bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor intrinsic mechanisms of immunosuppression in CD8 T cell function and response to lung cancer
Authors: Melese Etienne Sylvain , Franks Sarah Elizabeth , Cederberg Rachel , Seo Jung Hee , Lam Wan , Bennewith Kevin L , Lockwood William , Abraham Ninan
Published: January 2023

Immunotherapies are effective in a subset of patients with non-small cell lung cancer (NSCLC), however the majority do not respond which highlights the ongoing need for novel therapeutics and biomarkers. Tumor cells can produce cytokines and chemokines that influence both the recruitment and function of immune cells. In patients with NSCLC, the microRNA-17~92 (miR-17~92) cluster was shown to be upregulated, and high levels of microRNA-17 in serum was associated with decreased survival. We aimed to identify whether tumor cell intrinsic mechanisms, like upregulation of miR-17~92, regulate chemokine or cytokine production to induce an immunosuppressive tumor microenvironment and inhibit CD8+ T cell anti-tumor function in the lung. We used the CIBERSORTX program to evaluate alterations to immune cell populations and survival in NSCLC patients with increased chemokine or miR-17~92 expression. We found high expression of miR-17 and low expression of IL-7Rα correlated with decreased survival in lung adenocarcinoma (LAC) patients. Furthermore, LAC patient’s tumor biopsies with high miR-17 had decreased IL-7Rα and increased populations of CD8+ T cells by CIBERSORTX. We will use in vitro and in vivo models of lung cancer to determine whether miR-17~92 upregulation and tumor cell chemokines and cytokines production drives immunosuppression in the tumor microenvironment. Understanding how lung tumor cells regulate cytokines and chemokines, and their putative control of exhaustion in CD8 T cells in the tumor microenvironment would guide selection of efficacious immunotherapy treatment.
The Development, Function, and Plasticity of the Immune Macroenvironment in Cancer
Authors: Allen Breanna M , Hiam Kamir , Burnett Cassandra E , Venida Anthony , DeBarge Rachel , Carmi Yaron , Spitzer Matthew H
Published: January 2023

Harnessing immune defense mechanisms has revolutionized cancer therapy, but our understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. Here, we use mass cytometry to define the organism-wide immune landscape in response to tumor development across five tissues in eight tumor models. Systemic immunity was dramatically altered across mouse models and cancer patients, with changes in peripheral tissues differing from those in the tumor microenvironment and taking place in phases during tumor growth. This tumor-experienced immune system mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Disruptions in T cell responses were not cell-intrinsic but rather due to reduced responses in antigen-presenting cells (APCs). Promoting APC activation was sufficient to restore T cell responses to orthogonal infection. All systemic immune changes were reversed with surgical tumor resection, revealing remarkable plasticity in the systemic immune state, which contrasts with terminal immune dysfunction in the tumor microenvironment. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.
Reprogramming the tumor microenvironment with next-generation chaperone-based immunolytic agent for cancer immunotherapy
Authors: Guo Chunqing , Liu Wenjie , Yu Xiaofei , Subjeck John R. , Repasky Elizabeth A , Wang Xiang-Yang Shawn
Published: January 2023

The tumor microenvironment (TME) represents a major barrier for successful cancer immunotherapy due to tumor-associated immunosuppressive pathways that facilitate immune evasion and therapeutic resistance. We recently created a multifunctional chimeric chaperone (i.e., Flagrp170) by incorporating the flagellin-derived NF-κB-stimulating sequence for superior antigen cross-presentation and co-stimulation. Upon intralesional administration, this immunolytic agent results in profound inhibition of multiple mouse tumors including melanoma, prostate cancer, colorectal cancer, and mammary tumor. Strikingly, reprogramming the TME with Flagrp170 induces elevation of multiple Th1 cytokines such as GM-CSF, IL-12 and IFN-γ. Mechanistic studies reveal that GM-CSF is mainly produced by CD8+ T cells and is indispensable for the antitumor efficacy of in situ Flagrp170 therapy. Neutralizing GM-CSF abrogates the antitumor activity of the Flagrp170, which is associated with reduced mobilization and recruitment of dendritic cells (DCs) as well as IFN-γ-expressing T cells, suggesting that GM-CSF-derived from tumor-infiltrating T cells is essential for expansion and/or function of DCs within the TME. Our findings elucidate an immunological mechanism of action of the Flagrp170 in skewing the TME towards a Th1-polarized immunostimulatory phenotype and provide a novel approach to reprogramming the immune landscape of the TME for improved immunotherapeutic outcomes.
CD317-mediated cell cytoskeleton regulation as a novel means of tumor immune evasion
Authors: Zhang Guizhong , Cheng Jian , Liu Zhao , Deng Tian , Adeshakin Funmilayo Oladunni , Wan Xiaochun
Published: January 2023

