Tfh cells were largely unaffected in the absence of SAP. These findings suggest that prolonged T-B lymphocyte conjugates maintained by SAP are not required for retaining Tfhs; however, these interactions maybe essential for the genesis of pathogenic Tfhs when functionally silent B lymphocytes recognize a beta cell autoantigen.
CD8αα expression increases during demyelinating disease progression
Authors: Thyagarajan Hiran Maye , Cornwall Douglas H. , Evavold Brian D.
Published: February 2023

Experimental autoimmune encephalomyelitis (EAE) is a well-described mouse model for multiple sclerosis (MS), a demyelinating autoimmune disorder of the central nervous system (CNS). While the role of CD4 T cells is well established, other immune cell types, particularly CD8 T cells, are key contributors to disease pathogenesis. Our study aims to categorize the functional contributions of CD8 T cell subsets to EAE kinetics and disease outcomes. The use of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide alongside an adjuvant to inoculate mice, provides a reproducible disease model. Demyelinating disease severity was analyzed at defined timepoints including, onset (d10–14), peak (d17–21), early chronic (d28–35) and late chronic (d80–120) stages. The total number of CD3+ T cells in the CNS remained consistent during EAE. However, it goes from a CD4 T cell dominated response at peak EAE to an equalized 1:1 ratio of CD4 and CD8 T cells at late chronic stage. Furthermore, differentiation of CD8 T cell subsets based on CD8α and CD8β protein expression revealed that CD8αα T cells numbers increase to comprise ~40% of the CD8+ population at late chronic EAE. In addition, CD8βLO cells become a prominent subset during late chronic stage, with a concomitant decrease in CD8αβHI T cells. Moreover, CD8αα T cells are activated and exhibit a central memory phenotype along with downregulation of exhaustion markers by FACS analysis, and yet have significantly reduced cytokine expression. These results are consistent with single cell RNA sequencing data obtained at the peak and late chronic time points. Thus, CD8αα T cells constitute a phenotypically distinct class of CD8 T cell subsets, with a blunted effector role in demyelinating disease outcome.
Inhibition of cholesterol pathway intermediates attenuates IL-33 mediated mast cell cytokine production
Authors: Jackson Kaitlyn G. , Dailey Jordan , Kankaria Roma , Sebti Said , Ryan John J
Published: February 2023

Asthma is a growing problem in developed countries, affecting over 24 million individuals in the US alone. Allergic asthma is denoted by a Th2 response that supports IgE-mediated mast cell function and is enhanced by the cytokine IL-33. Although corticosteroids and other treatments are available to help control asthma, many patients have persistent symptoms. In order to meet this unmet need, we investigated the utility of repurposing statin drugs. Statins decrease serum cholesterol by inhibiting HMG-CoA reductase. These drugs have anti-inflammatory effects largely ascribed to loss of isoprenyl lipids downstream of HMGCR. We previously found that statins could decrease IgE-mediated mast cell function. Our current data demonstrate that HMGCR inhibition by simvastatin or isoprenylation blockade with the novel compound FGT-2734 attenuates IL-33 induced mast cell cytokine production in vitro. Statin efficacy for the treatment of allergic asthma has been inconsistent in clinical trials. However, our data suggest that inhibiting isoprenylated signaling molecules may provide a feasible therapeutic target in mast cell-driven disease, and supports the use of statins as a potential adjunct or alternative treatment for individuals with asthma.
Activation of human skin mast cells by vancomycin via MrgX2: Comparison to the effects of brimonidine
Authors: Fukuoka Yoshihiro , Schwartz Daniel L , Ward Brant R
Published: February 2023

