bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanistic role of eosinophils in the initiation and progression of pancreatitis pathogenesis
Authors: Manohar Murli , Verma Alok K , Venkateshaiah Sathisha Upparahalli , Mishra Anil
Published: January 2023

Several clinical reports indicated that IL-18 and eosinophils are induced in patient with chronic pancreatitis and pancreatic malignancy. However, the etiology and pathogenesis of eosinophilic pancreatitis (EP) is poorly understood and termed as rare disease. Accordingly, we tested the hypothesis that IL-18 and IL-5 synergy may be critical in promoting EP pathogenesis. We performed qPCR, ELISA, western blot, immunofluorescence and immunohistochemical analyses to detect eosinophils, mast cells, collagen, associated cytokines and chemokines in experimental and human pancreatitis. Herein, we show the evidence of tissue eosinophilic microabscesses, mast cells and collagen accumulation in non-malignant and malignant human pancreatitis. Additionally, we observed more prominent collagen accumulation, fibrotic lesions and degranulated eosinophils in malignant pancreatic tissue samples compare to non-malignant pancreatic samples. A similar characteristics such as acinar cells atrophy, increased accumulation of tissue eosinophils, mast cells and induced levels of IL-5, IL-18, Eotaxin-2, TGF-β1, Collagen-1, Collagen-3, fibronectin and α-SMA was observed in cerulein-induced chronic mouse model of pancreatitis. Mechanistically, we show that IL-5 deficiency in mice protect pancreatic pathogenesis including fibrosis in cerulein-induced mouse model of pancreatitis, indicating a critical role of eosinophils in disease pathogenesis. Taken together, our combined clinical and experimental data indicates that eosinophils accumulation and degranulation may be critical in promoting pancreatic fibrosis that may leads to pancreatic malignancy.
Correlation of NK Cell Phenotypic Properties with Individual Differences in Human Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
Authors: Hudig Dorothy , Tang Jennifer J-J , Sung Alexander P. , Guglielmo Micheal , Smith-Gagen Julie , Anderson Stephen K. , Bateman Lucinda , Barao Isabel , Redelman Doug D.
Published: January 2023

Antibody-dependent cell-mediated cytotoxicity (ADCC) supports anti-viral protection and monoclonal antibody anti-tumor therapies. We used two assays to compare ADCC of healthy donors: CX@1:1 (the percentage cells killed at a 1:1 ratio of CD16Apositive NK cells to ‘target’ cells that were pre-labeled with saturating antibody); and EC50 (the effective concentration of antibody that supports 50% of maximal ADCC). CX@1:1 measures lytic capacity while the EC50 measures cellular recognition; there was no correlation between the 2 assays though we observed 4.5-fold differences among donors in each assay. We correlated ADCC with 5 cytometric parameters (the median fluorescent intensities [MFIs] of the CD16A IgG Fc-receptor; the %NK cells positive & MFIs of the adhesion molecule CD2; the MFIs of perforin; and the %CD16Apositive of CD56positive NK cells) and with CD16A V&F genotypes that affect FcR affinity. For CX@1:1, the best donor killed 73% of targets while the worst killed 16% and perforin levels correlated weakly (P=0.11). Unexpectedly and inexplicably, the %CD16Apositive cells among NK cells correlated (P<0.05). CD16A and CD2 were dissociated from CX@1:1 as might be expected for the high antibody concentrations. For EC50s, there were no statistical correlations except for the expected difference between V/V &V/F vs. F/F genotypes, though the P=0.11. We conclude that substantial individual differences in ADCC per CD16Apositive NK cell are determined by parameters other than cell surface receptors and perforin. The unanticipated correlation of %CD16Apositive NK cells with CX@1:1 has potential as a surrogate marker for ADCC function.
Molecular mechanism for adenosine regulation of dendritic cells
Authors: Cekic Caglar , Kayhan Merve , Koyas Altay , Akdemir Imran , Savas Ali Can
Published: January 2023

Extracellular adenosine concentrations and expression adenosine receptors are elevated after cell death, injury and inflammation. Antigen presenting cells are one of the primary targets for adenosine to suppress T and NK cell responses. However, molecular mechanisms for adenosine regulation of dendritic cells are poorly understood. Here we show that adenosine receptor stimulation strongly suppresses dendritic cell activation. Adenosine did not affect phosphoactivation of inflammatory signaling pathways such as MAPKs, NF-κB and IRF3. Adenosine receptor signaling increased intracellular cAMP accumulation. Specific cAMP analogs for EPAC and PKA pathways phenocopied the effects of adenosine on dendritic cells especially when used together. Adenosine-mediated suppression of inflammatory/effector cytokines is associated with increased expression of anti-inflammatory c-Fos and NR4A receptors. NR4A and cFos expression were also increased by EPAC and PKA-specific cAMP analogs. Addition of conditioned mediums from dendritic cells prestimulated with stable adenosine analog or PKA and EPAC specific cAMP analogs together inhibited the IFNγ production by activated T cells. Overall our data suggest that adenosine can potentially target both PKA and EPAC pathways and increase the expression of NR4A nuclear orphan receptors and cFos to regulate dendritic cell responses. Our findings have important implications for the development of novel therapies for immune related diseases such as cancer by targeting adenosine receptors.
Tissue-resident liver NK cell responses to MCMV infection
Authors: Dodard Garvin , Erick Timothy , Tata Angela , Brossay Laurent
Published: January 2023