CD317 is frequently overexpressed in various human cancers. Although our previous study had demonstrated its pro-proliferative role in HCC development, the functions of CD317 in tumorigenesis, especially in tumor immune evasion, remain unclear. Herein we report that CD317 protects cancer cells from immune cell-mediated cytotoxicity. CD317 knockdown in cancer cells affects neither cell-cell conjugation nor effector cell activation, but markedly increases the vulnerability of cancer cells by which renders cancer cells more sensitive to YTS- or CAR-modified NK cell-mediated cytotoxicity. This sensitivity alteration cannot be restored by adding recombinant CD317 extracellular domain (317ECD) protein, excluding the possibility that CD317 acts as a traditional immune checkpoint protein that provides inhibitory signaling to effector cells. Mechanically, immunoprotective function of CD317 is mainly dependent on the interaction with RICH2, a known CD317-binding partner that is involved in the cytoskeleton regulation. Knocking down RICH2 significantly rendered CD317 incapable of protecting cancer cells from immune cell-mediated killing, suggesting CD317 facilitates tumor immune evasion by regulating tumor cell cytoskeleton to confront immune killing. Taken together, our findings reveal a novel means of tumor immune evasion and suggest CD317 as an attractive target for improving the efficiency of cancer immunotherapy.
TLR2 Signaling Promoted Tumor Development through Enhancing Inflammation and Regulatory T cells to Suppress Anti-Tumor Immunity
Authors: Shen Chia-Rui , Lee Meng-Hua , Kuo Ming-Ling
Published: January 2023

Inflammation is a relevant physiologic process necessary for immune protection, tissue repair and remodeling. However, chronic inflammation is known to contribute tumor development, including tumor initiation, promotion and progression. Recent studies have shown that up-regulation of Toll-like receptors (TLRs) in tumor microenvironment induced the inflammatory responses. Here, we hypothesized that the TLR2 signaling within tumor microenvironment elicits an inflammatory response and such facilitates tumor development. A murine B16F10 melanoma model was utilized to investigate the role of endogenous TLR2 signaling in tumor development. Anti-tumor immunity and the presence of tumor-associated inflammatory mediators were analyzed by flow cytometry and ELISA in the TLR2 knockout (TLR2 KO) and wild-type (WT) tumor-bearing animals. When compared to the TLR2 deficient mice, WT mice had a significantly aggressive-appearing pathology, such as elicited tumor growth and metastasis. When the splenocytes cultured with tumor-conditional medium, WT produced elevated levels of pro-inflammatory cytokines, whereas TLR2KO or the WT receiving anti-TLR2 antibody treatment blocked the inflamed-like phenomenon and consequently showed a better anti-tumor IFN-γ response. Additionally, tumors from WT mice exhibited comparably higher infiltration levels of CD4+CD25+Foxp3+ regulatory T cells, but much less CD8+ T cells. These results support that cancer cells-derived components through TLR2 signaling generate an inflammatory microenvironment hospitable for tumor growth, and it indicates TLR2 as a potential therapeutic target for cancer immunotherapy.
Immune checkpoint molecule V-set Ig domain-containing 4 (VSIG4) expression is associated with poor prognosis in advanced gastric cancer patients
Authors: Kim So-Woon , Roh Jin , Jung Jiwon , Pak Hyo-Kyung , Lee A-Neum , Park Young-Soo , Park Chan-Sik
Published: January 2023

Background

New therapeutic approaches are needed for the treatment of advanced gastric cancer (AGC). V-set Ig domain-containing 4 (VSIG4) is an immune checkpoint molecule that may have prognostic or predictive utility in the treatment of AGC. The aim of this study was to investigate expression of VSIG4 and its clinical significance in AGC and other major cancers.

Methods

We analyzed VSIG4 expression and its correlation with survival rate in various carcinomas including 1,034 surgically resected AGCs from two academic hospitals. Patients were divided into a discovery (N = 572), internal validation (N= 230), and external validation (N= 232) sets.

Results

VSIG4-positive AGC tumors were significantly associated with overall survival (OS) (hazard ratio (HR) = 3.02, 95% confidence interval (CI) = 2.32–3.94, P<0.001,) and event-free survival (HR = 2.8, 95% CI=2.18–3.72, P <0.001). These findings was successfully validated in the independent cohorts. VSIG4-positive status was also significantly correlated with low intratumoral CD8+ T-cell infiltration (P = 0.036) and high FoxP3+/CD8+ intratumoral T-cell infiltration ratios (P = 0.016). VSIG4 expression in other cancers was not associated with higher 5-year OS rates (colon, P = 0.459, lung, P = 0.275, kidney, P = 0.121, breast, P = 0.147).

