bluuebunny/crossref_metadata_2025 huggingface dataset:
Screening immunotherapy cancer drugs in a scalable, physiologically relevant 3D in vitro tumor microenvironment model
Authors: Chiovaro Francesca , Strebel Silvan , Wolf Armin , Moritz Wolfgang , Agarkova Irina
Published: February 2023

Interaction of tumor cells with surrounding stromal and immune cells, secreted cytokines, and extracellular matrix proteins, collectively known as the tumor microenvironment (TME), is crucial for cancer progression and metastasis. Here, we present a novel 3D in vitro TME model to assess candidate drugs for cancer therapy.

For this study, we prepared lung carcinoma spheroids from GFP-A549 cells co-cultured with cancer-associated fibroblasts (CAFs) and PBMCs. To simulate a pro-inflammatory tumor microenvironment, we activated PBMCs with cytokines and/or anti-CD3/CD28. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. We then developed a tumor model using melanoma PDX cells labeled with CellTracker to monitor tumor cell viability after exposure to activated PBMCs. We evaluated killing activity and effector function of T cells by measuring tumor microtissue size by automatic stage fluorescence microscopy and release of IL-6, TNFa, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune cell infiltration and its effects on tumor were assessed by histological analysis using tumor, immune cell and apoptosis markers.

Our results show combination treatment with cytokines and anti-PD-1 antibody led to higher secretion of IL-6/TNFa/IFNγ/GM-CSF, reduced tumor size, increased infiltration of the model by CD8+ T cells, stronger expression of Granzyme B resulting in enhanced tumor apoptosis and killing. This confirms our novel 3D in vitro TME model is a promising tool for studying cell-cell interactions and allows for HCS of novel candidates for anti-cancer drugs.
The clinical considerations for early cancer detection using ctDNA within the Pacific setting
Authors: Sika-Paotonu Dianne , Walton Mischa , Finau Eseta , Faavae Sepola , Vaimoli Jessica , Guilford Parry
Published: February 2023

Pacific Peoples living in Aotearoa/New Zealand and in the Pacific Region, remain disproportionately affected by some cancers. In addition to ensuring effective treatments and therapies are available and continue to improve, continued early cancer diagnostic efforts are also critical for supporting better health outcomes for Pacific Peoples within the cancer setting. A novel and promising early cancer detection method known as circulating tumour DNA (ctDNA) analyses small fragments of tumour DNA found in the blood circulation to provide information about a tumour. This approach is more commonly associated with and utilised for monitoring patients being treated for their cancers and is being adapted to support early cancer detection efforts. To support the possible acceptability of this approach for early cancer diagnosis for Pacific Peoples, it is imperative to better understand the clinical considerations relevant to the Pacific setting. To identify the clinical considerations needed for early cancer detection with ctDNA approaches within the Pacific setting for Pacific Peoples living in Aotearoa/New Zealand, researchers and Pacific Health Professionals were consulted. Relevant considerations included the immediate need to support early cancer diagnostic efforts to avoid late presentations, better understanding of the cultural context for Pacific Peoples within the healthcare setting more broadly, with strong support expressed for a simple approach for early cancer detection such as that being proposed with ctDNA technology. These findings highlight the critical need to consider the clinical context within the cancer setting for Pacific Peoples.

Health Research Council of New Zealand
Control of antigen-mediated proliferation and activation of CD4+ T cells by the RNA binding protein HuR
Authors: Ellis Jason S , Fattahi Fatemeh , Atasoy Ulus
Published: February 2023

The RNA binding protein HuR (elavl1) stabilizes target mRNAs and aids in their transport to the heavy polysomes, ultimately resulting in increased protein expression. We have previously shown that HuR plays a critical role in T helper cell fate decisions, with HuR deficient mice having significant impairment in Th2 differentiation. Interestingly, HuR deficiency also leads to large increases in IL-2 production. We hypothesized that HuR is important for normal T cell activation and expansion. To investigate this, we used mouse models in which HuR was conditionally ablated during T cell development either early at the double positive stage (CD4-Cre ROSA HuRfl/fl) or late (distal Lck-Cre ROSA HuRfl/fl). Upon activation in vitro, HuR KO T cells had significant reductions in CD25 (IL-2Ra) protein and RNA. An in vivo OVA challenge model was used to determine ascertain the effects of HuR KO on airway inflammation. Despite only half of CD4+ T cells in dLck-Cre HuR KO mice lacking HuR, there was a significant decrease in total inflammation, with essentially no T cell infiltrate. These mice also had significantly reduced BAL IL-13. Furthermore, mice were immunized with either OVA or KLH, boosted 10 days later, and antigen specific proliferative responses measured in vitro. T cells lacking HuR had profound proliferation defects compared to control cells, regardless of the antigen system employed. Interestingly, non-specific CD3/CD28 stimulation only partially restored these proliferative defects. RNA-Seq analysis of activated HuR deficient T cells indicated altered expression in multiple genes associated with T cell activation and function. We conclude HuR function is vital for normal antigen-specific activation and proliferation in CD4+ T cells.
Profiling CIS progression and stability using RNA-seq single-cell analysis in peripheral blood mononuclear cells
Authors: Giarraputo James , Jain Vaibhav , Arvai Stephanie , Smilek Dawn E , Harris Kristina M , Gregory Simon
Published: February 2023

Objective:

To use single-cell mRNA analyses to identify immune cell profiles in CIS patients associated with a higher risk of developing an MS relapse or new central nervous system (CNS) disease activity on brain MRI.