Natural Killer (NK) cells belong to the ILC1 subgroup, which contains conventional NK (cNK) cells, ILC1 and tissue-resident NK (trNK) cells. The murine liver is home to both cNK cells and trNK cells. In this organ, cNK cells and trNK cells can be distinguished based on the expression of the transcription factors Eomes and T-bet. cNK cells are double positive for Eomes and T-bet while trNK cells do not express Eomes. During viral infection, the fate of the activated cNK cell population is relatively well defined. However, the fate of the trNK cell population remains unexplored. Using the MCMV model of infection, we found that a contraction phase precedes a replenishment of the trNK cell subset, which inversely correlates with cNK cell kinetics. We sought to determine whether the trNK cell response was a direct result of MCMV infection or an indirect response to inflammatory milieu. In order to help distinguish between these two possibilities, mice were injected with the iNKT ligand a-galactosylceramide (a-GalCer). We found that treatment with a-GalCer lead to an initial contraction phase of liver trNK cells, indicating that the phenotype observed is independent of MCMV infection. Altogether, these data suggest that liver cNK and trNK cell subsets, which arise from distinct developmental pathways, perform different functions in this organ.
CoCr micro-particles induce a robust type 2 inflammation in knee synovial tissues leading to fibrosis and tissue damage
Authors: Palma Mark J , Mishra Pankaj Kumar , Millman Ariel , Hallab Nadim J , Beebe Kathleen , Benevenia Joseph , Gause William C
Published: January 2023

Cobalt chrome (CoCr) is a major component of orthopaedic implants and a source of metallic wear debris, which may contribute to harmful inflammation and implant failure. In this study we show that the presence of M2 macrophages (CD68+, CD206+, and CD163+) and tissue fibrosis is observed in human peri-prosthetic tissue samples obtained from patients undergoing revision joint arthroplasty. We further examined the molecular mechanisms that drive the sterile inflammatory response to CoCr microparticles in 2 clinically relevant murine articular models. Bilateral intra-articular knee injections of CoCr microparticles were given to C57BL/6 mice and innate immune cell infiltrates were examined in both acute (2 day) and chronic (2 week) models. At the acute time point, CoCr microparticles induce inflammation characterized by an innate type 2 response in synovial tissues with marked increases in neutrophils, eosinophils, and M2 macrophages. This response in articular tissues is dependent on both SYK and BTK signaling as pharmacologic blockade of either pathway abrogates the innate response. Studies in our chronic model demonstrates that repeated exposure of articular tissues to CoCr microparticles triggers an inflammatory response that is characterized by persistent innate cell infiltrates of neutrophils and macrophages, as well as the development of tissue fibrosis. Additionally we show that the innate response and generation of tissue fibrosis in our chronic model is independent of the Caspase-1 inflammasome. Taken together, our findings suggest that CoCr mediates a robust type 2 inflammation that leads to fibrosis and identifies novel potential targets for control of microparticle induced sterile inflammatory responses.
Activation of innate sensing cGAS-STING pathway by Sindbis Virus
Authors: Fung Sin-Yee , Yuen Chun-Kit , Zhu Wuyang , Liang Guodong , Kok Kin-Hang , Chan Chi-Ping , Jin Dong-Yan
Published: January 2023

The cGAS-STING pathway for cytosolic DNA sensing induces type I interferon response during the course of infection with DNA viruses, retroviruses or bacteria. In addition, cGAS detects cytosolic self-DNA, which could be resulted from the reactivation of endogenous retroviral elements or leaked from mitochondria or the nucleus under autoimmune conditions. Cytosolic RNA species can also serve as cGAS ligand in the form of DNA:RNA hybrid. However, the relevance of cGAS-STING pathway to RNA viruses remains unclear. In this study we report the activation of cGAS-STING pathway by Sindbis virus with a positive-sense single-stranded RNA genome. cGAS transcript was induced during Sindbis virus infection and this induction was independent of type I interferon signaling. Loss of either cGAS or STING augmented Sindbis virus replication and prevented viral induction of type I interferons, suggesting a possible role of cGAS-STING signaling pathway in the sensing of RNA viruses. Reconstitution of cGAS and STING in cGAS- and STING-null cells exhibited a protective effect against Sindbis virus infection. Analysis of cytosolic nucleic acid species enriched from Sindbis virus-infected cells indicated the presence of a cGAS ligand. Taken together, our findings reveal an unrecognized role of cGAS-STING pathway for cytosolic DNA sensing in RNA virus infection.
Proteasome inhibition ameliorates experimental Sjögren’s syndrome by suppressing Th17 and B cell responses
Authors: XIAO Fan , LIN Xiang , LU Liwei
Published: January 2023

Primary Sjögren’s syndrome (SS) is a chronic autoimmune disease that mainly affects salivary and lacrimal glands. Currently, there is a lack of effective therapies for SS patients. Increasing evidence indicates that Bortezomib (BTZ), the first proteasome inhibitor approved for treating multiple myeloma, shows beneficial effects in treating lupus. Although clinical findings have revealed increased expression of proteasome in SS patients, it remains unclear whether proteasome inhibition is an effective approach for treating SS. In this study, we evaluated the therapeutic effects of BTZ on experimental SS (ESS) mouse model. Mice were immunized with proteins extracted from salivary glands (SG) to induce ESS and received either vehicle or BTZ treatment. Compared with vehicle-treated counterparts, BTZ-treated mice displayed markedly higher saliva flow rates and ameliorated tissue destructions accompanied with less lymphocytes infiltration in SG. Notably, mice with BTZ treatment showed significantly decreased Th17 but not Th1 responses, and reduced germinal center B cells and plasma cells in peripheral lymphoid organs. In culture, BTZ treatment significantly suppressed Th17 cells differentiation and proliferation. Moreover, adoptively transferred naïve CD4 T cells differentiated into Th17 cells and induced salivary dysfunction in immunized Il17a−/− mice. However, the BTZ-treated recipient mice exhibited suppressed Th17 responses with ameliorated reduction in salivary secretion. Taken together, our findings have shown that BTZ treatment can suppress Th17 and B cell responses and effectively ameliorate ESS development in mice, which may contribute to the validation of BTZ as a promising candidate for treating SS.
GMCSF-MOG abrogates experimental autoimmune encephalomyelitis (EAE) through the induction of MOG-specific regulatory T-cells.
Authors: Moorman Cody D , Curtis Alan Dale , Mannie Mark D
Published: January 2023