Conclusions

VSIG4 is an independent prognostic factor in AGC. Notably, VSIG4 expression was significantly associated with low CD8+ T-cell infiltration and high regulatory T cell/CD8+T cell ratios, implying a cancer tolerating immune environment. Our results may be an important basis for future gastric cancer-specific immunotherapeutic drug development.
A TruCulture Whole Blood Assay to Evaluate Annual Flu Vaccine Recall Responses
Authors: Hwang Shen-An , Eisinger Dominic , LaBrie Samuel
Published: January 2023

Evaluation of vaccine efficacy to protect against future infection remains a challenge. One of the difficulties lies in developing an antigen specific recall assay that can be applied across clinical sites. TruCulture is a whole blood collection and culture system that was designed for ex vivo/in vitro evaluation of immune responses for clinical trials. This abstract demonstrates a TruCulture tube that is highly effective in eliciting recall responses against Influenza A hemagglutinin (HA) proteins after the annual flu vaccine. Whole blood from 10 healthy donors were collected at 2 days pre- and 14 days post-vaccination (Afluria, Sequiris). Blood was collected in TruCulture tubes containing 5mg recombinant HA antigens (Immune Technology Corporation). After incubation at 37°C for 48 hours, the supernatants were collected and analyzed for 13 analytes by OptiMAP and 47 analytes by TruCulture MAP (Myriad RBM). Several analytes (such as IFNγ, IL-1β, IL-3, IL-4, IL-6, IL-10, IL-12, IL-17, and IL-23) demonstrated an increase at 14 days after the flu vaccination compared to samples taken prior to vaccination. This increase is specific to the recombinant HA antigens from flu strains included in the flu vaccine administered. The increase in these analytes were either not observed or at significantly reduced levels when the recall response was conducted using recombinant HA antigens from flu strains not included in the vaccine administered. TruCulture preloaded with HA antigens can be a valuable tool to evaluate human immune responses to the annual flu vaccine and indicates that a similar strategy using antigen-specific TruCulture tubes can be developed for the evaluation of any novel vaccine.
Evaluating Influenza A virus vaccines in a dirty mouse model better mimics the human immune response
Authors: Fiege Jessica K , Block Katharine E , Pierson Mark J , Pape Kathryn A , Jenkins Marc K , Hamilton Sara E , Masopust David , Langlois Ryan A
Published: January 2023

Current seasonal influenza A virus (IAV) vaccines often exhibit reduced efficacy to seasonal strains and offer limited protection to novel pandemics. A number of therapies, including vaccines, that are successful in mice often fail to translate to humans. This could be due to housing animals in specific pathogen free (SPF) conditions. Humans are exposed to a variety of natural host pathogens that SPF mice are protected from. Unlike SPF mice, the immune system of pet store mice more closely recapitulates the immune system of humans. When SPF mice are co-housed with pet store mice harboring natural mouse pathogens, termed dirty mice, these co-housed mice obtain phenotypes observed in the human immune system. In order to determine if dirty mice are an improved mouse model for vaccine development/testing, we sought to study the adaptive immune response to IAV in dirty mice. We infected SPF housed mice or dirty mice with IAV and assessed viral replication, clearance and adaptive immune responses. When we assessed memory responses to IAV in control or dirty mice, we observed altered B and T cell responses in dirty mice. These data demonstrate the importance of studying IAV in the dirty mouse model to facilitate more effective vaccine development and testing before human studies are performed.
The humoral immune response elicited upon immunization with recombinant BCG vaccines protects against hRSV and hMPV in a murine preclinical model
Authors: Galvez Nicolas M. S. , Soto Jorge A. , Bueno Susan M. , Kalergis Alexis M.
Published: January 2023

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are among the main etiological agents responsible of acute lower respiratory tract infections in vulnerable populations worldwide. Despite their clinical relevance, the licensing of a safe and effective vaccine has not been achieved. Considering this, our laboratory developed two different recombinant Mycobacterium bovis BCG strains as vaccine candidates. These recombinant BCGs express either the hRSV-Nucleoprotein (rBCG-N) or the hMPV-Phosphoprotein (rBCG-P). Both vaccines prototypes were able to promote cellular protection against their respective virus. However, the humoral response elicited upon immunization with these vaccines has not been characterized. Here we show that, upon immunization and challenge of mice with the respective rBCG and virus, the induced humoral response was able to protect against the viral infection, promoting the secretion of specific antibodies against each virus and several of their proteins, as measured in sera samples by ELISA assays. We also described that immunization and challenge promoted the induction of antibodies with enhanced neutralizing capacities, as seen during in vitro assays. Moreover, we show that sera transferred from immunized and challenged mice into naïve mice is enough to protect from the infection, when these naïve mice were challenged with the viruses. This protection was characterized by a decreased in the viral replication and pulmonary disease. These results support the notion that the use of rBCG vectors could be an effective vaccine platform against these two major respiratory pathogens and places our vaccine candidates as suitable approaches for the treatment against both hRSV and hMPV.
Ursolic acid moderates Th17-mediated immunity in respiratory syncytial virus infections of young cattle
Authors: Slate Jamison Ray , HONG SUYEON , McGill Jodi L
Published: January 2023