Vancomycin (VCM) is a glycopeptide antibiotic that inhibits bacterial peptidoglycan biosynthesis and is used for the treatment of certain Gram-positive bacterial infections of the eyes. However, the use of VCM is associated with several potential adverse reactions. We examined the mechanism of activation of human mast cells by vancomycin (VCM) and compared to brimonidine, an alpha2-adrenergic receptor agonist used to treat in glaucoma. VCM induced degranulation and cytokine secretion by SMCs, which were suppressed by pertussis toxin (PTX) (similar to MrgX2 ligands). Brimonidine induced less degranulation than VCM that was partially inhibited by PTX. MrgX2 expression on the surface of SMCs was detected by FACS analysis. VCM enhanced FceRI-mediated degranulation and cytokine secretion in a dose-dependent manner, whereas brimonidine failed to induce cytokine production and suppressed FceRI-mediated cytokine secretion. VCM induced maximal ERK-phosphorylation by 5 min similar to compound 48/80, an MrgX2 ligand and inhibited by PTX. VCM- but not brimonidine-induced degranulation of MCs was significantly inhibited by QWF, an MrgX2 antagonist. This study indicates that VCM but not brimonidine activates human SMCs via MrgX2 and that MCs become more responsive to VCM when co-stimulated by aggregating FceRI, which may occur when sensitive patients exposed to aeroallergen and VCM.
A history of microbial exposures has a temporally limited inhibitory effect on mouse lung ILC2 responses to a fungal allergen
Authors: Block Katharine E , Iijima Koji , Pierson Mark , Walsh Daniel A , Kita Hirohito , Jameson Stephen C
Published: February 2023

The ability of some microbes and their products to restrain the immune response to allergens has been extensively studied and is a key element of the hygiene hypothesis. However, it is unclear whether a modulated immune response relates to recent microbial exposure, or whether a diverse infectious history promotes sustained changes in reactivity to allergens. Thus, we explored how prior microbial experience in “dirty” mice affected innate immune responses to an airway fungal allergen. Dirty mice were generated by cohousing laboratory mice with pet store mice for at least 60 days to allow for physiological transmission of microbes. Despite changes induced in many immune cell populations, lung type 2 innate lymphoid cell (ILC2) numbers and phenotype were unaltered by cohousing. Short term (4.5 hour) production of the type 2 cytokines IL-5 and IL-13 in response to Alternaria alternata extract was consistently depressed in dirty mice relative to specific pathogen free (SPF) and germ-free animals. This correlated with diminished release of IL-33, an alarmin that activates ILC2, in dirty mice. Nevertheless, at a later timepoint (24 hours) or after repeated Alternaria exposure, the influx of eosinophils, neutrophils and T cells into the lungs was similar in dirty and SPF mice. Our data therefore indicate that a history of robust and diverse microbial exposure delays but ultimately does not restrain development of a type 2 response to airway allergens, in contrast to the established inhibitory effects of acute exposure to certain microbes and their products.
Engineering T cells with improved TCR-CD3 interaction for optimal tumor killing
Authors: Yogambigai Velmurugu , Natarajan Aswin , Ge Chenghao , Yuan Zhou , Li Kaitao , Kim Joseph , Shin Hye Won , Rookwood Richard , Beesam Saikiran , Nyovanie Samantha T. , Patskovsky Yury , Zhu Cheng , Krogsgaard Michelle
Published: February 2023

T cell activation requires extracellular stimulatory signals mainly mediated by T cell receptor complex (TCR/CD3). Obtaining detailed mechanistic knowledge of the TCR-mediated signaling pathway holds significant importance in understanding immune diseases, formulating new immunotherapies, and overcoming immunosuppression in current therapies. Here, we used a novel strategy to overcome ineffective anti-tumor responses by modifying the TCR interaction with CD3 subunits without affecting the binding affinity and/or specificity of the TCR-antigen interaction. Based on NMR and mutational studies, we introduced specific alanine mutations in the Cβ helix 3 and Cβ helix 4 – F strand of the TCR extracellular domain. We demonstrated varied functionality in terms of IL-2 production as well as altered bond lifetime measured by biomembrane force probe assay (BFP). Selected mutations retained or displayed enhanced CD3 binding ability in a CD3 tetramer assay. Based on these results we generated retroviral TCR libraries by random mutagenesis at specific extracellular TCR-CD3 interaction sites, expressed them in a T cell hybridoma system and selected TCRs with novel binding specificity using soluble CD3γɛ- and CD3δɛ-tetramers. We are currently determining the correlation between CD3 tetramer binding, bond lifetime, and functionality (kinase activity, IL-2 production, and tumor rejection) of selected TCR mutants. By integrating binding data, T cell signaling data, and functional outcomes, we expect to be able to manipulate the human immune system via the extracellular TCR-CD3 interaction site to be utilized in novel human T cell therapies.
Precise control of immune modulation using DNA scaffold-mediated biomaterial functionalization
Authors: Huang Xiao , Tang Qizhi , Lim Wendell A. , Desai Tejal A.
Published: February 2023