FoxP3+ regulatory T-cells (Tregs) play a crucial role in maintaining peripheral tolerance by suppressing auto-reactive T conventional cells (Tcon). Developing a safe therapeutic approach to induce autoantigen-specific Tregs could provide an effective treatment for Multiple Sclerosis. Previous studies have shown that single-chain fusion proteins comprised of murine GM-CSF and the dominant MOG35-55 neuroantigen (NAg), when injected subcutaneously in saline, were potent tolerogens that suppressed experimental autoimmune encephalomyelitis (EAE). To assess the potential involvement of FoxP3+ NAg-specific Tregs in the tolerogenic mechanisms of GMCSF-MOG, we used 2D2-FIG mice that express a transgenic “2D2” T cell repertoire specific for MOG35-55 and express a GFP reporter of FoxP3 expression. The results showed that a single subcutaneous administration of GMCSF-MOG in saline elicited a major population of FoxP3+ Tregs comprised of approximately 10–50% of all circulating 2D2-FIG T cells. This GMCSF-MOG-induced FoxP3+ Treg (GMiTreg) subset appeared within 3 days, was sustained over several weeks, and was associated with a desensitized phenotype within the Tcon compartment including a decrement in circulating transgenic 2D2 T cells, and down-regulation of TCR, CD3, and CD4 on a per cell basis. Covalent linkage between GM-CSF and MOG35-55 was required for the induction of FoxP3+ Tregs and the desensitized phenotype. The tolerogenic activity of GMCSF-MOG was dependent upon CD25+ GMiTregs, because treatment of mice with the anti-CD25 mAb PC61 abrogated GMiTregs and completely reversed vaccine-induced tolerance. This study indicates that immunization with GMCSF-MOG induces antigen-specific Tregs, which inhibit EAE in mice.
Potential Role of Nitrobiolome in Autoimmune Disease: Efficacy of GSNO in EAE.
Authors: Singh Inderjit , Saxena Nishant , Singh Avtar K , Won Jeseong
Published: January 2023

S-nitrosoglutathione (GSNO) is a physiological nitric oxide (NO) carrier molecule important in modulation of NO-signaling. Cellular GSNO homeostasis is maintained by its synthesis by nitric oxide synthase (NOS) as well as its catabolism by GSNO reductase (GSNOR). Treatment of EAE mice (wild-type /WT) with exogenous GSNO or GSNOR inhibitor (N6022) significantly alleviated the clinical disease and associated myelin damage. In addition, more profound alleviation of EAE disease was observed in mice deficient in GSNOR (GSNOR−/−). However, EAE disease was further exacerbated in mice deficient in inducible NOS (iNOS−/−) as compared to WT mice. As expected, the spleen levels of S-nitrosothiol proteins were higher in GSNOR−/− EAE mice but lower in iNOS−/− EAE mice compared to WT EAE mice indicating a negative correlation of spleen GSNO levels with EAE disease severities. Both GSNO and N6022 treated EAE mice had decreased TH17 and increased CD4+/CD25+/FOXP3− Treg differentiations without any significant alterations in other CD4+ T cell subset differentiation. Accordingly, CD4 T+ cells from GSNO or N6022 treated EAE mice produced lower amount of IL-17 and higher amount of IL-10 compared to the cells from untreated WT EAE mice. GSNOR−/− EAE mice also exhibited decreased T 17 and increased CD4+/CD25+/FOXP3− Treg differentiations. On the other hand, iNOS−/− EAE mice exhibited increased TH17, but decreased Tregs differentiations compared to WT EAE mice. In summary, these studies document that nitrobiolome (GSNO) participates in differentiation of autoreactive TH17 and Treg subsets during the course of EAE, and that GSNO as a potential therapeutic drug for multiple sclerosis. Supported in-part by VA(BX002829) and NIH(NS037766).
Vector-Free Engineering of Antigen Presenting Cells for Adoptive Immunotherapies for the Treatment of Cancer
Authors: Myint Melissa , Loughhead Scott , Merino Amy , Tian Lina , Bernstein Howard , Sharei Armon
Published: January 2023

In recent years, ex vivo manipulation of primary cells has shown immense clinical potential with the advent of adoptive T cell therapies. Conventional methods for ex vivo manipulation, however, are not without limitations. They typically rely on the application of electrical fields or exogenous materials such as viral vectors and plasmids, which can increase the potential for cellular toxicity and off-target effects. To overcome such limitations, we have developed an approach using our CellSqueeze Technology that causes temporary membrane disruption as cells are passed through a microfluidic constriction. While the membrane is disrupted, material in the surrounding buffer can diffuse directly into the cytosol. This system has demonstrated efficacy in patient-derived cells, such as stem cells and immune cells, and with a variety of molecules that are difficult to address with alternative methods.