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and young children. Bovine RSV is a common cause of respiratory disease in young cattle with high genetic and immunological similarity to human RSV, making it a relevant clinical model. While RSV is highly prevalent in both humans and cattle, the disease pathogenesis remains poorly understood and therapeutic options are limited. Th17-mediated immunity is intrinsically linked to RSV infections and characterized by the production of the inflammatory cytokine IL-17A; however, excessive IL-17 production may contribute to immunopathology by exacerbating neutrophil recruitment and activation in the lungs. Herein, we sought to elucidate the role of Th17-mediated immune responses by inhibiting Th17 cell differentiation with ursolic acid (UA), a small molecule agonist of the RORγt transcription factor. Calves were prophylactically treated with UA before being challenged with aerosolized bovine RSV. Challenged calves were monitored for clinical disease, gross and microscopic lung pathology, and immunologic responses in the periphery and respiratory tract. Local and systemic neutrophils and monocytes were analyzed for phagocytic and oxidative burst functions. Cellular adaptive immune responses were measured in the peripheral blood and airways by antigen recall assays. Our results suggest that UA-treatment may result in reduced RSV-associated immunopathology by shifting the immune response from Th17-mediated immunity to a more productive Th1-mediated immune response.
Glioblastoma infiltration of both tumor- and virus-antigen specific cytotoxic T cells correlates with experimental virotherapy responses
Authors: Nakashima Hiroshi , Alayo Quazim , Chiocca E. Antonio
Published: January 2023

The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetric to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing “exhaustion” markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume, oHSV intratumoral gene activity and increased infiltration of both viral and tumor antigen-specific CD8+ T cells. These findings imply that both intratumoral viral replication and CD8+ T cell cytotoxicity against both tumor and viral antigens are important in oHSV-mediated GBM therapy.
Phenotypic small-molecule screening identifies JAK inhibitors as pharmacologic modulators of T cell exhaustion
Authors: Marro Brett S. , Zak Jaroslav , Pratumchai Isaraphorn , Lairson Luke , Oldstone Michael B.A. , Teijaro John R
Published: January 2023

Immune checkpoint blockade (ICB) therapy has emerged as viable first-line treatment for cancers such as melanoma. Despite the striking long-term clinical benefits that were previously unattainable, the effectiveness of ICB therapy remains limited in a majority of patients and cancer types. This is due in part to insufficient activation and/or restoration of anti-tumor T cell responses. Thus, understanding the signaling pathways that potentiate T cell exhaustion is essential for developing pharmacologic drugs that reactivate T cells and overcome the limitations of current ICB therapy. To this end, we developed a biologically relevant phenotypic screening system using the LCMV-CL13 infection model coupled with high-throughput flow cytometry to identify small-molecules that resurrect hypofunctional T cells. We discovered 19 hits following a screen of a manually curated collection of 12,000 repurposed drugs. Among the lead compounds identified were janus kinase (JAK) inhibitors including the anti-myelofibrosis drug ruxolitinib. Phenotypic rescue of exhausted T cells in vitro following ruxolitinib treatment resulted in a unique transcriptional signature that diverged from anti-PD-L1 treatment. Lineage-defining genes known to preserve precursor exhausted CD8+ T cells, including transcription factor 7 (Tcf7), were significantly upregulated following exposure to ruxolitinib. Mechanistically, ruxolitinib attenuated the cumulative pSTAT signaling signature within resurrected cells to enhance their survival. Collectively, these results demonstrate a disease-relevant framework for identifying small-molecule modulators of dysfunctional T cells and suggest that JAKs are viable targets for cancer immunotherapy.
T helper differentiation of CAR T cells augments function and target cell killing
Authors: Nelson Andrew Scott , Khan Ibrahim , Kaplan Mark H
Published: January 2023

Chimeric antigen receptor (CAR) T cell therapy involves genetically manipulating T cells to express an antigen receptor specific for tumor protein, resulting in a powerful anti-cancer therapeutic. However, trafficking to tumor sites and the suppressive nature of the tumor microenvironment have prevented effective treatment of solid tumors. Previous work has shown that antigen specific T helper (Th) subsets have variable tumor killing capabilities, however, it is not completely defined how different transcriptional differentiation programs effect the fitness and function of CAR T cells. It was hypothesized that differentiation of T cells transduced with CAR improves cancer cell killing in an antigen specific manner. To test this hypothesis, splenic CD4+ and CD8+ T cells were cultured under T cell activating (T0), Th2 (T2), or Th9 (T9) cell differentiation conditions and transduced with a 2nd generation CD19-specific CAR. Control cells were transduced with CD19-specific CAR lacking the intracellular domain. CAR T cells were co-cultured with CD19+ B cells and target cell killing was measured by flow cytometry. Preliminary results show that CAR T9 cells, but not T2 cells, kill target cells similar to the clinic relevant T0 cells. FACS analysis of T cells after co-culture with target cells revealed antigen-experience promoted production of lineage specific cytokines as well as granzymes A and B. T9 conditions maintained a larger proportion of CD8+ T cells than T2 conditions, suggesting that T9 conditions promote expansion of effector CD8+ T cells. These results suggest that the Th9 transcriptional program promotes target cell killing, transduction efficiency, and generates a better ratio of CD4:CD8 T cells for adoptive cell therapy.
CD4+ T cell help is required for the formation of a cytolytic CD8+ T cell subset that protects against chronic viral infection and cancer
Authors: Zander Ryan A , Schauder David , Xin Gang , Nguyen Christine , Wu Xiaopeng , Zajac Allan J , Cui Weiguo
Published: January 2023

