bluuebunny/crossref_metadata_2025_split huggingface dataset:
Non-cellular mechanism of peripheral immunosuppression as a novel feature in experimental glioblastoma
Authors: Khadka Roman H. , Ayasoufi Katayoun , Jin Fang , Malo Courtney Sarah , Desai Nidhi , Johnson Aaron J. 
Published: January 2023

Abstract
Peripheral immune suppression is a hallmark of highly malignant Glioblastoma multiforme (GBM). Given that effective immunotherapy for GBM relies on a functional immune system, it is crucial to define the underlying mechanism of peripheral immunosuppression. We therefore evaluated immunosuppression using the GL261 model of GBM. We first examined cytokine production by microglia and brain-infiltrating lymphocytes for immune suppressing factors. We observed relatively unchanged levels of IL-10 and TGF-β in glioma bearing mice compared to controls. Therefore, we next investigated cellular components of the blood for evidence of suppression. However, we did not observe enhanced frequencies of Tregs (Foxp3+ CD4+) in tumor bearing mice. Finally, we analyzed cytokine levels in the serum of mice harboring gliomas compared to glioma negative animals. This assay revealed fluctuations in chemokine and cytokine levels between these two groups, prompting the analysis of a potential serum derived factor in promoting immunosuppression. We therefore evaluated T cell activation and proliferation in the presence of serum isolated from glioma harboring mice and healthy controls. CFSE-labeled T cells stimulated with CD3/CD28 beads in the presence of glioma negative mouse serum had normal proliferation. In contrast, CD4+ and CD8+ T cell proliferation was completely inhibited when cultured with serum from glioma bearing mice. These studies demonstrate the importance of a serum derived factor in mediating immunosuppression. Defining and inhibiting this circulating factor could be the first step towards improving immunotherapy strategies for GBM.
Identification of dysfunctional CD8+ T-cell subsets rescued by PD-L1 blockade in the tumor microenvironment
Authors: Yamauchi Takayoshi , Hoki Toshifumi , Odunsi Kunle , Ito Fumito 
Published: January 2023

Abstract
While depletion studies using mice clearly show that CD8+ T cells are of crucial importance in anti-tumor efficacy, gaps in understanding the exact mechanisms that regulate CD8+ T cell responses in the tumor microenvironment (TME) are a major impediment in efforts to improve immunotherapy. We have evaluated phenotypical and functional heterogeneity of tumor-infiltrating lymphocytes (TILs) after adoptive transfer of ex vivo primed Pmel-1 T-cell receptor transgenic CD8+ T cells specific for the gp100 antigen expressed on B16 melanoma. We found that the chemokine receptor, CX3CR1 identifies distinct effector CD8+ T-cell subsets, CX3CR1 negative (−), intermediate (int), and high (hi) in the TME. A CX3CR1hi subset contained terminally-differentiated CD8+ T cells that express higher levels of killer-cell lectin like receptor G1 (KLRG1), TNF-related apoptosis-inducing ligand (TRAIL), perforin, and granzyme A. Significantly more CX3CR1int CD8+ T cells expressed CD25 compared to other subsets, suggesting this is the subset that is rapidly proliferate and generate terminally-differentiated T cells. Unexpectedly, despite their terminally differentiated status, a CX3CR1hi subset expressed significantly lower levels of co-inhibitory receptors, PD-1, LAG3, and TIGIT compared to CX3CR1− and CX3CR1int subsets in the TME. In line with this, proliferation and cytokine production of CX3CR1− and CX3CR1int subsets were significantly decreased in the TME compared to a CX3CR1hi subset. Importantly, functional burst after PD-L1 blockade came almost exclusively from CX3CR1− and CX3CR1int subsets. These findings provide a better understanding of dysfunctional CD8+ TILs and can be exploited to develop more effective cancer immunotherapy.
Inhibition of intrinsic coagulation improves immune checkpoint-targeted gene delivery of cationic solid lipid nanoparticles
Authors: Sun Tianmeng , Wu Xuan , Yang Yong Guang 
Published: January 2023