Synthetic materials displaying multi-modal ligands with exact chemiophysical properties for immune modulation are valuable research tools and a promising therapeutic platform. However, precise chemical conjugation of multiple proteins on biomaterial surfaces is challenging. Through DNA hybridization-mediated biomolecule loading, we achieved high density and precise ratiometric control of multiple ligands on nano-/micro-particles. We found increasing ratios of anti-CD3 to anti-CD28, 1:5, 1:3, 1:1, 3:1 to 5:1, on microparticles yielded a linear increase of ex vivo expansion of primary human CD4+ and CD8+ T cells until reaching a plateau at 3:1 ratio. For CD8+ T cells, the ratio of 3:1 resulted in the highest percentage of central memory cells, 51.4 ± 7.2% vs. 14.4 ± 7.6% for 1:5 ratio (n = 5 donors). Particle surface presentation of IL-2 using an anti-IL-2 antibody (in trans to CD3/28 particles) yielded ~3 fold more ex vivo expansion of CD4+ and CD8+ T cells in 14 days when compared to the equivalent dose of soluble IL-2. Using intratumoral injection of microparticles presenting a ligand for a synthetic Notch receptor, we locally induced chimeric antigen receptor (CAR) expression on systemically infused engineered T cells and observed CAR T cell killing of the injected tumors, while sparing the uninjected identical tumors in the contralateral flank. These results highlight the potential of this platform in achieving better control of therapeutic cell manufacture and local tuning of immunotherapies. Ongoing work is using DNA origami-mediated patterning to dissect spatial requirements of various T cell activating ligands to better understand critical parameters of T cell activation with the goal to improve the design of immunotherapies.
Single-cell analyses characterize circulating anti-tumor CD8+ T cells in mice and humans and identify markers for their enrichment
Authors: Pauken Kristen E , Shahid Osmaan , Lagattuta Kaitlyn A , Mahuron Kelly M , Luber Jacob M , Lowe Margaret M , Huang Linglin , Delaney Conor , Long Jaclyn , Fung Megan E , Newcomer Kathleen , Tsai Katy K , Chow Melissa , Guinn Samantha , Kuchroo Juhi R , Burke Kelly P , Schenkel Jason M , Rosenblum Michael D , Daud Adil I , Sharpe Arlene H , Singer Meromit
Published: February 2023

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential, but is currently challenging due to the limited number of reagents to track antigen-specific T cells. Here, we used paired single-cell RNA sequencing and T cell receptor (TCR) sequencing to detect and characterize “tumor matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared to non-matching T cells in blood, and appeared less exhausted than matching counterparts in tumor. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells in both mice and humans. By leveraging the transcriptome, we identified candidate cell surface marker panels for TM cells in mice and melanoma patients, and validated CX3CR1, NKG2D, and CD39 proteins in mice. Combinations of markers performed better than single markers in identifying TM cells from non-TM cells in the blood, providing a platform to potentially track TM cells based on surface markers instead of the TCR. These data demonstrate that the TCR can be used to identify tumor-relevant populations for comprehensive characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.
Impact of neutrophil extracellular traps (NETs) in primary graft dysfunction following human lung transplantation
Authors: Bonneau Steven , Landry Caroline , Bégin Stéphanie , Adam Damien , Clavet-Lanthier Marie-Élaine , Dasilva Ariane , Charles Elcha , Dumont Benjamin , Menaouar Ahmed , Neagoe Paul-Eduard , Brochiero Emmanuelle , Stevens Louis-Mathieu , Nasir Basil , Ferraro Pasquale , Noiseux Nicolas , Sirois Martin
Published: February 2023