CD8 T cells responding to chronic viral infection can be classified into two major subsets, with a TCF-1+ subset serving as a self-renewing progenitor population that can give rise to a more terminally exhausted PD-1hi subset. However, whether additional heterogeneity exists amongst virus-specific CD8 T cells remains unclear. Here, using single cell RNA-sequencing and flow cytometric approaches, we demonstrate that in addition to the progenitor and exhausted subsets, CD8 T cells responding to persistent LCMV infection also encompass a previously unrecognized T cell subset that exhibits potent cytolytic activity in vitro, and is required for viral control in vivo. This cytotoxic subset is characterized by high expression of killer cell lectin-like receptors, the chemokine receptor CX3CR1, and the transcription factors T-bet and Zeb2. Notably, we further demonstrate a critical role for CD4 help via IL-21 production in facilitating the differentiation of this cytotoxic subset, as depletion of CD4 T cells or deletion of IL-21R signaling in P14 cells (which express a transgenic TCR specific for GP33–41) wholly abrogated the development of CX3CR1+CD8 T cells. Moreover, PD-L1 blockade, an immunotherapy known to partially reverse T cell exhaustion, failed to rescue this differentiation defect of “un-helped” CD8 T cells. Lastly, using a B16 melanoma model, we found that provision of IL-21+CD4 T cells was sufficient to induce over a 2-fold increase in the development of CX3CR1+CD8 TILs, which was associated with enhanced control over tumor growth. Collectively, our findings demonstrate a critical role for CD4 help in the generation of protective CX3CR1+CD8 T cells, and that this differentiation checkpoint may be exploited for immunotherapy.
HSV-2 infects T follicular helper cells to promote HIV reactivation
Authors: Pierce Carl A , Loh Lip Nam , Preston-Hurlburt Paula , Herold Kevan C , Herold Betsy C
Published: January 2023

HSV-2 is one of the most significant factors fueling the global HIV epidemic and coinfection is associated with higher HIV plasma viral loads and increased rates of HIV transmission and HSV-2 recurrences. The mechanisms underlying this syndemic likely reflect local and systemic effects. To explore potential systemic effects, we characterized PBMC from a cohort of HIV-1+ women who were or were not HSV-2 seropositive (HSV-2+) and identified phenotypic changes in CD4 T cells including increased frequencies of activated cells and cells expressing co-receptors for HIV entry in HIV+/HSV-2+ vs. HIV+/HSV-2 seronegative (HSV-2−) women. Surprisingly, IL-32, a proinflammatory cytokine, was lower in CD4 T cells from HSV-2+ women. Addition of IL-32γ to CD4 T cells from HIV+ women blocked HIV reactivation following stimulation with phytohemagglutinin, suggesting that HSV-2 induces phenotypic changes in CD4 T cells to promote HIV reactivation. Subsequent RNAseq analysis of CD4 T cells infected ex vivo with HSV-2 also showed lower levels of IL-32, as well as elevated bcl6, itch, and irf4 transcripts along with decreased ccr7, psgl1, and s1pr1, suggesting a transcriptional landscape consistent with T follicular helper (TFH) cells. Flow cytometry studies using CD4 T cells isolated from PBMC from HIV− or HIV+ donors, as well as from tonsils, showed an increased frequency of TFH cells in HSV-2 infected CD4 T cells, suggesting preferential infection of this subpopulation, which is known to harbor latent HIV. HSV-2 infection of CD4 T cells isolated from HIV+ donors or latently infected T cell lines triggered HIV reactivation. These findings demonstrate that HSV-2 directly infects CD4 T cell subpopulations including TFH cells to promote HIV reactivation.
Concurrent microglial activation in CD8 T cell-mediated CNS vascular permeability during Theiler’s virus infection
Authors: Khadka Roman , Zheng Jiaying , Ayasoufi Katayoun , Jin Fang , Tritz Zachariah Peter , Wu LongJun , Johnson Aaron J
Published: January 2023

Immune-mediated blood brain barrier (BBB) disruption is a prominent feature of various neurological conditions for which an emerging role of CD8 T cells is being realized. Our laboratory has developed a unique model of CD8 T cell-mediated BBB disruption which employs a variation of the Theiler’s murine encephalomyelitis virus infection (TMEV) infection. Upon intravenous administration of VP2121–130 viral peptide during peak anti-viral adaptive response, Db:VP2121–130 epitope specific CD8 T cells induce BBB disruption in a perforin dependent manner. In this model, we addressed the role of microglia in tandem with CD8+ T cells. Using real time two-photon in vivo imaging, we demonstrate that microglia adopt distinct morphological features including enlarged cell body and fewer ramified processes as early as 6 hours post administration of VP2 peptide. Notably, perforin-deficient mice failed to display BBB disruption and had significantly diminished microglial expression of MHC-II and CD68 in this model. This study demonstrates CD8 T cell promote microglia activation through a perforin-dependent process during BBB disruption. Importantly, microglia are activated concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.
Biophysical characterization of the adhesion between enveloped virus and phosphatidylserine-binding proteins
Authors: Zhang Xiaohui , Cao Wenpeng , Jagota Anand
Published: January 2023