In this work, we describe the use of our vector-free technology to deliver antigens directly to the cytoplasm of antigen-presenting cells (APCs) to drive potent antigen-specific CD8 T cell responses. Conventional methods for eliciting T cell responses with APCs typically rely on cross-presentation, which can be inefficient and require lengthy cultures. Our results show that murine APCs processed with our CellSqueeze Technology can stimulate enhanced antigen-specific T cell responses in vitro and in vivo by at least 3-fold, when compared to responses stimulated by endocytosis controls. Additionally, we show translation of these significant advantages of the CellSqueeze Technology to human APCs, reinforcing the exciting clinical potential of CellSqueeze for adoptive cell therapy.
Peptide-based polymer Toll-Like Receptor 7/8 agonist nanoparticles increase the breadth of anti-tumor CD8 T cell immunity
Authors: Ramirez-Valdez Andrei , Blobel Nicolas J , Ishizuka Andrew S , Pan Jenny , Vodnala Suman , Restifo Nicholas P , Lynn Geoffrey M , Seder Robert A
Published: January 2023

Targeting tumor specific neoantigens through personalized therapeutic vaccination is a promising approach to induce T cell immunity against a variety of tumors. Current vaccine formulations induce CD8 T cell responses against a limited subset of pre-selected neoantigens. We developed a novel vaccine platform to enhance the breadth and magnitude of CD8 T cell responses by chemically coupling synthetic neoantigen peptides to a synthetic polymer containing a toll-like receptor-7/8 agonist that self-assembles into nanoparticles (termed SPP-7/8a). To test the efficiency of the SPP-7/8a platform for inducing antigenic breadth, 173 transcribed non-synonymous single nucleotide variants were identified in a mouse melanoma tumor line (SB-3123). Mice were vaccinated twice with a random set of 96 SB-3123 neoantigens with SPP-7/8a. The binding affinity was predicted for each neoantigen using the Immune Epitope Database and Analysis Resource (IEDB.org) Consensus algorithm, which ranged from the 0.15 to 3.05 percentile. Overall, CD8 T cell responses were detectable at frequencies ranging from 0.1% to 16% of total CD8 T cells against 10 of the 96 neoantigens (~10%). However, by stratifying the 96 neoantigens according to high affinity binders (Consensus < 0.5), 7/14 (50%) induced CD8 T cell responses. These data show that the SPP-7/8a platform efficiently induces CD8 T cell using peptides that have high binding affinity. Ongoing studies will determine whether the increased breadth of CD8 responses following SPP-7/8a vaccination confers protection against established tumors.
Optimization of Gene Editing in Human Primary T Cells with Neon® Transfection System
Authors: Chang Sean , Andronikou Nektaria , de Mollerat du Jeu Xavier
Published: January 2023

The successful cases of autologous CAR-T cell therapy in leukemia have highlighted the promising future of cell therapy. However, most studies in this field have been using viruses to engineer T cells, which lead to safety concerns. On the other hand, there has been a growing interest in developing allogeneic immune cell therapies to tackle the challenges of production scale up. Hence, there is a need for a better delivery system to maximize gene editing efficiency in immune cells.

Neon® Transfection System has been widely used in a variety of cell models and especially hard-to-transfect cells, such as primary cells and stem cells. Here we demonstrate how Neon® is fully capable of supporting all spectrums of research in cell therapy, including RNA delivery, DNA delivery, and CRISPR/Cas9 gene editing. Human primary T cells from four different donors were isolated from individual fresh Leukapheresis Pak, followed by activation/expansion in OpTmizer media with 2% human serum and CD3/CD28 Dynabeads. The optimal delivery time was between 3–5 days after activation. The transfection efficiency can be reached over 95% for RNA (EGFP mRNA) and 85% for DNA (pcDNAEF1a-emGFP) with high viability using certain Neon® programs. For CRISPR/Cas9 gene editing, more than 30% knock-in efficiency was observed while cleavage efficiency at HPRT locus can be reached more than 70%.

The promising results of gene delivery and editing in primary T cells from Neon® could facilitate the progress of immunotherapy. Meanwhile, we have been dedicating in developing a next generation cGMP-certified large-scale electroporation system, which enable researchers to seamlessly scale-up from Neon®.
Detection and tracking of public, broadly neutralizing antibodies against group 1 and group 2 Influenza A viruses in healthy donors and post-seasonal flu vaccine.
Authors: Gittelman Rachel M , Yusko Erik , Sanders Catherine M , Vignali Marissa , McDermott Adrian , Robins Harlan
Published: January 2023

Influenza A viruses form two diverse clades that co-circulate and cause seasonal infections each year. This diversity has made it challenging to develop a universal flu vaccine that effectively targets epitopes across both clades. One approach to developing a universal flu vaccine is to identify broadly neutralizing antibodies that can be reproducibly elicited across individuals by novel immunogens or immunogens specifically designed to enhance these sequences. These public antibodies may represent common and accessible routes to protection against influenza in the general population. Next generation sequencing can be used to monitor vaccine trials for the presence of these sequences. In this work, we leverage Adaptive Biotechnologies’ IGH sequence database and immunoSEQ® assay to track and characterize a set of recently identified public IGH motifs that are capable of neutralizing group 1 and group 2 flu viruses1. We first characterized the incidence and basal frequency of these motifs in blood from 3 healthy individuals, as well as bone marrow from 97 patients following immune reconstitution. Next, we monitored clonal expansion and contraction of these motifs in 15 healthy subjects immediately prior to, and at days 3, 7, 14, 28, and 120 post seasonal flu vaccination. Finally, we placed these motifs within the broader context of immune response following seasonal flu vaccination through additional deep sequencing of the IGH and TCRB loci in these subjects. These data show that immunoSequencing can be used as a highly quantitative and sensitive tool to track the production and maintenance of specific antibody sequences.
Adapting the innate immune system to develop long-lived vaccines to bacterial pathogens
Authors: Super Michael , Doherty Edward , Cartwright Mark , Stafford Alexander , Ali Omar , White Des , Graveline Amanda , Urena Frank , MacDonald Chloe , Dinis Alexandre , Duffy Shannon , Duckless Benjamin , Seiler Benjamin , Ingber Donald , Mooney David
Published: January 2023