Methods:

Cryopreserved peripheral blood mononuclear cells were collected at study entry and six months from six CIS participants in the ITN020AI STAyCIS trial of atorvastatin. Three participants met the primary endpoint defined as ≥ 3 new T2 lesions on MRI or an MS relapse within 12 months, while the other three did not. RNA from about 10,000 PBMC per sample were identified at the single-cell level using the 10X Genomics Chromium platform and analyzed using Cell Ranger software. Seurat R software package was used for downstream analysis.

Results:

31 clusters of differentially expressed genes were assigned as specific cell types; baseline frequencies of clusters enriched >2-fold for macrophage, NK, NKT, CD4+ T cell, and B cell markers were associated with disparate outcomes at six months. Several clusters of B cells, CD4+ and CD8+ T cells were expanded from baseline at six months in participants with CNS disease activity at that time point, while no notable changes were observed in those with stable disease.

Conclusion:

Our results show varied frequencies of innate and adaptive leukocyte populations in participants with disparate clinical outcomes, suggesting a predictive biomarker for aggressive early intervention. Expanded clusters of CD4+ and CD8+ T cells and B cells in blood at the time of disease activity may identify biomarkers leading to acute demyelination. Follow-up studies are underway to increase sample size and incorporate protein data to better define cell subtypes associated with disparate outcomes in the STAyCIS trial.
Analysis of immunogenic cell death in the tumor microenvironment of tumor spheroids
Authors: Demuynck Robin , Efimova Iuliia , Krysko Olga , Declercq Heidi , Krysko Dmitri
Published: February 2023

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment. Immunogenic cell death (ICD) has the potential to be exploited in immunotherapy by activating the host’s own immune system against the cancerous cells, but analysis of ICD induction (including ferroptosis) in 3D cultures has not been performed so far. In this work, we have optimized cell death induction in tumor spheroids. Our cell death analysis method (3DELTA) uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. The 3DELTA method was able to detect cell death type without the need to disaggregate spheroids. Moreover, in this work we also demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. Ferroptotic cell death induction in spheroids lead to 50% of cell death whereas in 2D cultures, this caused 90% of cell death. The tumor microenvironment is an important factor that can affect the outcome of anti-cancer therapy. By adding endothelial cells (EC) to cancer cells (CC) in an optimized ratio (EC:CC), we were able to form vascularized spheroids. In conclusion, these spheroids will be a more relevant model for in vivo tumors by analyzing interactions between the different cell types and can be further used for analysis of their immunogenic potential.
IL-33 activates mTOCR1 and modulates glycolytic metabolism in CD8+ T cells
Authors: Wang Xiaofang , liang Yuejin , Sun Jiaren
Published: February 2023

Interleukin (IL)-33, a member of cytokines in the IL-1 family, plays a central role in innate and adaptive immunity. However, the underlying mechanism of IL-33-mediated T cell regulation and the associated downstream signals remain elusive. In this study, we found the increased IL-33 expression in T cells following cell activation. However, extracellular, but not nuclear IL-33 contribute to effector CD8+ T cell activation and proliferation. IL-33 not only induced its receptor ST2 expression, but also up-regulated GATA3 and T-bet levels in CD8+ T cells. Importantly, IL-33 induced mTORC1 activation in CD8+ T cells as evidenced by increased p-S6 levels both in vitro and in vivo. Meanwhile, this IL-33-induced T cell activation was suppressed by mTORC1 inhibitor rapamycin. Move over, IL-33 elevated glucose uptake and lactate production in CD8+ T cells in a dose- and time-dependent manners. Our mechanistic study further revealed that IL-33 was capable of promoting the expression of glucose transporter and glycolytic enzymes via mTORC1, leading to accelerated aerobic glucose metabolism and increased effector T cell activation. Together, our data provide a new insight into IL-33-mediated regulation of T cells, and might be beneficial for therapeutic strategies of inflammatory and infectious diseases in future.
Tropomyosins are critical mediators of CD8+ T cell synaptic actomyosin organization.
Authors: Schrock Dillon C
Published: February 2023

CD8+ T cells are a critical arm of the adaptive immune system because they kill virally-infected and transformed cells. Their function is critically dependent on their ability to form stable interactions with antigen-presenting cells (APCs). These interactions are mediated by a highly-organized structure at the T cell: APC interface termed the immunological synapse (IS). The IS itself is organized largely by the underlying actin cytoskeleton. Perturbation of either the Arp2/3-dependent branched network of the distal region or of the formin-derived arc network of the peripheral region dampens TCR signaling and impairs subsequent T cell activation. Tropomyosins are actin-binding proteins that form head-to-tail polymers along the actin filament. Several tropomyosin isoforms associate preferentially with linear formin-generated filaments, such as those in the peripheral region of the IS, where they promote the recruitment and activation of myosin 2 and thwart cofilin-mediated severing. We have found that mouse CD8+ T cells express two prominent tropomyosin isoforms (Tpm3.1 and Tpm4.2) and that these isoforms are strongly upregulated following naïve cell activation. Interestingly, both of these isoforms have been associated with human pathology in proteomics experiments. Fluorescent-tagged versions of these tropomyosins colocalize with pSMAC actomyosin arcs and migrate toward the center of the IS. Importantly, treatment with an inhibitor of Tpm3.1, ATM3507, severely impacts the organization of T cell synaptic actin structures. Our aim is to examine the role that tropomyosins play in the organization of T cell synaptic actin and actomyosin structures and to determine their importance for effector function.
Determining the impact of HLA type and chronic viral infection on peripheral T-cell receptor sharing between unrelated individuals
Authors: Johnson Sarah , Seale Spencer , Gittelman Rachel M , Sanders Catherine M , Robins Harlan , Fields Paul
Published: February 2023