Enveloped viruses are viruses that have a lipid membrane. Many enveloped viruses, such as the emerging virus Ebola, are pathogenic and of great clinical importance. Recent studies have shown that the outer lipid leaflets of viral envelopes contain phosphatidylserine (PS), which serves as an important attachment factor for enveloped virus entry into host cells. The envelope PS interacts with cellular PS receptors in a manner that mimics apoptotic body-PS receptor interactions during the engulfment of dead cell debris by phagocytes. The PS receptors utilized by enveloped viruses for viral internalization are designated phosphatidylserine-mediated virus entry enhancing receptors (PVEERs). While the importance of PVEERs in mediating viral entry has been demonstrated, the biophysical properties of the PVEER-virus interactions, including the detailed kinetics of PVEER-virion-associated PS binding, have not been comprehensively evaluated and compared. Using Ebola virus (EBOV) as the model system, this study investigated and compared the interactions between model Ebola viral particles and PVEER family proteins that are known to mediate enveloped virus attachment and entry. We used single-molecule force spectroscopy to quantify the specific interaction forces between PS or model EBOV viral particles and PVEERs. Based on the dynamic force spectra of various PS-receptor interactions, our results suggest that PS-PVEER interactions are mechanically comparable to antibody-antigen interactions, and that Gas6 and MFG-E8 may generate more force-resistant intersections than TIM-1 and ProteinS. The findings can help explain the biophysical mechanisms underlying the PVEER-mediated entry of enveloped virus into cells.
Evaluation of miRNAs role in the immunopathogenesis of Microcephaly caused by ZIKV in experimental models
Authors: Polonio Carolina Manganeli , Brandão Wesley Nogueira , Cugola Fernanda , Zanluqui Nagela , Oliveira Lilian , Garcia Marilia , Braga Patricia , Peron Jean Pierre S
Published: January 2023

Viral infections have always been the cause of serious human diseases, usually increasing rates of morbidity and mortality worldwide. In 2015, the flavivirus Zika virus (ZIKV) caused an alarming increase in the number of babies born with microcephaly. Genetic differences, mainly related to immune response, are known to influence susceptibility to infection, once is very important in blocking viral replication and may be modulated by several different factors, in which, miRNAs play a key role. Still very little is known about the involvement of miRNAs during ZIKV infection. In this context, we evaluated the role of miRNAs during ZIKV experimental infection. We performed miRNAs analysis in vitro using CNS cells and, in vivo, with SJL animals susceptible to infection. Analyzing human neurons, we observed that ZIKV was able to reduce miR-9 and this reduction is known to cause cerebral cortex thickness reduction and ventriculomegaly, characteristics similar to microcephaly. In human neuronal precursor cells, ZIKV was able to reduce miR-194, miR-302b and miR-302c expression and consequently increased MXD1, a transcriptional repressor. On the other hand, ZIKV infection increased miR-295 and miR-302d expression leading to a reduction of AHR repressor and molecules involved in neurodevelopment, neuroD4, and neuroD6. In SJL fetal brain a miR-146 increase leading a decrease of IRAK1, favoring viral replication. Taken together, these data are indicative that ZIKV is able to modulate miRNA profile to regulate important molecules involved in neurodevelopment and antiviral immune response, evidencing the importance of these regulatory molecules, which may help us to better understand immunopathogenic mechanisms of microcephaly caused by ZIKV
The divergent evolution of cytomegalovirus-encoded CD48 homologs contributes to the expansion of the immunevasin repertoire
Authors: Angulo Ana , Martínez-Vicente Pablo , Farré Domènec , Engel Pablo
Published: January 2023

The genesis of gene families by capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a GPI-linked cell surface protein containing two extracellular immunoglobulin (Ig) domains. Via its N-terminal Ig domain, CD48 interacts with the cell surface receptor 2B4, modulating leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, derived from a common host CD48 ancestor gene acquired by retrotranscription. Recently, we examined one member of this family, A43, showing that it acts as a functional viral decoy receptor, impairing 2B4-mediated NK cell cytotoxicity. Here, we have characterized the rest of the vCD48s. We show that they are highly glycosylated type I transmembrane proteins which display remarkably distinct structures, biochemical properties, locations, and temporal kinetic classes. Molecular modeling of the N-terminal Ig domains of these vCD48s evidence significant changes in the number and length of their β-strands and inter-sheet loops that participate in the interaction of CD48 with 2B4, and accordingly we found that none of these molecules binds to 2B4. Interestingly, we determined that two of them, S30 and S31, have new targets in T or B lymphocytes. In particular, S30 tightly interacts with CD2, a T- and NK-cell adhesion and costimulatory molecule whose primary ligand is CD58. Thus, altogether our results show how a key host immune receptor captured by a virus can be subsequently remodeled during viral evolution to yield new molecules with shifted binding specificities to additional immune targets, expanding the repertoire of viral immunevasins.
Zika virus evolution in the presence of dengue virus-elicited cross-reactive immunity
Authors: Nava Jose Angel Regla , Wang Ying-Ting , Fontes-Garfias Camila R , Syed Thasneem , Gonzalez Andrew , Viramontes Karla , Valentine Kristen M , Terskikh Alexey V , Gleeson Joseph G , Shi Pei-Yong , Shresta Sujan
Published: January 2023