Abstract
Tumor-targeted delivery of CRISPR/Cas9 plasmid or siRNA to inhibit immune checkpoint protein expression in tumor cells has the potential to improve anticancer immune responses. Cationic solid lipid nanoparticles (cSLNs) are promising carrier for the delivery of negatively charged nucleic acids. cSLNs with high surface charge and low PEG density have been demonstrated to enhance transfection efficiency in vitro, but for unknown reasons fail to improve gene delivery in vivo. We prepared 9 kinds of cSLNs with varying surface charge and PEG density and detected the interactions of these cSLNs with blood cells. We found that increasing surface charge and/or reducing PEG density present a risk for systemic platelet aggregation and activation. Thrombosis was clearly detected in liver, lung and kidney from mice treated with cSLNs with high surface charge and low PEG density. Mechanistic studies revealed that the intrinsic coagulation pathway was responsible for cSLN-induced thrombus formation. Furthermore, pretreatment of recipient mice with heparin, a clinically-approved intrinsic coagulation inhibitor, was highly effective in preventing toxicity, prolonging the circulation time, improving tumor accumulation and enhancing the tumor cell uptake in tumor-bearing mice. These results indicate that the thrombotic toxicity of cSLNs is an important factor affecting their drug delivery efficiency in vivo, and that heparin treatment offers a useful strategy for improving cSLN-based cancer immunotherapies.
Porcine SIRPA binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient (SCID) pigs
Authors: Boettcher Adeline N. , Cunnick Joan E. , Powell Ellis J. , Egner Timothy K. , Charley Sara E. , Loving Crystal L. , Tuggle Christopher K. 
Published: January 2023

Abstract
Xenotransplantation success with human hematopoietic stem cells into non-obese diabetic (NOD)-derived severe combined immunodeficient (SCID) mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47 (hCD47). Interaction between NOD mouse SIRPA and hCD47 results in higher phagocytic tolerance compared to other SCID mouse strains. In the context of developing a humanized SCID pig, we investigated whether porcine SIRPA binds hCD47 and subsequently inhibit phagocytosis of human cells. Porcine SIRPA bound to hCD47 in vitro. Porcine monocytes phagocytosed human and porcine red blood cells (RBCs) at significantly lower levels than mouse RBCs. When human RBCs were pre-incubated with CD47 blocking antibody B6H12 or non-blocking antibody 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity requires interaction between porcine SIRPA and hCD47. Collectively, we provide the first evidence that porcine monocytes may be phagocytically tolerant towards human cells, suggesting that porcine SCID models have the potential to support engraftment of human stem cells.
Analysis of T cell antigen receptor expression by porcine natural killer T cells
Authors: Yang Guan , Artiaga Bianca Libanori , Jayaprakash Anitha , Sachidanandam Ravi , Driver John 
Published: January 2023

Abstract
Invariant natural killer T (iNKT) cells are an innate-like T cell subset expressed by some, but not all mammals. Unlike conventional T cells, iNKT cells express a semi-invariant T cell receptor (TCR) (Vα24-Jα18/Vβ11 in human and Vα14-Jα18/Vβ8.2, Vβ2, or Vβ7 chains in mice) that recognizes glycolipid antigens presented by the non-polymorphic molecule CD1d. Once activated, iNKT cells generate an extensive array of immune responses that may be useful for treating cancer, autoimmunity and infectious disease in humans. In addition, there may be potential to target iNKT cells for veterinary applications. Pigs express iNKT cells that can be detected using mouse or human CD1d-tetramer reagents loaded with the prototypic iNKT cell ligand, α-galactosylceramide (α-GalCer). We exploited this phenomenon to define the TCR repertoire of iNKT cells (CD3α+CD1d tetramer+) compared to conventional T cells (CD3α+CD1d tetramer−) sorted from the peripheral blood of commercial mixed-breed pigs. We found that iNKT cells are highly enriched for the α-chain and CDR3α loop from Vα87*-Jα60* that possess a high level of amino acid sequence homology with human Vα24 (85%) and Jα18 (86%) as well as mouse Vα14 (74%) and Jα18 (79%). Particularly striking was that the most abundant CDR3α sequence was a near perfect match for the human invariant CDR3α. Porcine iNKT cells were also biased for Vβ84* which is homologous to Vβ11 (74%) in humans, as well as Vβ8.2 (71%) and Vβ2 (80%) in mice. Our findings indicate that the porcine iNKT cell TCR is highly homologous to mouse and particularly human iNKT cell TCR, which suggests that iNKT cells among these species play a similar role in immunity and disease.
Characterization of the immune reagent chicken IL-16
Authors: Grant Ar’Quette , Kim Woohyun , Lillehoj Hyun S. 
Published: January 2023