Background:

Primary graft dysfunction (PGD), the major complication associated with lung transplantation in the peri-operative period, is characterized by vascular and alveolar inflammation and results in hypoxemia and lung edema. Up to 30% of recipients develop the most severe form of PGD (Grade 3; PGD3), leading to 50% mortality. Animal studies suggest that neutrophils can contribute to the inflammatory process through the release of neutrophil extracellular traps(NETs). NETs are composed of DNA filaments decorated with granular proteins, which can contribute to vascular occlusion associated with PGD. Our main objective is to correlate NETosis with recipient outcomes.

Methods:

Clinical data and blood samples were prospectively collected from donors and recipients (n=32) pre-, intra- and post-operatively (up to 72hrs). Inflammatory markers inducing NETs’ synthesis (CRP, IL6, IL8) and markers of NETs (myeloperoxidase [MPO], MPO-DNA complexes, cell-free DNA) were quantified by ELISA. When available, histology and immunochemistry techniques were used on donor lung samples collected before transplantation to evaluate the presence of activated neutrophils and NETs.

Results:

In lung samples from which PGD3 developed in recipients, there was a marked increase of vascular occlusion composed of activated neutrophils and NETs before transplantation. Also, in donors and recipients pre- and intra-operatively, circulating levels of inflammatory (CRP, IL6, IL8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3.

Conclusion:

Elevation of these NETosis biomarkers might serve to elaborate an algorithm to better delineate the recipients at risk of developing severe PGD.
SARS-CoV-2 vaccines induce potent immune responses in infant rhesus macaques
Authors: Curtis Alan D , Garrido Carolina , Dennis Maria , Gao Hongmei , Montefiori David , Van Rompay Koen KA , Tomai Mark A , Fox Christopher , Edwards Darin , Carfi Andrea , Corbett Kizzmekia , Graham Barney S , Permar Sallie , De Paris Kristina
Published: February 2023

SARS-CoV-2 has caused > 100 million infections, > 2 million deaths and disproportionally affects older and comorbid patients. Given authorized adult vaccines against SARS-CoV-2 together with the success of childhood vaccine programs for infectious diseases such as polio, early life vaccination has the potential to decrease transmissibility and mitigate ongoing public health and socioeconomic impacts of SARS-CoV-2.

Two groups of 8 infant monkeys were immunized at week (W) 0 & W4 with 15 μg prefusion SARS-CoV-2 S-2P spike protein (S) formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (Protein) or 30 μg mRNA encoding S in lipid nanoparticles (mRNA-LNP). Dosage decisions were made in the context of infant immunogenicity and safety. Both regimens were well tolerated.

Protein group IgG binding for RBD, NTD, S1 & S2 peaked at W6. ACE2 to RBD blocking (1:10 dilution) was 100% and D614G pseudovirus neutralizing antibody geometric mean titer (GMT) was 9,792 at W6. These responses were stable through W14. S-specific memory B cells were detected by W4. Vaccine-elicited S-specific T cell responses were dominated by IL-17 or IFNγ at W14. CD8 responses were variable.

mRNA-LNP-induced RBD, NTD, S1 & S2 binding responses peaked at W6. Plasma (1:10 dilution) blocked 100% of ACE2-RBD interactions and D614G neutralizing antibody GMT was > 103 at W6. Blocking and neutralization responses persisted through W14. S-specific memory B cells were detected at W4. By W14, 100% of mRNA-LNP monkeys had S-specific CD4 T cells that expressed IL-2, IL-17, IFNγ or TNFα. CD8 responses were highly varied. This study is a proof-of concept for SARS-CoV-2 vaccine-induced immune responses in a pediatric setting and provides evidence that immunity may persist.
Development of a gp96-Ig vaccine for COVID-19
Authors: Strbo Natasa , Fisher Eva , Padula Laura , O’Neill Katelyn Elizabeth
Published: February 2023