T cells generate highly diverse T-cell receptors (TCRs) to recognize antigens, most of which are private to a given individual. TCRs with low junctional complexity have a higher likelihood of being generated and thus shared across multiple individuals. Additional factors that may further limit diversity and increase the likelihood that particular TCRs are public include: (a) thymus selection for maturation of TCRs that bind antigens presented by an individual’s human leukocyte antigen (HLA) proteins, and (b) T-cell proliferation upon exposure to cognate antigens, which remain within the periphery as memory T cells. Numerous public TCRs have been associated with particular HLA restrictions and antigen specificities1,2. Chowell et al. also recently showed that HLA diversity is important for mounting an effective T-cell response against solid tumors3. However, little is known about the overall impact of these forces on the diversity of the T-cell repertoire and the expected TCR sharing across individuals. Here, we leverage the TCR repertoires of 666 healthy individuals sequenced with the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle, WA) to address these questions. We investigate (a) how the similarity of HLA alleles between individuals impacts the number of TCRs shared between individuals and (b) the impact of shared antigen exposure by determining how age and CMV status impacts clone sharing between individuals. These data show that HLA type, age, and CMV exposure shape the immune repertoire and influence the sharing of clones between unrelated individuals.
Tlr3 interacts with ESCRT-I components Tsg101 and Hcrp1 in mouse astrocyte cell line
Authors: Mielcarska Matylda Barbara , Struzik Justyna , Toka Felix Ngosa
Published: February 2023

It is established now that ESCRT (endosomal sorting complex required for transport) complexes participate in the trafficking of endosomal Toll-like receptors (TLRs), which are relevant in the regulation of the TLR-mediated immune response. TLR3 plays a vital role in the innate immune control of herpes simplex type 1 virus (HSV-1) infection in the brain, however, the process of TLR3 delivery to the ligand recognition site and fate as endosomal cargo including the receptor degradation await explication. Proximity ligation assays in TLR3 agonist, poly(I:C) stimulated or unstimulated murine astrocytes, confirmed the association of Tlr3 with Tsg101 and Hcrp1, ESCRT-I subunits essential for the lysosomal sorting of ubiquitinated membrane receptors. The interaction of Tlr3 and ESCRT-I components was prominent in the first hour and 24 h after poly(I:C) addition. TLR3 was highly ubiquitinated in cells at all studied times, while the interaction of Hrs, a core component of ESCRT-0 and ESCRT-I, and Tlr3, as well as Hrs and Tsg101, and Hrs and Hcrp1, increased in the first hour of stimulation, but 24 h following stimulation returned to levels observed in unstimulated cells. Results suggest a possible role of ESCRT-I in prolongation of TLR3 signaling and/or receptor degradation, prompting investigation whether Tsg101 and Hcrp1 influence Tlr3 transportation in cells of the central nervous system, which would portray ESCRT-I as a possible modulation target regarding TLR3-mediated antiviral response in the brain.
The leaked mitochondrial C1QBP inhibits activation of the DNA sensor cGAS
Authors: Song Kun , Wu Yakun , Fu Bishi , Wang Lingyan , Hao Wenzhuo , Hua Fang , Sun Yiwen , Dorf Martin E , Li Shitao
Published: February 2023

Cytosolic DNA released from microbial pathogens triggers the activation of DNA sensor cyclic GMP-AMP synthase (cGAS) which catalyzes the synthesis of the cyclic GMP-AMP (cGAMP), a second messenger that triggers a signaling cascade leading to type I interferon (IFN) production. The cGAS cannot discriminate the origin and sequence of dsDNA; thus, it is also activated by self-DNA leaked from the nucleus or the mitochondria. Recent studies have shown that DNA virus infection triggers mitochondrial stress, leading to the release of mtDNA to the cytosol and activation of cGAS; however, the regulatory mechanism of the mtDNA-mediated cGAS activation is not well elucidated. To seek potential regulators for cGAS, we used proteomics to map the protein interactome of cGAS in macrophages and found C1QBP as a novel binding partner of cGAS. C1QBP is predominantly localized in the mitochondria matrix and leaked to the cytosol upon DNA virus infection. C1QBP binds to the NTase domain of cGAS and inhibits the cGAS activity in cells and in vitro. Overexpression of the cytosolic form of C1QBP inhibited the cytosolic DNA-induced immune response and facilitated HSV-1 infection. By contrast, deficiency of C1QBP leads to elevated innate immune responses and impaired HSV-1 infection. Taken together, our study suggests that C1QBP is a cGAS inhibitor hidden in the mitochondria.
Distinct type I interferon (IFN) signaling is involved in damage and fungal clearance during Aspergillus pulmonary infection
Authors: Shepardson Kelly , Roemer Julia , Robinson Ema , Johns Laura L , Hall Monica , Rynda-Apple Agnieszka
Published: February 2023