Zika virus (ZIKV), belongs to Flavivirus, causes neurological disorders including Guillain Barré syndrome and fetal microcephaly, and is responsible for widespread epidemics in countries where other flaviviruses, such as dengue (DENV), and yellow fever viruses, are endemic. However, how the cross-reactive, pre-existing immunity impacts the evolution of flaviviruses is poorly understood. To mimic the natural transmission cycle of ZIKV between vertebrate (human) hosts with pre-existing flavivirus immunity and invertebrate hosts (mosquito), ZIKV was subjected to 10 alternating cycles of passaging between DENV-immune mice and mosquito cells. We identified two mutations in the passaged ZIKV strains- one in the NS2B gene (I39V), both after passage in DENV-naïve and DENV-immune mice, and a mutation in the E gene (T470M), only after passage in DENV-immune mice. These mutations increase ZIKV virulence and pathogenesis in non-pregnant and pregnant Ifnar1−/− mice and abrogate cross-protection mediated by the pre-existing DENV immunity. In addition, ZIKV strain with the I39V mutation was more virulent than the parental ZIKV strain in human fetal neural progenitor cells (NPCs), and ZIKV variants in position 39 of the NS2B protein were observed in recent clinical isolates of ZIKV. Our data thus identify a mutational hotspot in the ZIKV NS2B protein and a mutation in the ZIKV E protein that arose as a consequence of the pre-existing immune pressure. Our results uncover the evolutionary strategies by which ZIKV can become pathogenic in both DENV-naïve and DENV-immune populations. Mechanistic studies of evolution in the presence and absence of cross-reactive immunity will benefit the development of safe and efficient flavivirus vaccines.
Features of Neuropsychiatric Lupus in Nba2 mouse model
Authors: Browne Kim , Zhang Emily , Sullivan James , Evonuk Kirsten Scarlett , Liu Jessica , DeSilva Tara M , Jorgensen Trine N
Published: January 2023

Background

Neuropsychiatric lupus (NPSLE) is a poorly understood manifestation of the autoimmune disease systemic lupus erythematosus (SLE). NPSLE is characterized by symptoms including anxiety and depression, and recent data have suggested a pathogenic role for interferon-alpha (IFNα). Additionally, IFNα can diminish the production of the neurotransmitter serotonin, suggesting a causative link between IFNα and NPSLE. B6.Nba2 mice develop SLE in an IFNα-dependent manner making it a valuable model for the study of NPSLE.

Methods

Age-matched female B6.Nba2 and B6 mice were tested. Behavioral studies included open field testing (OFT), elevated plus maze (EPM), and fear conditioning (FC) tests for detection of anxiety-like behavior, and forced swim test (FST) for detection of depression-like behavior. Serum serotonin and dopamine levels were measured by ELISA and serotonin receptors were enumerated by IHC.

Result

Results from both OFT (p=0.001), EPM (p<0.05) and FC (p<0.05) tests supported a strong anxiety phenotype in B6.Nba2 female mice, while data from FST (p<0.05) suggested a depressive phenotype as well. B6.Nba2 females also showed significantly less serum serotonin (p<0.0001) and dopamine (p<0.05) levels than age-matched B6 females.

Conclusion

Our data suggests that the B6.Nba2 mouse model of SLE expresses an anxiety and depressive phenotype resembling NPSLE. Along with our previous data showing that IFNα levels are upregulated and required for disease development, our results suggest a positive correlation between IFNα, reduced serotonin, and an anxiety phenotype. Current studies are evaluating the effect of genetic IFNα-receptor manipulation in brain cells and the direct relationship between IFNα and NPSLE.
T-cells from NOD-PerIg mice transfer aggressive peripheral neuritis to NOD. scid recipients
Authors: Racine Jeremy J , Chapman Harold D , Doty Rosalinda , Cairns Brynn M , Hines Timothy J , Tadenev Abigail L.D. , Anderson Laura C , Green Torrian , Dyer Meaghan , White Jacqui , Ettinger Rachel , Burgess Robert W , Serreze David V
Published: January 2023