There is an urgent need to develop vaccines against bacteria due to the rise of Multi-drug resistant (MDR & XDR) organisms. To date, it has been difficult to produce protective vaccines against bacterial pathogens; there is a danger of outgrowth of fast growing attenuated bacterial strains while polysaccharide cell walls are poorly immunogenic and subunit vaccines are ineffective, generating TI (T-independent) immune responses, characterized by low affinity, short lived, non-class switched IgM antibodies.

We are developing technologies for generating vaccines in mice against multiple pathogen species, including bacterial; MDR E. coli, MRSA, MTb LAM, Viral; HIV gp120, parasitic; P. falciparum and fungal; C. albicans. We capture pathogens using FcMBL Opsonin technology (which binds more than 90 different pathogen species), and present the killed pathogens in an immune modulating biomaterial system. This lyophilized product provides long-lasting protection with a single dose through a novel self-boosting mechanism of action.

Our vaccines can protect mice against MDR strains of E. coli which are lethal within 12 hours. We have raised antibodies with titers which are sustained beyond 90 days (to date) with a single vaccination (the biomaterial system has demonstrated titers beyond 1 year in other indications). Using this opsonin and immune modulating technology, E. coli can be captured from the blood and tissues of one animal and used to vaccinate other animals.
Investigating the roles of TNFα and TGF-β1 in Ly-6A-mediated CD4+ T cell death
Authors: Patel Akshay G , Bamezai Anil K
Published: January 2023

Ly-6A, a member of the Ly-6 family of proteins, negatively regulates the antigen receptor-driven clonal expansion of CD4+ T cells by an unknown mechanism. Ly-6A is a 12–14 kDa protein that lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane with a glycosylphosphatidylinositol (GPI) anchor. On immune cells, this cell surface protein also has adhesion and signaling properties. To explore the mechanism of Ly-6A-mediated CD4+ T cell growth inhibition, we have used immortalized CD4+T cell lines as in vitro experimental model. Engaging Ly-6A caused growth inhibition, apoptosis, and cytokine secretion in culture. Ly-6A activation also upregulated p27kip1, a cell cycle inhibitory protein. Additionally, we observed mitochondrial destabilization as gauged by the release of cytochrome C in the cytoplasm, and detection of active Caspases 3 and 9. The simultaneous observance of cytokine transcription and cell death appeared enigmatic. To examine this issue, we hypothesized that CD4+ T cells generate death and growth inhibitory factors after engaging the Ly-6A protein with a monoclonal antibody. The cytokines TNFα and TGF-β1, growth inhibitory and pro-apoptotic molecules, respectively, were examined. We observed that engaging Ly-6A results in growth inhibition and production of TNFα at higher concentrations than what is generated by engaging the antigen receptor. Importantly, blocking antibodies against TNFα decreased cell death in a concentration-dependent manner as evaluated by flow cytometry. Levels of total and free active forms of TGF-β1 were significantly upregulated by CD4+ T cells to stimulation through the antigen receptor complex, but no changes in secretion of TGF-β1 were found after engaging Ly-6A.
Direct ODN ligation on CD4+ T cells results in the activation of TLR9 signaling
Authors: Sharma Ravi Kumar , Sharma Jyoti , Pattekar Ajinkya , Jain Pooja , Sachdeva Naresh
Published: January 2023

Oligodeoxynucleotides (ODNs) are established ligands for TLR9, however functional response via ODN in CD4+ T cells has been believed to be independent of TLR9 and MyD88. Here, we studied the uptake of ODN2216 in human peripheral CD4+ T cells and assessed its effect on TLR9 signaling. We observed that ODN2216 was taken up intracellularly by endocytosis in CD4+ T cells, leading to increase in gene expression of TLR9 in a time dependent manner (p=0.029). The inhibition of endocytosis by dynasore and promethazine resulted in lower uptake of ODN2216 by CD4+ T cells. Inhibition of MyD88 resulted in decreased uptake of ODN2216 (p=0.0009) by CD4+ T cells, while there was no significant effect of inhibiting IRAK1 or IRAK4. Post uptake, we observed a significant increase in gene expression of TLR9, MyD88, IRAK1, IRF-7, and TRAF3 but unlike canonical TLR9 signaling, there was no significant increase in expression of IRAK4, IRF-3, and TRAF6 (p<0.0001). Moreover, TLR9 expression on CD4+T cells also decreased significantly upon treatment with endocytosis inhibitor (p=0.0001), MyD88 inhibitor (p=0.016), IRAK1 inhibitor (p=0.0001) but not IRAK4 inhibitor as compared with untreated CD4+ T cells. Further the uptake of ODN2216 led to an increase in TGF-β synthesis in TLR9 signaling dependent fashion by CD4+ T cells (p<0.0001). Collectively, our results demonstrate a direct but distinct TLR9 agonist ligation induced signaling pathway in CD4+ T cells which is independent of antigen presenting cells. This TLR9 signaling pathway may have a significant role in modulation of T-cell responses in exaggerated inflammatory responses.
Delayed neutrophil apoptosis is regulated by cancer associated a2 isoform vacuolar ATPase
Authors: Ibrahim Safaa A. , Kulshrestha Arpita , Katara Gajendra K , Beaman Kenneth D
Published: January 2023