Human fungal infections with Aspergillus fumigatus (Af) are becoming more prevalent. Over 4 million cases of Af occur globally, with 300,000 cases/year due to invasive pulmonary aspergillosis in immune suppressed patients. Recent increases in number/severity of cases of influenza and SARS-CoV-2-infected patients acquiring Af suggests that viral infections create suppressed immune environments permissive to fungal infection. Influenza infection was also shown to increase Af susceptibility in mice and was associated with anti-viral type I interferon (IFN) signaling. Importantly, we found that type I IFN signaling, via IFNAR2 of the IFNAR1/2 receptor, contributes to regulation of susceptibility to and damage from influenza in mice, while others have found that IFNAR2 expression correlates with SARS-CoV-2 infection severity. As clinical outcome to Af is associated with host tissue damage, this suggested involvement of IFNAR2 signaling in Af susceptibility. We found that absence of IFNAR2 (Ifnar2−/− mice) resulted in increased damage in the lungs (from myeloid and epithelial/endothelial cells), morbidity, and inflammation in response to Af, while absence of IFNAR1 (Ifnar1−/− mice) did not. We also found that Ifnar2−/− mice developed invasive disease with increased hyphal growth compared to Ifnar1−/− and WT mice and this was associated with epithelial/endothelial damage. Our results also indicate that altered myeloid cell function in Ifnar2−/− mice, rather than differences in cell recruitment, may enhance damage responses and fungal growth. Together, our results begin to establish IFNAR2’s role in regulating host damage responses to Af and suggest that aberrant type I IFN signaling contributes to an Af permissive environment.
Sialophorin is necessary for vaccine-induced CD8+ T cell responses and immunity against lethal fungal pneumonia in CD4+ T-cell deficient hosts
Authors: Mudalagiriyappa Srinivasu , Nanjappa Som G.
Published: February 2023

Increased opportunistic fungal infections in immunocompromised patients and lack of effective therapeutic modalities caused millions of deaths worldwide. Further, there are no licensed vaccines available for such and for immune-sufficient individuals. Previously, we have shown that in the absence of CD4+ T cells, vaccine-induced CD8+ T cells can mount sterilizing fungal immunity. However, molecules that have an immunomodulatory effect on CD8+ T cells are still to be explored. Using a mouse model of CD4+ T cell lymphopenia and experimental fungal vaccine, we demonstrate that Sialophorin (CD43), a molecule deficient in WAS patients, plays a critical role in mounting anti-fungal immunity. Genetic ablation of CD43 resulted in blunted CD8+ T cell responses associated with reduced expression of prototypic lineage-specific transcription factors following vaccination. Cell-intrinsic expression of CD43 on CD8+ T cells was indispensable for their expansion. Poor vaccine-induced effector CD8+ T cell responses under CD43-deficiency caused the failure to control fungal pneumonia following lethal challenge, independent of trafficking defect of the CD43-deficient effector cells. Further, we demonstrate antigen-specific CD8+ T cells preferentially expressed glycosylated-CD43. Notably, the numbers of glycosylated-CD43+ CD8+ T cells were directly correlated with vaccine-immunity. Thus, the glycosylated-CD43 expression on the CD8+ T cells could serve as a biological marker of vaccine efficacy and clinical outcome of fungal pneumonia. In conclusion, we report that CD43 is required for effective vaccine-immunity against lethal fungal pneumonia, and glycosylated-CD43 could be a novel phenotypic T-cell marker of fungal immunity.
Prime-Trap immunization with protective liver stage antigen, MIF-4G and Kb17 peptide, induces liver CD8 TRM cells and protection against Plasmodium berghei malaria
Authors: Krzych Urszula , Althubaiti Nouf , Pichugin Alexander V , Duffy Patrick E , Zarling Stasya N
Published: February 2023

Exposure to Plasmodium sporozoites (spz), the causative agent of malaria, induces anti-disease immunity, but fails to protect against repeated Plasmodium infections. In contrast, multiple immunizations with radiation-attenuated Plasmodium spz (RAS) induce sterile lasting protection in humans, non-human primates, and murine models. The mechanisms of protective immunity are multifactorial and CD8 T cells are considered sine qua none of protection. The Plasmodium berghei (Pb) RAS-induced protection in C57Bl/6 mice correlates with a network of liver stage antigens (LS Ags)-activated liver CD8 T cell subsets including resident memory (RM) CD8 TRM cells. A protective Pb LS Ag, termed MIF-4G, induced liver CD8 T cells that recognize H-2Kb-restricted epitope, Kb17. One of the current efforts to improve malaria vaccines focuses on maximizing the induction of liver CD8 TRM cells by Prime-Trap immunization regimen. Here we asked if priming with DNA-MIF-4G followed by trapping with Adenovirus serotype 5 (Ad5) vectored-Kb17 would induce Kb17-specific liver CD8 TRM cells as well as protection against Pb spz challenge. Liver Kb17-specific CD8 TRM cells (CD69+CXCR6+) exceeded the number of these cells in the spleens, 27% vs 2%, respectively. Protection measured by liver parasite burden was CD8 T cell dependent. In contrast, prime:boost immunization with DNA/Ad5 MIF-4G induced only 8% liver CD8 TRM cells, as did control immunization with DNA/Ad5 empty vector. Utilizing LS Ags and the corresponding CD8 T cell-inducing epitopes in Prime-Trap immunization lends itself for further explorations as an ancillary approach to malaria vaccine regimens where liver CD8 TRM may be crucial in preventing virulent blood stages by targeting LS parasites.
Early Life Imprinting of a Th2-Stromal Cell Niche in Skin
Authors: Boothby Ian C , Boda Devi P , Kwan Elaine Y , Cohen Jarish N , Habrylo Ireneusz , Molofsky Ari B , Rosenblum Michael D
Published: February 2023