Abstract
Interleukin-16 has been characterized as a pro-inflammatory cytokine that mediates an immune response in human and mouse monocytes and peripheral blood mononuclear cells (PBMC), and it plays a role in proliferating B-cells and mylomas. The function of chicken IL-16 ortholog (ch-IL-16) is far less understood and only shares 52% and 49% sequence homology with human and mouse, respectively. Although it has previously shown chemotaxis abilities in chicken splenocytes, the low sequence homology to their mammalian counterparts, the current study was undertaken to characterize the function of yeast-expressed ch-IL-16 as an immune reagent in pro-inflammatory cytokine production and cell proliferation. In a dose dependent manner, ch-IL-16 was applied to 5 × 106 cells/mL PBMC and HD11 chicken monocytes, and the extracted RNA was used to determine the pro-inflammatory cytokine production. Also in a dose dependent manner, ch-IL-16 was applied to primary bursal lymphocytes, PBMC, and RP-9 chicken myeloma cells. Ch-IL-16 produced pro-inflammatory cytokines: IL-6, IL-1β, and TL1A which is the chicken equivalent to TN1-α at an optimal concentration between 40 and 50 ng/mL. We are still in the process of analyzing the proliferation effects of ch-IL-16 on bursal lymphocytes, PBMC and RP-9. Future directions also include the production of monoclonal antibodies to neutralize the function of ch-IL-16. These results would be useful in the further characterization of the chicken immune system, and the role of ch-IL-16 as a mediatory cytokine.
Resistance of field-isolated porcine epidemic diarrhea virus to interferon and neutralizing antibody
Authors: Park Jung-Eun , Shin Hyun-Jin 
Published: January 2023

Abstract
Porcine epidemic diarrhea virus (PEDV) infects pigs and causes enteric disease characterized by watery diarrhea and dehydration. Despite the extensive vaccination, PEDV strains with variations in the spike gene have been reported in several countries, including Korea, China, and the United States. Variant PEDV belonging to genogroup G2 has higher pathogenicity and morbidity than conventional PEDV belonging to genogroup G1. To understand the pathogenesis of the variant PEDV, we examined the susceptibility of the variant PEDV to interferon and PEDV specific neutralizing antibodies, which are known to important for the control of PEDV infection. We found that variant PEDV (WG strain) showed higher resistance to interferon than conventional PEDV (KPEDV-9 strain). Similarly, KPEDV-9 strain grew well in interferon-deficient Vero cells but did not grow in interferon-releasing porcine alveolar macrophages, whereas WG strain showed similar infectivity in both cells. We also found that WG strain is not blocked by the neutralizing antibodies against KPEDV-9, suggesting the differences in the antigenicity of two strains. To elucidate the possible mechanism of resistance to interferon and neutralizing antibody in variant PEDV, sequencing analysis was performed. We found huge variations in spike and ORF3 proteins. Our results indicate that the recently introduced variant PEDV exhibits higher resistance to the interferon than the existing PEDV and avoids the neutralizing antibody against the existing PEDV. These results also provide understanding of the occurrence of variant PEDV and its pathogenesis.
Identification of a DRB3*1101-restricted CD4 T cell response against bovine respiratory syncytial virus
Authors: Hofstetter Amelia R. , Yadav Akshay , Norimine Junzo , Sacco Randy E. 
Published: January 2023

Abstract
A better understanding of the immune response elicited by bovine respiratory syncytial virus (BRSV) vaccines is needed for vaccine improvement. Although killed-BRSV vaccines are available as part of multivalent products, their efficacy is controversial. We screened for BRSV-specific T cell responses in cows with a history of annual vaccinations. We defined the minimum BRSV F peptide 261–270 (INDMPITNDQ), which elicits IFN-γ from CD4 T cells in four of fourteen cows tested. The response is restricted by the bovine class II MHC molecule DRB3. All four cows share the DRB3*1101 allele. We are currently developing a specific DRB3*1101:INDMPITNDQ tetramer to facilitate further characterization of this response.
Host antigen modulated anti-influenza immunity with antigen level dependent distinct mechanisms of severe influenza
Authors: Huang Ching-Tai , Dutta Avijit , Huang Chi-Wei , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin , Chen Ting-An 
Published: January 2023