Neutrophils are short life span cells but they can live for weeks in cancer tissues and promote tumor progression. Identifying cancer derived factors that enhance neutrophil survival will provide new targets for cancer therapy. A peptide from the N-terminal domain of a2- isoform vacuolar ATPase (a2NTD) is specifically secreted from cancer cells and promotes the neutrophil protumorigenic properties which in turn, enhance tumor progression. This gives a2NTD the propensity to regulate neutrophil survival. In fact the treatment of human neutrophils with recombinant a2NTD led to 2.1 fold increase in the percentage survival of neutrophils (P < 0.001). a2NTD treatment modulates different apoptosis pathways. Executive pathway; a2NTD treated neutrophils (a2Neuφ) showed a significantly decreased gene expression of caspase-3, -6, -7, that corresponded with decreased activity of these caspases. Intrinsic pathway; a2NTD treatment upregulates the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL and downregulates the pro-apoptotic factors; BAX and Apaf-1. Extrinsic pathway; a2NTD treatment dowenregulats the gene expression and the activity of caspase-8 and upregulates the gene expression of c-Flip. a2NTD treatment also stimulates reactive oxygen species generation that activate NF-kB p65 in neutrophils. Moreover, NF-kB inhibition negated that promoted neutrophil survival that was associated with downregulation of the gene expression of Bcl2-A1, Bcl-xL, G-CSF and upregulation of BAX. Thus, a2NTD enhances neutrophil survival by activating the NF-kB pathway in neutrophils. Together, these data demonstrate a novel role of the cancer associated a2-vacuolar ATPase on regulating neutrophil survival by the action of a2NTD.
Versican proteolysis is associated with robust CD8+ T-cell infiltration in human mismatch repair-proficient and -deficient colorectal cancers
Authors: Asimakopoulos Fotis , Hope Chelsea , Emmerich Philip , Pagenkopf Adam , Matkowskyj Kristina , Van De Hey Dana , Payne Susan , Clipson Linda , Papadas Athanasios , Johnson Michael G , Deming Dustin
Published: January 2023

Colorectal cancer (CRC) originates within immunologically complex microenvironments. To date the benefits of immunotherapy have been modest except in neoantigen-laden mismatch repair (MMR)-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. We recently reported that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN), in myeloma tumors, generates a bioactive fragment, versikine, with putative immunostimulatory activities. We show that VCAN proteolysis strongly correlated with CD8+ T-cell infiltration in CRC. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T-cell infiltration. The correlation between VCAN proteolysis and CD8+ T-cell infiltration was maintained in MMR-proficient and -deficient CRCs. Tumor-intrinsic WNT pathway activation was associated with CD8+ T-cell exclusion and correlated with VCAN accumulation. Our findings indicate VCAN proteolysis may shape CRC immune contexture and provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker. Therapeutic manipulation of the VCAN-versikine axis may augment immunotherapy efficacy against CRC.
Ectopic B cell response to Ehrlichia muris in the liver
Authors: Trivedi Nikita H , Kader Muhamuda , Diniro Roberto , Ismail Nahed , Shlomchik Mark J
Published: January 2023

The development of high-affinity, isotype-switched Abs and memory cells is generally facilitated by Germinal Center reactions (GCs) that form in the secondary lymphoid organs within 4–5 days of antigen encounter. However, in the case of Ehrlichia muris (E. Muris), an obligate intracellular tick borne pathogen, GCs are not detectable up to day 35. Instead a robust B cell response dominated by IgM antibody forming cells (AFCs) originates from extrafollicular (EF) sites in the spleen, with somewhat lower, but still large frequencies of IgG AFCs. Interestingly, E. Muris is known to infect the liver, and we found both AFCs as well as histologic collections of plasmablasts in the liver parenchyma. In both cases the number of total AFCs was far larger than that which bound to lysates that contain E. Muris antigens, suggesting either a non-specific or very low affinity response. We hypothesize that these EF sites to be locations where B cells undergo somatic hypermutation (SHM) and acquire mutations that increase affinity to E. muris. Such mutations would allow what appear to be low affinity non-specific responses to gain detectable affinity for E. Muris. To test this, we performed laser microdissection of these plasmablast patches followed by sequencing of BCR variable regions. These studies revealed that somatic hypermutation occurred in the spleen as well as liver and thus demonstrated that liver is a novel locus for generating B cell immune responses that include V region mutation and affinity-based selection.
HVEM-BTLA interactions stimulate lung innate immunity following S. pneumoniae infection.
Authors: Hitomi Kaori , Shui Jr-Wen , Del Rio Maria Luisa , Rodriguez-Barbosa Jose-Ignacio , Kronenberg Mitchell
Published: January 2023