Tissue inflammation early in life can be imprinted on the immune system, causing lasting changes in immunologic tone that confer disease protection or susceptibility in adults. The cellular and molecular mechanisms responsible for immune imprinting in many nonlymphoid tissues remain largely unknown. We find that time-limited neonatal inflammation induced by transient reduction of regulatory T cells (Tregs) causes a dramatic dysregulation of skin stromal cells, accompanied by the selective accumulation of Th2 cells within a distinct microanatomic tissue niche. Th2 cells are maintained into adulthood through interactions with a previously uncharacterized stromal population in skin fascia that we refer to as Th2-interacting fascial fibroblasts (TIFFs), which expand following Treg reduction, respond to Th2 cytokines, and produce IL-33. Formation of the Th2-TIFF niche imprints skin with increased reparative capacity after wounding. Taken together, these data define a novel Th2 niche in skin and suggest a mechanism of immunologic imprinting whereby inflammation early in life creates networks between adaptive immune cells and parenchymal cells, establishing an immunological set-point in tissues that is maintained throughout life.
γδ T cells control gut pathology in a chronic inflammatory model of colorectal cancer
Authors: Dasgupta Suryasarathi , Liu Hong , Bailey Brandi , Wyrick Corey , DeBoever Chris , Grieves Jessica , Faustin Benjamin , Murphy Craig
Published: February 2023

Gut gd T cells are important intraepithelial lymphocytes with critical roles in maintenance of gut barrier integrity against infection and cancer, through unknown mechanisms. In addition to their role in barrier function, absence of gd T cells has been reported to lead to faster polyp formation in an inflammation unassociated model of colorectal cancer (CRC). The significant linkage between Inflammatory Bowel Disease and CRC is of strong relevance in gut translational research, and it necessitates a deeper understanding of the underlying cell-molecular mechanism causing inflammation associated CRC. In a new disease-relevant, chronic inflammation associated Colorectal cancer (CRC) model, we observed enhanced chronic colon pathology for both inflammation and polyp formation in mice lacking gd T cells. RNAseq analysis of colon from TCR d−/− mice treated with AOM-DSS revealed an early and profound dysregulated transcriptional landscape with reduced tumor suppressive genes, upregulated tumor induction and epithelial mesenchymal transition genes, and increased expression of inflammatory markers. As tumorigenesis progressed, colons lacking gd T cells harbored enhanced potentially immunosuppressive CD4 T cells in the epithelial layer but not in the lamina propria. Consistently, the gut gd T cell dependent tissue signature identified is positively correlated with different stages of colon adenocarcinoma in patients. Taken together, we demonstrate that gd T cells display broad tissue functions critical in gut homeostasis by controlling both epithelial and immune functions, and maintain tissue integrity against CRC.
Regulatory mechanism of fucoidan-mix AG in intestinal mucosal immune cells
Authors: Miyazaki Yoshiyuki , Bak Juneha , Nakano Hayato , Takeuchi Shugo , Takeuchi Hideaki , Tachikawa Daisuke
Published: February 2023

Fucoidan is a series of natural sulfated polysaccharides contained in brown sea algae, and their beneficial physiological activities such as anti-tumor, anti-virus and immune regulatory effects have been reported. In previous studies, we proved that fucoidans derived from Cladosiphon okamuranus (C. okamuranus) and Undaria pinnatifida effectively improved anti-tumor immunity and tumor vaccine efficiency in combination with an Agaricus blazei mycelium extract. It is thought that the fucoidan-agaricus mix (named fucoidan-mix AG) stimulate innate immune cells to activate intestinal and mucosal immune reaction, but actual target immune tissues and cells by fucoidan have not been evidenced yet. In this study, we investigated action mechanisms of fucoidan in regulation of mucosal immune activities. At first, immune cells were isolated from Payer’s patches, mesenteric lymph node (MLN) and intestinal epithelia of Balb/c mice, then stained with fluorophore-labeled fucoidan. As the results, it was revealed that fucoidan directly interact with lymphocytes besides dendritic cells and macrophages existing in these intestinal immune tissues. Furthermore, immune cells from Payer’s patches and MLN stimulated with fucoidan from C. okamuranus and fucoidan-mix AG produced more TNF-α, IL-12 and Th1 cytokine IFN-γ. The production of an immunosuppressive cytokine IL-10 also was augmented in the fucoidan-treated immune cells, which suggesting that fucoidan balanced immune system to prevent occurrence of excessive reaction. In addition, it was newly proved that fucoidan directly stimulated T lymphocytes to induce enhanced IFN-γ production.
Fibroblastic reticular cell lineage convergence in Peyer’s patches governs intestinal immunity
Authors: Onder Lucas , Prados Alejandro , Koliaraki Vasiliki , Mörbe Urs Michael , Lütge Mechthild , Cheng Hung Wei , Ludewig Burkhard , Kollias George
Published: February 2023