The rapid spread of the global COVID-19 pandemic has put pressure on the development of a SARS-CoV-2 vaccine to address global health concerns. We generated a gp96-Ig-secreting vaccine expressing full-length spike or “S” glycoprotein of SARS-CoV-2 via a cell-delivered platform. Vaccine is based on the secreted heat shock protein, gp96-Ig, vaccine strategies that induces an antigen specific CD8+ cytotoxic T lymphocytes (CTLs). Secreted heat shock proteins were successfully used to induce immunogenicity against tumors, HIV/SIV, ZIKA, malaria in different animal models. Importantly, this vaccine strategy has proven to be effective in delaying virus acquisition as well as improving survival of non-small cell lung cancer patients in clinical trials. The principle of this vaccine relies on a cell-based, secreted gp96-Ig chaperoning antigenic proteins that are efficiently taken up and cross presented by activated dendritic cells (DC) to CD8 cytotoxic T lymphocytes (CTL), thereby stimulating an avid, pathogen-specific, cytotoxic CD8+ T cell response. We showed that our vaccine platform effectively stimulates a robust cellular immune response against protein S. Moreover, we confirmed that gp96-Ig, secreted from allogeneic cells expressing full-length protein S, generates powerful, protein S polyepitope-specific CD4+ and CD8+ T cell responses in both lung interstitium and airways. These findings were further strengthened by the observation that protein-S-specific CD8+ T cells were induced in human leukocyte antigen (HLA)-A2-02-01 transgenic mice thus providing encouraging translational data that the vaccine is likely to work in humans, in the context of SARS-CoV-2 antigen presentation.
Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein
Authors: Suryadevara Naveen Chandra , Shrihari Swathi , Gilchuk Pavlo , VanBlargan Laura A. , Binshtein Elad , Zost Seth J. , Nargi Rachel S. , Sutton Rachel E. , Winkler Emma S. , Chen Elaine C. , Fouch Mallorie E. , Davidson Edgar , Doranz Benjamin J. , Carnahan Robert H. , Thackray Larissa B. , Diamond Michael S , Crowe James E.
Published: February 2023

Most human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2 isolated to date recognize the spike (S) protein receptor-binding domain (RBD) and block virus interaction with the cellular receptor angiotensin converting enzyme 2 (ACE2). We isolated a panel of human mAbs binding to diverse epitopes on the spike protein N-terminal domain (NTD) from SARS-CoV2 convalescent donors and found that a minority of these possessed neutralizing activity. Two mAbs (designated COV2-2489 and COV2-2676) inhibited infection of both authentic SARS-CoV-2 and recombinant chimeric VSV/SARS-CoV-2 reporter viruses. We mapped their binding epitopes by alanine scanning mutagenesis and selection of functional SARS-CoV-2 S protein variants with mutations in the NTD that conferred resistance to recombinant chimeric VSV/SARS-CoV-2 reporter virus. Mechanistic studies showed that the antibodies neutralize in part by inhibiting at a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 prevented infection and disease in mice when administered either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, a subset of human NTD-specific mAbs protects against infection using multiple functional attributes including neutralizing activity and Fc-mediated activities.
Role of aging-associated Treg cell accumulation in experimental autoimmune encephalomyelitis (EAE) — a model of late-onset multiple sclerosis (MS)
Authors: Wang Weikan , Oh Jiyoung , Thomas Rachel , Su Dongming
Published: February 2023