Streptococcus pneumoniae (S. pneumoniae) is the leading cause of bacterial pneumonia in children and the elderly and it kills close to half a million children under 5 years old worldwide every year. Recent reports indicate that epithelial cells act not only as a physical barrier to pathogen invasion, but also as an extension of the immune system to prevent infection or eliminate pathogen. The herpes virus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor super family. HVEM polymorphisms are a risk factor for several inflammatory and infectious diseases. Although lymphocytes and myeloid cells express HVEM, our previous data suggested that HVEM expression by epithelial cells was critical for innate immunity in the lung. However, the mechanism of HVEM-mediated host defense against S. pneumoniae is still unknown. To address the issue, we have analyzed mice with a lung epithelial cell-specific deletion of HVEM (Cc10-cre x Hvemfl/fl). We found that Cc10-cre x Hvemfl/fl mice were highly susceptible to S. pneumoniae, with an increased bacterial load by 24h and reduced survival. Mice treated with an antibody that blocks HVEM had a similar outcome, confirming that HVEM expression is required during the infection. HVEM has three ligands: the TNF super family member LIGHT and the Ig super family members BTLA and CD160. We determined that BTLA-deficient mice showed increased bacterial load in the lung and reduced survival, similar to HVEM deficiency. Moreover, CD11c-cre x BTLA fl/fl mice were highly susceptible to S. pneumoniae. Our data show that interaction between BTLA expressed by a CD11c positive myeloid cell type and HVEM expressed by lung epithelial cells leads to a protective innate response in the lung after infection.
Atypical activation of human primary dendritic cells by Plasmodium falciparum
Authors: Goetz Anton , Tang Mei San , Ty Maureen , Rodriguez Ana
Published: January 2023

Malaria is characterized by high levels of inflammation and while an early inflammatory response is important for parasite clearance, excessive and persistent inflammation can contribute to severe forms of the disease. At the same time, P. falciparum infections fail to induce sterile immunity. Reports about the role of dendritic cells (DCs) in the immune response to P. falciparum and how they contribute to the activation of CD4+ T cells have been contradictory. To analyze this critical part of the malaria immune response we enriched primary human DCs from peripheral blood of naïve donors and co-incubated them with P. falciparum-infected red blood cells. Although DCs up-regulated surface expression of HLA-DR, co-stimulatory markers, and secretion of chemokines, they did not secrete significant amounts of inflammatory cytokines. Surprisingly, these parasite-activated DCs were able to activate and polarize naïve autologous CD4+ T cells into Th1-like cells secreting high levels of IFNγ and TNF. A re-stimulation with autologous P. falciparum-activated DCs increased proliferation and cytokine secretion of primed CD4+ T cells, indicating these DCs are able to induce an antigen-specific response. We further analyzed the activation phenotype of the two major DC subsets, myeloid and plasmacytoid DCs. Contact-dependent co-culture of both subsets was essential to up-regulate parasite-induced chemokine secretion while plasmacytoid DCs were dispensable for CD4+ T cell activation. RNA-seq of myeloid DCs revealed a distinct transcriptional profile induced by the parasite. Our findings might contribute to a better understanding of the mechanisms involved in the initiation of the adaptive immune response against P. falciparum.
The Kallikrein-kinin system: a novel player of IL-17-driven anti-Candida immune response in the Kidney
Authors: Biswas Partha Sarathi , Ramani Kritika , Garg Abhisek V , Jawale Chetan V , Conti Heather R , Whibley Natasha , Kolls Jay , Gaffen Sarah L
Published: January 2023

Disseminated candidiasis is the fourth most common hospital acquired infection and is associated with a 40–60% mortality rate. During disseminated candidiasis, Candida albicans form invasive hyphae that damage kidney, leading to renal failure. Treatment of candidiasis is hindered by drug toxicity, the development of antifungal drug resistance and lack of vaccines against fungal pathogens. Previous studies have identified a key role of interleukin-17 (IL-17) in controlling systemic infection. The mechanisms of IL-17-mediated renal immunity have so far been assumed to occur solely through the regulation of antimicrobial mechanisms. Here, we discovered an unanticipated role for IL-17 in inducing the Kallikrein (Klk)-Kinin System (KKS) in the infected kidney, and we show that the KKS provides significant renal protection in candidiasis. Consequently, overexpression of Klk1 or treatment with bradykinin rescued IL-17RA−/− mice from candidiasis. Therapeutic manipulation of IL-17-KKS pathways restored renal function and prolonged survival by preventing apoptosis of renal cells following disseminated infection. Moreover, combining a minimally effective dose of fluconazole with bradykinin strikingly improved survival compared to either drug alone. These findings have potential translational significance, as agonists of the KKS are in routine clinical use. Therefore, these results not only identify downstream mediators that could serve as novel drug targets, but could possibly be used to guide decisions on whether targeting these mediators could be a useful therapeutic option in conjunction with current antifungal drugs.
A single nucleotide polymorphism in an AP2 transcription factor encoded in the malaria-causing Plasmodium berghei alters the development of host immunity
Authors: Sheehan Patrick William , Akkaya Munir , Bansal Abhisheka , Arora Gunjan , Otto Thomas Dan , Qi Cheng-Feng , Pena Mirna , Yazew Takele , Billker Oliver , Miller Louis , Pierce Susan K
Published: January 2023

The Apetela2 (AP2) family of transcription factors is a major regulator of Plasmodium parasite gene expression. Twenty seven members of this family have been identified since their discovery in 2005. However, our understanding which AP2 members regulate key features of Plasmodium biology is incomplete. Here we describe the impact of a single nucleotide polymorphism (SNP) in the active site of the AP2 protein, PBANKA_011210, on the outcome of infection in mice. Plasmodium berghei Anka (PbA), the only rodent parasite that causes experimental cerebral malaria (ECM), codes for a phenylalanine at position 1823 whereas all other Plasmodium parasite strains, none of which cause ECM in mice, including PbNK65, have a serine in this position. Using CRISPR, we modified the PbNK65 parasite (PbNK65-WT) to encode the PbA SNP (PbNK65-PbA). The course of infection with PbNK65-WT or PbNK65-PbA were similar and showed no ECM pathology indicating that the PbA AP2 SNP is not sufficient to cause ECM. However, mice infected with PbNK65-PbA generated dramatically higher germinal center responses and produced higher titers of parasite-specific antibodies. When infected mice were treated with sub-therapeutic doses of the anti-malarial, chloroquine, to lower the parasitemias PbNK65-PbA-infected mice cleared the infection whereas mice infected with PbNK65-WT were unable to control the infection resulting severe anemia and death. These results reveal a novel role of AP2 proteins in controlling host immunity to Plasmodium infections.
Neuraminidase-mediated TGF-β activation facilitates evolution of protective Th17 immunity
Authors: Dutta Avijit , Huang Ching-Tai , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin
Published: January 2023