Fibroblastic reticular cells (FRCs) determine the organization of lymphoid organs and control immune cell interactions. While the cellular and molecular mechanisms underlying of FRC differentiation in lymph nodes and the splenic white pulp have been elaborated to some extent, in Peyer’s patches (PPs) they remain elusive. Using a combination of single-cell transcriptomics and cell fate-mapping in advanced mouse models, we found that PP formation in the mouse embryo is initiated by an expansion of perivascular FRC precursors, followed by FRC differentiation from subepithelial progenitors. Single-cell transcriptomics and cell fate-mapping confirmed the convergence of perivascular and subepithelial FRC lineages. Furthermore, lineage-specific loss and gain-of-function approaches revealed that the two FRC lineages synergistically direct PP organization, maintain intestinal microbiome homeostasis and control anti-coronavirus immune responses in the gut. Collectively, this study reveals a distinct mosaic patterning program that generates key stromal cell infrastructures for the control of intestinal immunity.
Antigen-specific T cell tolerance in the prevention of post-infectious myocarditis induced with Coxsackievirus B3
Authors: Reddy Jay , Arumugam Rajkumar , Lasrado Ninaad , Renu Sankar , Gourapura Renukaradhya J
Published: February 2023

Of the many types of heart disease, myocarditis is a predominant cause of heart failure in adolescents, and 10–20% of those affected can develop dilated cardiomyopathy (DCM) with infiltrates in their hearts. Autoimmunity resulting from exposure to infectious agents such as Coxsackievirus B3 (CVB) has been suggested as a possible underlying mechanism of chronic myocarditis in the development of DCM. We recently demonstrated that CVB infection could lead to the generation of pathogenic autoreactive T cells leading to a suggestion that their effector functions can be suppressed by developing antigen-specific T cell therapies. To this end, we sought to generate induced Treg cells by using biodegradable nanoparticle delivery system made of chitosan and poly(lactic-co-glycolic acid) (PLGA) entrapped with interleukin-2 and transforming growth factor termed, nanocytokines. Our data suggest that the PLGA nanocytokines can stably induce Forkhead box protein P3, a molecular marker of Treg cells accompanied with complete suppression of interferon-γ production. Our preliminary data indicate that the Treg cells induced with nanocytokines can suppress non-Treg cells in vitro leading to a suggestion that they could be used to prevent the expansion of autoreactive T cells generated in mice affected with viral myocarditis.
Tissue-specific complement inhibition by antibody-Complement Regulatory Protein (CRP) fusions utilizing Therapeutic Autoimmune reguLatOry proteiN (TALON™) platform
Authors: Jung Jiwon , Larkin Bridget M , Rajani Gaurav , Borah Minasri , Nagy-Domonkos Celesztina , Zhou Weijie , Otipoby Kevin L , Viney Joanne L , Higginson-Scott Nathan , Mascanfroni Ivan , Shaheen Hussam H
Published: February 2023

The complement system plays a major role in humoral innate immunity. Upon activation, complement proteins trigger a cascade of enzymatic events leading to the production of inflammatory mediators and membrane attack complexes. Tight regulation of complement proteins is essential for immune homeostasis due to their potency and abundance in fluid phase. Complement Regulatory Proteins (CRPs) that are membrane-bound (e.g., CD59), or in fluid phase (e.g., Factor H), prevent aberrant activation of complement pathways against self and mitigate autoimmunity. The importance of CRPs is underscored by the finding that genetic variations in the CRPs can lead to autoimmune diseases such as paroxysmal nocturnal hemoglobinuria (PNH) characterized by the lysis of host red blood cells from dysregulated complement system.

Systemic complement inhibition via anti-C5 antibody has clinically been shown to benefit some patients with complement-mediated disorders, but the therapy requires a high, frequent dosing scheme and supplemental vaccinations to compensate for systemic immunosuppression. In addition, other diseases such as C3 glomerulopathy affect specific tissues and involve pathways beyond C5, limiting treatment options. To this end, local immunomodulation through targeted delivery of CRPs to specific tissues is an attractive strategy to restore homeostasis in complement-mediated autoimmune diseases while circumventing the need for high dose systemic inhibition of complement factors. Here, we present a proof-of-concept study for the design, generation, and evaluation of tissue-tethering antibodies fused with CRPs. Also, we demonstrate the pharmacokinetics and pharmacodynamics of tissue-targeting CRP fusions in mouse models.
4-Ethylguaiacol modulates neuroinflammation and Th1/Th17 differentiation to ameliorate disease severity in experimental autoimmune encephalomyelitis
Authors: Kuo Ping-Chang , Weng Wen-Tsan , Brown Dennis A. , Scofield Barbara A. , Paraiso Hallel C. , Yu I-Chen , Yen Jui-Hung
Published: February 2023