Recently, we created NOD mice transgenically expressing a peripherin reactive B cell receptor (BCR) inducing rapid onset T1D (NOD-PerIg). We unexpectedly discovered that in addition to T1D, NOD.scid recipients receiving transferred NOD-PerIg T-cells or whole splenocytes developed a visible hind leg weakness caused by aggressive limb neuritis. Neurobehavior phenotyping failed to uncover any measurable clinical metric that could predict onset of visible hind leg symptoms. Nerve conduction velocities were measured on hind limbs in mice that made it to the critical 15-weeks post transfer window, and revealed that most mice with clinical symptoms of neuritis had little to no discernible nerve conduction, while those without clinical symptoms of neuritis had near normal NOD.scid-like levels of nerve conduction. A longitudinal flow cytometry analysis of sciatic nerves at onset of diabetes, onset of visible hind leg symptoms, or at the end of incidence revealed that mice with clinical symptoms of neuritis had increased CD45+ cells amongst live cells compared to controls, and increased T-cells amongst CD45+ cells compared to both controls and mice without clinical symptoms. Depletion of either CD4 or CD8 T-cells after transfer of whole NOD-PerIg splenocytes led to the complete prevention of diabetes development. Interestingly, CD4 but not CD8 T-cell depletion led to the absence of any clinical manifestation of neuritis by 20 weeks post transfer. Finally, we have observed lymphocyte infiltration in both primary NOD-PerIg and NOD mice, likely explaining the occasional NOD mouse that has developed neuritis in The Jackson Laboratory’s extensive NOD collection.
IL-27-producing B cells synergize with IFNγ to promote IgG2a and IgG1 antibody responses
Authors: Yan Hui , Wang Rui , Wang Jingwei , Wu Shuai , Fernandez Maria , Rivera Carlos Eduardo , Moroney Justin B , Li Xiaodong , Zhang Nu , Zan Hong , Meng Xiangzhi , Zhang Fushun , Kedl Ross , Min Booki , Hunter Christopher A , Xiang Yan , Casali Paolo , Xu Zhenming
Published: January 2023

How B cells integrate innate receptor and adaptive T cell signals in the class-switched antibody response needs be better understood. By using cytokine gene knockout and reporter mice, in vitro B cell cultures and single-cell analyses, we have identified a subset of B cells that produces IL-27 as signature cytokine upon priming by TLR ligands, which up-regulate CD40 expression, and exposure to Tfh cell stimuli CD154 and IL-21. B cell IL-27 induction depends on NF-kB activated by linear ubiquitin assembly complexes and up-regulated Baft3 transcription factor, which are recruited to the Il27p28 locus, as made accessible by CD154 and IL-21 stimulation. IL-27-producing B cells are bystander B cells that are inefficient in secreting antibodies, but express high levels of adhesion molecules which engage other activated B cells. Together with IFNg, they boost proliferation, survival and class-switching to IgG2a of B cells stimulated with CD154 and IL-21. Accordingly, mice with B cell-specific ablation of IL-27 and IFNg receptors display severe defects in germinal center B cell and plasma cell differentiation, leading to abrogation of IgG2a and markedly reduced IgG1 responses. Thus, IL-27-producing B cells function as “helper” cells to promote differentiation of antigen-specific B cells for optimal IgG responses. As such, they play a critical role in the production of effective anti-viral antibodies and, possibly, modulation of anti-tumoral immunity.
Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α
Authors: Alrashed Fatema , Ahmad Zunair , Thomas Reeby , Alghaith Abdulwa , Melhem Motasem , Ahmad Rasheed
Published: January 2023

Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase 2 (nSMase2; product of the sphingomyelin phosphodiesterase 3 gene, SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether the nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase2 activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of IL-1b and MCP-1. However, inhibition aSMase activity did not attenuate CD11c expression and secretion of IL-1b and MCP-1. Phosphorylation of JNK, p38 and NF-κB resulting from TNF-α stimulation was also attenuated by the inhibition of nSMase. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. Our human data show that SMPD3 gene expression was not only found to be elevated in obese individuals but also positively corelate with TNF-α elevated expression. These findings indicate that nSMase acts, in a part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.
Elevated Interferon Alpha in the Serum of Laboratory Strains of Mice and Porcine Serum by ELISA
Authors: DeJesus Elfie , Schwartz Barbara , Kisiel Greggory , Clark William , Lavoie Thomas
Published: January 2023

We examined Interferon (IFN)-Alpha and -Beta levels in pooled sera and individual bleeds from C57/Bl, Balb/C and CD-1 mice. 20 Individuals from each strain from two independent colonies were evaluated in commercial assays. The IFN-Beta ELISA detected IFN in 1 of the 60 individuals while the IFN-Alpha assay which detects all 14 subtypes measured IFN in 13 of 60. 5 individuals exhibited greater than 4 pg/ml of IFN-Alpha detected. Specificity was demonstrated by knockdown of the signal using non-biotinylated detection antibody. 14 of 54 porcine sera samples from 4 independent sources were positive for IFN alpha by an ELISA under development, and similar knock-down experiments suggest specificity. This is in contrast to human sera/plasma samples where less than 3% of healthy donor samples have quantifiable IFN-Alpha or -Beta above 0.5 pg/ml. This study may raise questions about the regulation of the IFN system in different species and the potential of these differences to affect experimental results in a variety of models.
Adarza’s ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system
Authors: Schmuke Jon J , Mueller Ernie , Carter Alun , Cooper Matt , Kabakibi Ayman
Published: January 2023

We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. The flexibility of this system can also reduce the need to generate a series of sample dilutions in order to get results in a limited detection range by enabling the development of each assay with sensitivity expectations for each target on a chip. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.