The immune system responds to influenza virus infection for eradication of the virus but the associated inflammation may cause tissue damage to the host. In our antigen-specific mouse system, naïve influenza hemagglutinin (HA)-specific CD4+ T cells adoptively transferred with infection respond with Th1 phenotype and produce IFN-γ. Some of them secrete IL-17 as well. Second batch HA specific CD4+ T cells, adoptively transferred on day 4 after infection and the first transfer, respond to the infection with Th17 phenotype. They produce IL-17 but not IFN-γ. Both the Th1 and Th17 cells are activated with similar levels of ZAP-70 phosphorylation and T-bet induction. For the Th17 cells, IFN-γ deficiency is associated with excessive ROR-γt induction and altered T-bet dominance. Th17 cells display surface LAG-3 expression, suppress T cell activation in vitro and decrease lung inflammation in vivo. The evolution of Th17 cells is augmented in IL-10 deficiency, with IL-10 knockout mice as the recipients transferred with IL-10 knockout HA specific CD4+ T cells. There is increased neuraminidase-mediated TGF-β activation in this IL-10 deficient environment. Viral neuraminidase mediated TGF-β activation in the first batch of HA specific CD4+ T cells facilitates the Th17 evolution of the 2nd batch of cells. The 6.5 T cell receptor (TCR) transgenic mice of monotonous HA specific TCR repertoire respond to influenza virus infection with Th17 deviation and demonstrate survival benefit. Consecutive waves of naïve HA specific T cells from the thymus in 6.5 mice may evolve with mechanisms similar to consecutive adoptive transfers. Evolution of protective Th17 immunity is facilitated by neuraminidase-mediated TGF-β activation.
Effects of the PKC agonist Ingenol-B on HIV suppression by CD8+ T cells
Authors: Kwaa Rebecca Abena K. , Goldsborough Kennedy , Walker-Sperling Victoria , Gama Lucio , Blankson Joel
Published: January 2023

Shock and kill strategies have been proposed as a possible mechanism for HIV-1 eradication. These strategies involve the use of latency-reversing agents (LRAs) such as PKC-agonists, to “shock” latently infected CD4+ T cells and myeloid cells into producing viral proteins that could then be recognized by effector cells leading to the “kill” component of the strategy. In its wake, recent studies have shown that some LRAs inhibit HIV-specific CD8+ T cell responses, suggesting that LRAs should be screened for immunosuppressive properties before use in clinical trials. In a prior study to determine whether PKC agonists could inhibit the potent HIV-specific CD8+ T cell responses seen in patients who control HIV replication naturally (elite suppressors, ES), we found that Bryostatin-1, but not Prostratin, inhibited ES CD8+ T cell responses. Ingenol-B is another PKC agonist with potent LRA activity both by itself and in combination with the bromodomain inhibitor JQ1. In this study, we determined the effect of this drug on CD8+ T cells from 6 ES.

We show here that while Ingenol-B had no effect on the ability of CD8+ T cells to suppress viral replication (65.3% suppression versus 59.3% with DMSO, p > 0.05), its combination with JQ1 caused a modest, but significant, decrease in this suppressive capacity (39.9%, p= 0.033). The mechanism of this inhibitory effect could not be readily explained by differences in PD-1, TIM-3 or CD3 expression on CD8+ T cells. We conclude that since robust CD8+ T cell activity is essential for the eradication of reactivated latently infected cells, the potency of LRA combinations must be balanced against their effect on HIV-specific CD8+ T cell responses.
Natural killer cells support myeloid suppressor cell expansion during persistent viral infection
Authors: Ohayon David E , Brooks Taylor R , Mahl Sarah E , Cranert Stacey A , Waggoner Stephen N
Published: January 2023

Persistent viral infections in mice (e.g. lymphocytic choriomeningitis virus, LCMV) and humans (e.g. HIV, hepatitis C virus) are characterized by chronic inflammation. This sustained inflammatory milieu promotes T cell exhaustion, which limits harmful tissue damage but prevents effective viral control. We found that natural killer (NK) cells aid in the establishment of this tolerogenic state through cytolytic elimination of antiviral T cells. In parallel, others shown that chronic infection encourages expansion of immunoregulatory IL-10-expressing myeloid cells and bona fide myeloid-derived suppressor cells (MDSCs) that contribute to T cell inhibition immune exhaustion and cell injury. We now report that depletion of NK cells prior to persistent infection with the clone 13 strain of LCMV abrogates the expansion of immunosuppressive myeloid cells. In the absence of NK cells, the levels of myelo-stimulatory cytokines, including TNF-a, MCP-1, and IL-6 were also reduced. Furthermore, frequencies of myeloid precursor cells were reduced in the bone marrow during infection in the absence of NK cells. Our results suggest that NK cells support an inflammatory milieu promoting expansion of the myeloid niche and specifically suppressive myeloid cells during chronic infection. This cross-talk between NK cells and myeloid cells during inflammation has the potential to contribute to immune dysfunction during both chronic infection and in cancer.