Abstract
Some healthy individuals suffer from severe disease and even mortality with the same strain of influenza virus that causes self-limited illnesses in the majority of people. This fact exemplifies the importance of host factors in influenza disease manifestation. Our C3-HA transgenic mice express hemagglutinin (HA) as a self-antigen that is identical to the HA of PR8 influenza virus. Compared to wild type mice, C3-HA mice incurred severe disease with PR8, but not with HA-mismatched influenza virus. Self-HA in C3-HA mice altered the immune response to the infection and impaired clearance of the influenza virus. However, different mechanisms contribute for exacerbated disease in C3-HA mice with different level of HA. In C3-HALow mice, low-level self-HA modulated HA specific immunity upon infection with less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both HA-specific CD4+ and CD8+ T cells on day 4- post infection. The T cell activation status contracted further on day 7- post infection. Impaired immunity resulted in delayed virus clearance leading to severe disease and death. In C3-HAHigh mice, high-level self-HA also modulated impaired immunity and virus clearance on day 4- post infection. In contrast to further reduction on day 7- post infection in C3-HALow mice, the HA specific T cell immunity was higher on day 7- than day 4- post infection in C3-HAHigh mice. The boosted HA specific immunity resulted in severe disease with autoimmunity as a predominant feature in pathology. Our animal model exemplified the complicated interaction between cross-reactive self-antigen and influenza virus with severe disease as the results of antigen level dependent distinct mechanisms.
Single-cycle respiratory syncytial virus infection induces robust adaptive immune responses and reduces disease severity in mice
Authors: Schmidt Megan E. , Oomens Antonius G.P. , Varga Steven M. 
Published: January 2023

Abstract
Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. However, despite extensive efforts, there remains no licensed RSV vaccine. Recently, a recombinant infectious RSV strain lacking the gene for the matrix (M) protein (M-null RSV) was developed as a potential vaccine candidate. The M protein is required for reassembly of the virion following infection and gene expression within a host cell. Infection with M-null RSV produces viral proteins but does not result in the generation of infectious virus progeny resulting in a single-cycle infection. To determine its immunogenicity in vivo, mice were infected with either M-null RSV or a wild-type (WT) RSV. M-null RSV-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared to mice given WT RSV. Remarkably, M-null RSV infection induced CD4 and CD8 T cell responses in the lung and airways of similar magnitude to that of a WT RSV infection at acute time-points. Germinal center B cell numbers and serum antibody levels were also similar between M-null and WT RSV-infected mice. M-null RSV infection also generated robust memory immune responses. A substantial proportion of B cells, CD4 T cells, and CD8 T cells remained within the lung tissue of M-null RSV-infected mice 30 days post-infection. Importantly, RSV-specific memory CD8 T cells expressing CD69 and CD103, the canonical markers of tissue resident memory, were also generated by M-null RSV infection. Given the reduced disease severity and robust immune responses developed following single-cycle infection, M-null RSV may be an effective vaccine candidate.
Intranasal infection by MCMV reveals key roles for viral evasion of MHC-I to enable viral spreading and CD4 T cell help to promote functional CD8 T cell responses
Authors: Zhang Shunchuan , Snyder Christopher M. 
Published: January 2023