MS, a T cell-mediated autoimmune demyelinating disease of the central nervous system (CNS), has not been sufficiently studied in the elderly, despite instances of late-onset MS. Regulatory T (Treg) cells play an ameliorative role in MS disease onset and severity. Given that the aged immune system exhibits relatively enhanced thymic Treg cell generation and accumulated peripheral Treg cells, the role of Treg cell in aged MS patients remains to be elucidated. Using the mouse model EAE, we found that the aged mouse late-onset EAE occurs with two disease types. In Type I, EAE onset was delayed in the age mice, but the disease became more severe once onset. Alternatively, in Type II, some aged mice never developed severe symptoms than young mice until a second antigen challenge was given, which led to more a progressive disease course than their young counterparts. These features were potentially associated with a disparate distribution of Treg cells in the aged peripheral lymphoid tissues and CNS during the disease. The aged EAE mice had a higher proportion of polyclonal Treg cells in the peripheral lymphoid tissues, but a lower proportion of Treg cells specific for myelin oligodendrocyte glycoprotein (MOG) in the CNS, compared to young counterparts. Transient inhibition of accumulated polyclonal Treg cells in the periphery partially ameliorated disease severity in the aged mice, which was accompanied by increased MOG-specific Treg cells in the CNS. Therefore, we suggest that accumulated polyclonal Treg cells in the aged periphery are detrimental for CNS repair processes during neuronal inflammation in aged MS mice, potentially due to hampering trafficking of MOG-specific Treg cells into the CNS.
Dual IgA/IgG family plasmablast-derived mAbs from peripheral blood of individuals at risk for rheumatoid arthritis are polyreactive to autoantigens and intestinal bacteria
Authors: Chriswell Meagan , Bloom Michelle , Seifert Jennifer A , Feser Marie , Demoruelle M. Kristen , Deane Kevin D. , Norris Jill M. , Robinson William H , Holers V. Michael , Kuhn Kristine A
Published: February 2023

A wide array of data supports rheumatoid arthritis (RA) as a disease with mucosal origins, including presence of IgA RA-associated autoantibodies in peripheral blood and biomarkers of inflammation at mucosal sites. Individuals at-risk for developing RA, (serum autoantibody+, without joint disease), can be characterized by an expansion in dual-family IgA/IgG circulating plasmablasts. We examined the characteristics of mAbs generated from these plasmablasts in at-risk (n=4) and early RA (<1 year from diagnosis, n=2) subjects. The variable regions of the plasmablasts were sequenced and clonal families mapped. 94 total heavy and light chain sequences were cloned into a mouse IgG2a constant region. All 94 expressed mAbs bound RA-related citrullinated/uncitrullinated synovial targets in an antigen array, demonstrating polyreactivity. As these mAbs were derived from IgA-containing clonal families, we queried if they have commensal bacterial targets by exposing them to a human fecal bacterial pool. 61.7% of mAbs had bacterial targets, suggesting additional polyreactivity. Bound bacteria were 16S rRNA sequenced. Interestingly, 56.31% ±12.85 of bound bacteria were of families Lachnospiraceae/Ruminococcaceae. A smaller subset of Vh genes were utilized among bacterially reactive plasmablast mAbs as compared to the wider population, though mutations from germline remained static between groups. Specifically, mAbs utilizing IgHV4-4, V3-64, V3-66, and V3-74 were bacterially reactive, and mAbs utilizing IgHV4-39, V4-59, V3-33, and V3-43 were not bacterially reactive. This demonstrates a link between mucosal and systemic immune systems in RA, suggesting an early mucosal trigger for disease-specific autoantibody development.
A critical CTCF binding site of the Ifng-Il22 locus specifies cytokine expression and finetunes immune response
Authors: Shih Han-Yu , Liu Chunhong , wang ping , Signorella Sadie , Montgomery William , Jankovic Dragana , Nagashima Hiroyuki , Yao Chen , Kanno Yuka , Ryan Yijun , O’Shea John J
Published: February 2023