Multiple sclerosis (MS) is a progressive autoimmune disease characterized by the accumulation of pathogenic inflammatory immune cells in the central nervous system (CNS) that subsequently causes focal inflammation, demyelination, and neuronal damage. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. Presently, growing evidences show that phenolic compounds exert anti-inflammatory effects by inhibiting the production of inflammatory molecules. 4-ethylguaiacol (4-EG), a phenolic compound, which was detected in foods, such as green bell peppers, corns, sesames, and coffee has been shown to possess anti-inflammatory effects through attenuating peripheral immune responses. However, whether 4-EG exerts anti-inflammatory effects on modulating the CNS inflammatory immune responses remains unexplored. In this study we assessed the therapeutic effect of 4-EG in EAE using both chronical and relapsing-remitting animal models. Our results showed that 4-EG not only ameliorated disease severity in chronic EAE but also mitigated disease progression in relapsing-remitting EAE. Further investigations revealed that 4-EG suppressed MG activation, inhibited Th1 and Th17 infiltration, and mitigated BBB disruption in the CNS of EAE. Furthermore, 4-EG suppressed Th1 and Th17 differentiation in the periphery of EAE. Finally, 4-EG induced HO-1 expression in the CNS of EAE as well as in MG and macrophages. In summary, our work demonstrates for the first time that 4-EG confers a protection against autoimmune disease EAE through modulating neuroinflammation and inhibiting Th1/Th17 differentiation, suggesting that 4-EG could be developed as a therapeutic agent for the treatment of MS/EAE.
Generation of islet antigen-specific engineered Tregs for use in T1D therapy via homology-directed gene editing of conventional CD4+ T cells
Authors: Yang Soo Jung , Singh Akhilesh K , Tappen Tori , Honaker Yuchi , Mauk Kelsey , Smith Jessica , Sommer Karen , Rawlings David , Buckner Jane H
Published: February 2023

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in T1D mouse models. Notably, Tregs specific for pancreatic islets were shown more potent than polyclonal Tregs in blocking disease. However, the frequency of antigen-specific Tregs is extremely low and ex vivo expansion has the potential risk to destabilize Tregs leading to an effector phenotype. Here, we developed methods to generate durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells using a combination of lentiviral TCR transduction and FOXP3 homology-directed repair (HDR)-editing. Using TCRs derived from clonally expanded islet-reactive CD4+ T cells in T1D, we generated islet-specific EngTregs that exhibit a Treg-like phenotype. Islet-specific EngTregs effectively suppress proliferation and cytokine production by islet-specific effector T cells (Teff). Notably, EngTregs suppress Teff recognizing the identical peptide as well as bystander Teff recognizing alternative islet antigens. Importantly, islet-specific EngTregs suppress polyclonal endogenous islet-specific T cells derived from PBMC of individuals with T1D. To directly assess the capacity of EngTregs to modulate diabetes in vivo, we established an identical HDR-editing strategy in islet-specific murine cells. Following adoptive transfer, islet-specific mu-EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teff in recipient mice. Collectively, our approach enables the production of EngTregs specific to pancreatic islets with the capacity to efficiently suppress islet specific responses. This approach has the capacity to deliver targeted islet specific therapy to treat or prevent T1D.
Mannose Conjugated Chitosan-based Influenza Nanovaccine Intranasal Inoculation Enhances the Cross-reactive Immunity in Maternal Antibodies Positive Pigs
Authors: Gourapura Renukaradhya J , Feliciano-Ruiz Ninoshkaly , Patil Veerupaxagouda , Schrock Jennifer , Han Yi , Ramesh Anikethana , Dhakal Santosh , Krakowka Steven , Renu Sankar
Published: February 2023

Swine influenza A virus (SwIAV) causes respiratory tract infection in pigs. Parenteral administration of inactivated SwIAV vaccine in weaned piglets provides variable protection due to preexisting specific maternal derived antibodies (MDA). We developed killed SwIAV antigen (KAg) encapsulated mannose-conjugated chitosan nanoparticles (mCS NPs-KAg) vaccine which targets dendritic cells. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to control CS NPs-KAg vaccinates. In both mCS NPs-KAg and CS NPs-KAg vaccinates upon challenge with a heterologous virus observed augmented cross-reactive virus-specific sIgA antibody response in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid, and IgG antibody levels in lung lysate and BAL fluid samples. Whereas the multivalent commercial inactivated SwIAV vaccine administered intramuscular had increased only the specific serum IgG antibody response. Additionally, mCS NPs-KAg vaccine increased the specific recall lymphocyte proliferation, and the frequency of IFNγ secreting T cells, late effector and effector memory T cells, and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates. Both the mCS NPs-KAg and CS NPs-KAg vaccines efficiently cleared the challenge virus load from airways and reduced the lung lesions compared to commercial vaccine. Overall, mCS NP-KAg vaccine in MDA-positive pigs induced a robust cross-reactive immunity and offered cross-protection against influenza virus.
Immunogenetic variation at MHC class I loci in desert bighorn sheep (Ovis canadensis nelsoni) reflects natural and anthropogenic fragmentation
Authors: Dolan Brian P , Beechler Brianna , Dugovich Brian , Jolles Anna , Epps Clinton
Published: February 2023