Abstract
Natural Cytomegalovirus (CMV) infection occurs primarily through the oral/nasal mucosa. Recent evidence from murine (M)CMV infection suggested that MCMV is spread to salivary gland (SG) by dendritic cells after intranasal (i.n.) inoculation. In agreement with this, a recombinant MCMV, which cannot replicate in hematopoietic cells, could not spread to SG after i.n. infection. However, the mechanisms by which infected cells evade immune control while spreading the virus are unclear. In fact, previous work suggested that deleting all three genes blocking MHC-I antigen presentation results in only a subtle defect in total latent viral loads and SG replication in vivo. Surprisingly, we found that viral evasion of MHC-I antigen presentation was critical for MCMV to replicate persistently in nasal mucosa and spread to SG after i.n. inoculation, in contrast to intraperitoneal (i.p.) infection. Remarkably, depletion of CD4 T cells reversed this immune restriction on viral spreading, enabling even MCMV lacking all evasion genes to replicate in nasal mucosa and spread to SG after i.n. infection. This was not due to a direct anti-viral effect of CD4 T cells as delaying CD4 T cell depletion until day 7 post infection restored immune control of MCMV at later times. Instead, priming and expansion of functional CD8 T cells was impaired by lack of CD4 T cells after i.n. infection, in contrast to i.p. infection. Thus, intranasal inoculation revealed a critical role for CD4 T cell help in the promotion of protective CD8 T cell responses. Moreover, our data suggest since MCMV spreads only in infected hematopoietic cells after i.n. infection, viral evasion of MHC-I is necessary to protect CD8 T cells from killing infected cells carrying MCMV.
Elucidating the role of HLA-DO/H2-O in modulating the diversity of the MHC Class II self peptide repertoire
Authors: Nanaware Padma Pandurang , Jurewicz Mollie M. , Leszyk John , Shaffer Scott , Stern Lawrence J. 
Published: January 2023

Abstract
The classical MHC Class II molecules HLA-DP, DQ and DR proteins bind antigenic peptides in the endosomes of APCs and present them to CD4+ T cells whereas HLA-DM and DO are the non-classical proteins. DM catalyzes the exchange of CLIP peptides with other endosomal peptides whereas DO acts as an inhibitor of DM activity. The clear biochemical effect of DO in inhibiting DM is reported but its effect on the peptide repertoire is still not clear. We used Quadrupole Orbitrap mass spectrometry for characterizing peptides presented by the MHC Class II protein DR1 from CRISPR/Cas9 deleted LG2 cells. We identified 6139 peptides in wild type cells (WT) but only 5227 peptides from DO−/− cells (DO-KO). The label free quantitation analysis of WT Vs DO-KO showed DM sensitive CLIP peptides were present in lower abundance in DO-KO, consistent with cell surface staining experiments. We analyzed peptide diversity in the eluted peptidome using diversity measures like Shannon’s Entropy, Simpson’s diversity and Berger-Parker Index. In each case the diversity was higher for the WT peptidome as compared to DO-KO. We screened several peptides for binding affinity and DM susceptibility by analyzing their relative abundances in WT and DO-KO. Peptides from group WT &gt; DO-KO, WT only showed lower binding affinity and high DM sensitivity as compared to peptides in DO &gt; WT, DO only. The same phenomenon was observed for peptides eluted from I-Ab purified from B cells and thymus of BL/6 as compared to H2-O−/− mice. These results indicate that DO/H2-O broadens the peptide repertoire whereas its absence restricts the repertoire resulting in presentation of fewer different epitopes, and highlights the role of DO in regulating the MHC-II peptide repertoire by modulating DM peptide editing.
SMAD4-deficient Dendritic Cells in the Murine Lung shows an Activated Phenotype
Authors: Root Sierra Hope , Menoret Antoine , Cauley Linda S. 
Published: January 2023

Abstract
SMAD4 is a signaling intermediate that acts downstream of the TGFb receptor. This signaling pathway is known to regulate expression of the Itgae gene (CD103) in activated CD8 T cells. Whether SMAD4 plays a role in induction or maintenance of CD103+ dendritic cells (DCs) has not been investigated. To address this question, we crossed SMAD4 floxed mice with Cre under the control of the CD11c promoter. DCs in the lung, spleen and bone marrow of naïve CD11c-SMAD4 mice were analyzed by multi-parameter flow cytometry. The lungs of Smad4-deficient mice contained smaller numbers of CD11c+ cells than control animals, and the remaining cells expressed costimulatory molecules and CD11b at high levels. Lung suspensions were metal barcoded for time of flight cytometry (cyTOF) and viSNE maps were contracted using 25 parameters. Under these homeostatic conditions, Smad4-abalation caused a dramatic decrease in CD11c+ DCs with activation of pSTAT5, when compared to DCs from littermate controls. Activation of pSTAT5 in KO was specific to CD11c+ DCs as no difference was seen in other cell types including T cells. Our data indicate that the lungs of SMAD4-CD11c KO mice were depleted of migratory DCs, which migrated to the draining lymph nodes after spontaneous maturation. We currently are exploring whether DC-depletion undermines cell mediated immunity to respiratory virus infection.