Precise control of cytokine milieu plays an essential role in homeostasis and diseases, and dysregulated cytokine production leads to undesired inflammation and autoimmunity. Interferon gamma (IFN-γ) and interleukin-22 (IL-22), two key cytokines for against intracellular and extracellular pathogens, respectively, evolutionarily resides in close genomic proximity. Notably, these genes are exclusively expressed in type I and type III lymphoid cells via complex epigenomic regulation that remains largely unknown. Our ATAC-seq, ChIP-seq and PollI ChIA-PET datasets revealed a CTCF binding site 70kb upstream of Ifng setting a boundary of the enhancer landscapes. Therefore, we proposed that this CTCF site segregates Ifng and Il22gene enhancers for lineage-specific gene regulation. To investigate whether this CTCF binding contributes to bifurcated cytokine gene regulation at the Ifng-Il22 locus, we deleted this 17bp Ifng (−70kb)CTCF site in mice using a CRISPR strategy. We found that Ifng (−70kb)CTCFdel CD4+ T cells revealed deficiency in initial Th1 cell differentiation. Using single cell RNA-seq and Toxoplasma gondii infection as a model for type I immune response in vivo, we identified aberrant Th17 differentiation and increased T cells prolifereation in Ifng (−70kb)CTCFdel mice. To further understand the molecular mechanism, we measured CTCF associated loop formation in pre-differentiated Naïve CD4+ T cells and interestingly, this Ifng (−70kb)CTCF site formed a loop with a CTCF-enriched region on the other side of Il22 gene. Our data provide a novel pre-determined mechanism that prevents cross activation for IFN-γ enhancers to target IL-22 prior to differentiation.
Immunology education within an undergraduate program for Health Sciences education
Authors: Sika-Paotonu Dianne , Patel Jennika , Reed Katie , McTavish Arthur , Humphries Felix , Motu Lakin
Published: February 2023

Immunology education remains an essential component for undergraduate Health Sciences education across the health professions. The clinical relevance of Immunology teaching can be emphasised by utilisation and appropriate reference to pertinent health conditions affecting populations locally and across the global setting. Health issues of significance to Māori and Pacific populations groups in Aotearoa/New Zealand and in the Pacific Region include Cancer and Acute Rheumatic Fever (ARF) and Rheumatic Heart Disease (RHD). ARF is the body’s autoimmune response to untreated Group A Streptococcal (GAS) infection of the throat or skin. If left untreated, repeated or severe ARF episodes can lead to permanent cardiac damage known as RHD. RHD is a significant cause of morbidity and mortality worldwide. The growing global burden of cancer remains of concern, while Pacific Peoples and Māori are disproportionately affected by specific cancers. The utilisation of two areas with strong Immunological relevance for teaching examples in undergraduate Health Sciences education were shown to support improved understanding, class participation, topical interest, and research focus for Medical students engaged within Health Sciences education platforms in Aotearoa/New Zealand. This highlights the value of supporting Immunology education efforts by incorporating appropriate reference to pertinent health conditions relevant to populations groups locally and across the global setting.
Hematopoietic protein RELT stains prominently in breast cancer and binds the cytoskeletal protein Filamin A.
Authors: Cusick John K , Yanumula Anusri , Alhomsy Yasmeen , Bilasy Shymaa , Aghaians Dino , Gillespie Brittany , Senderovich Michelle , Zhou James , Wang Wenjia , Wong Stephanie , Shi Yihui , Hejazi Nazila
Published: February 2023

Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR superfamily member expressed most prominently in cells and tissues of the hematopoietic system. Mouse knockout studies indicate that RELT functions in part by negatively regulating T-cell activation. RELT has two homologous binding partners, RELL1 and RELL2, and collectively, these proteins are described as RELT family members. This study sought to identify novel protein interactions with RELT family members and to compare the expression of RELT in normal and diseased tissues. A yeast two-hybrid screen utilizing RELL1 as bait identified the cytoskeletal protein Filamin A (FlnA); physical interaction between FlnA and RELT family members was confirmed by in vitro co-immunoprecipitation. A truncated mutant of FlnA disrupts the ability of RELT family members to activate the p38 pathway. Previous reports indicate that autoantibodies against RELT are associated with breast cancer and that RELL2 prevents migration and invasion of the metastatic breast cancer cell line MDA-MB-231 (231), while multiple studies suggest a link between FlnA and breast cancer. We therefore sought to examine the association of RELT family members with breast cancer. Western blotting revealed significant expression of endogenous RELT family members in the breast cancer cell lines 231 and MCF-7. Immunohistochemistry (IHC) revealed a higher intensity of RELT staining in breast cancer tissues versus normal mammary tissue. Additionally, overexpression of RELT family members enhanced apoptosis in 231 cells. Collectively, these results further our understanding of signal transduction pathways associated with RELT family members and provide evidence that RELT is upregulated in breast cancer.
Nutrient partitioning in the tumor microenvironment
Authors: Madden Matthew Z , Reinfeld Bradley I , Wolf Melissa M , Chytil Anna , Cohen Allison S , Muir Alexander , Hongo Rachel A , Abraham Abin , Beckermann Katherine E , Manning H Charles , Rathmell W Kimryn , Rathmell Jeffrey C
Published: February 2023

The tumor microenvironment (TME) includes cancer and infiltrating immune cells. Tumors canonically consume glucose through Warburg metabolism, a process forming the basis of cancer imaging by positron emission tomography (PET). Activated immune cells also rely on glucose, and impaired immune cell metabolism in the TME contributes to tumor progression. It remains uncertain, however, if immune cell metabolism is dysregulated in the TME by cell intrinsic programs or by competition with cancer cells for limiting nutrients. Here we used PET tracers to measure access and uptake of glucose and glutamine by specific cell subsets in the TME. Surprisingly, myeloid cells had the greatest capacity to uptake glucose in vivo, followed by T cells and cancer cells across a range of cancer models. Cancer cells, in contrast, demonstrated high glutamine uptake. This distinct nutrient partitioning was cell intrinsically programmed through mTORC1 signaling and glucose and glutamine-related gene expression. Inhibiting glutamine uptake enhanced glucose uptake across tumor resident cell types, suggesting that glutamine metabolism suppresses glucose uptake without glucose being limiting in the TME. Thus, cell intrinsic programs dictate the preferential immune and cancer cell acquisition of glucose and glutamine. Cell selective partitioning of these nutrients may be exploited to develop therapies and imaging strategies to enhance or monitor the metabolism and activities of specific cell populations in the TME.
Developing natural killer cells producing hypoxia-inducible cytokines for effective immunotherapy against solid tumors.
Authors: Jo Dong-Hyeon , Lee Seung-Hwan
Published: February 2023

Natural Killer (NK) cells are a promising means for adoptive immunotherapy for cancers. NK cell therapies against hematological cancers such as acute myeloid leukemia have demonstrated good efficacy. However, the poor effector function of tumor-infiltrating NK cells limits the potential of NK cells against solid tumors. Accumulating data suggests that hypoxia in the tumor microenvironment (TME) suppresses the effector function of NK cells. One of the immunosuppressive features of hypoxia is the generation of adenosine, a metabolite that highly suppresses NK cell cytotoxicity and proliferation. Although engineering NK cells to overexpress stimulatory cytokines before the adoptive immunotherapy has demonstrated promising potential for cancer therapy, it raises a concern that high systemic levels of cytokines may lead to cytokine-mediated pathology. Therefore, we hypothesize that driving overexpression of the potent stimulatory cytokines exclusively in the TME would circumvent systemic toxicity. An attractive strategy to achieve this goal is to take advantage of the hypoxic condition, a key characteristic of the TME. Hypoxia-inducible factor 1 alpha (HIF1-α) plays a pivotal role in expressing hypoxia-responsive genes by associating with promoters containing hypoxia response element (HRE). By mimicking the biological system, HRE-induced cytokine expression vectors were generated and tested in NK cells. The expression was inducible under hypoxic conditions and was reversible upon re-oxygenated conditions. Our results suggest that the hypoxia-inducible vector system can be beneficial to trigger TME specific cytokine expressions in NK cells and provide insight into efficient immunotherapy against solid tumors.