Desert bighorn sheep (Ovis canadensis nelsoni) are a charismatic ungulate native to western North America that live in mountainous regions. In the desert of the southwest United States, populations of bighorn sheep can be isolated by both natural and anthropogenic barriers. Isolation limits gene flow, exacerbates loss of genetic diversity to drift, and thus could affect immune responses to infectious agents. We sought to develop a relatively rapid method for determining MHC class I gene diversity in individual sheep. Both DNA and RNA were isolated from leukocytes collected from 154 adult bighorn sheep from across different mountain ranges in Southern California. Previously identified primers from domestic sheep were used to amplify exons 2 and 3 from OMHC I genes while appending adapters for PacBio circular consensus sequencing. We successfully identified over 40 unique MHC class I sequences expressed by bighorn sheep. Using a combination of phylogenic tree analysis and mapping MHC sequences to the domestic sheep genome, we detect at least 4 unique classical MHC class I regions. Comparison of results using either genomic DNA or mRNA (cDNA) as a template for analysis suggest that certain MHC alleles are expressed at different levels within leukocytes, while many potential pseudogenes were identified when genomic DNA was used as a template. Our data suggests using messenger RNA (cDNA) as a template to identify MHC class I diversity is superior to genomic DNA. Finally, we found clear patterns of MHC class I genotypes differentiated by population, which suggests that both natural and anthropogenic-induced population fragmentation can limit the diversity of MHC genes within a given population.
Innate and Adaptive Responses to Bacillus Calmette-Guerin in Young Pigs
Authors: Byrne Kristen A , Loving Crystal L
Published: February 2023

Development of novel antibiotic alternatives is a critical One Health priority impacting both human and animal wellbeing. While the attenuated strain of M. bovis (Bacillus Calmette-Guerin, BCG) was developed as a vaccine against tuberculosis; recently, BCG has been observed to alter circulating monocytes leading to enhanced inflammatory immune responses to heterologous agonists. These changes can persist for months after the initial BCG exposure, and may serve to enhance innate immune health and reduce disease without the use of antibiotics. Following in vitro observation of innate training in pig monocytes, we wanted to study in vivo BCG responses in pigs as a potential path to enhance innate immunity. Young pigs were inoculated with BCG and weeks later ex vivo cytokine responses to innate TLR agonists and IFNγ response to M. bovis purified protein derivates (PPDb) were measured. PPDb stimulated PBMCs from pigs inoculated subcutaneous with 107 CFU of BCG produced the highest amount of IFNγ as compared to intramuscular inoculated or 106 CFU inoculated pigs. No differences were observed in PBMCs or monocytes stimulated ex vivo with TLR agonists between control and BCG inoculated pigs regardless of route or dose administered. Unlike cattle and humans, in pigs BCG does not seem to readily disseminate from the inoculation site, which may explain the lack of altered innate immune responses observed. Differences in adaptive immune responses indicated alterations to dose or route of BCG administration impacted immunogenicity. Future studies will employ intravenous BCG administration to understand if peripheral exposure to BCG is required to alter innate responses in circulating pig monocytes.
Coronavirus induces diabetic macrophage-mediated inflammation via IFN-beta regulation of SETDB2
Authors: Melvin William James , Audu Christopher , Joshi Amrita , Davis Frank , Barrett Emily , Mangum Kevin , Obi Andrea , Moore Bethany B , Gallagher Katherine
Published: February 2023

COVID-19 induces a robust inflammatory ‘cytokine storm’ that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the ‘cytokine storm’ that has been shown in T2D to promote excess inflammation in response to infection. Using sera from human patients with SARS-CoV-2 and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mϕ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, increasing inflammation. Mϕs with a myeloid-specific deletion of SETDB2 displayed increased inflammation following coronavirus infection. Further, IFNβ directly regulates SETDB2 in Mϕs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that SETDB2 mediates a heightened inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mϕs with IFNβ reversed the inflammatory cytokine production via upregulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mϕ-mediated ‘cytokine storm’ in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNβ/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.
Combined blockade of IFN-Type I and TNF alpha receptors provides protection against respiratory syncytial virus (RSV)-induced disease and bronchoconstriction
Authors: Morris Dorothea R , Ansar Maria , Qu Yue , Ivanciuc Teodora , Casola Antonella , Garofalo Roberto P
Published: February 2023

While the anti-viral/protective role of interferon type I (IFN-I) and tumor necrosis factor (TNF)-α in naturally acquired RSV infections remains controversial, human and animal studies suggest the existence of a cross-regulation between these two innate pathways, which may contribute to disease and airway inflammation characteristics of this infection. In this study, we investigated the role of IFN-I:TNF-α axis in a mouse model of RSV infection, which provides robust experimental evidence of clinical disease, airway bronchoconstriction and viral replication, recapitulating aspects of the human infection. BALB/c mice were treated with antibodies targeting the IFN-I receptor (IFNR) and either TNF receptor (TNFR)1 or TNFR2 prior to RSV infection. Significant improvements to body-weight loss, illness score and bronchoconstriction measured by plethysmography were observed in mice treated with either antibody combination vs untreated mice (RSV/IgG control). Additionally, several cytokines were significantly reduced in both RSV-IFNR-TNFR mice including IL-1α (p=0.02), IL-6 (p=0.0002), IL-9 (p=0.003), IL-17 (p=0.01), IFN-γ (p=0.005), Ccl3 (p=0.01), Ccl4 (p=0.0003) and Ccl5 (p=0.02). While lung viral load in RSV-IFNR-TNFR1 mice remained comparable to the RSV control (p=0.30), RSV-IFNR-TNFR2 mice had a significant increase in viral replication (p=0.001). Interestingly, this increase in viral replication was not appreciated in previous works blocking IFNR or TNFR2 alone in a similar experimental RSV model. This data collectively demonstrates a synergism between IFN-I and TNF-α during RSV infection as well as highlights the importance of selective receptor blockade when considering RSV therapeutic development.