bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of CD91 in HSP-mediated innate immune responses (101.29)
Authors: Pawaria Sudesh , Binder Robert 
Published: January 2023

Abstract
Immunogenic Heat shock proteins (HSPs) are known to chaperone antigenic peptides and elicit strong immunity against tumors and infectious agents. The peptide bound form of the HSP, gp96, interacts with the antigen presenting cells (APCs) through the receptor, CD91 (LRP1), resulting in endocytosis of the complex and the presentation of the peptides on MHC I and MHC II to T-cells. Independent of the peptides, the binding of HSPs to the APCs results in the translocation of NF-kB into the nucleus and the elaboration of a number of pro-inflammatory cytokines. Binding of gp96, HSP70 or calreticulin to CD91 results in the phosphorylation of its beta-chain, association with the Shc adaptor molecule and activation of NF-kB. By selective mutation of the two phosphorylation sites of CD91, we observed that HSPs appear to utilize different sites resulting in distinct downstream signaling pathways. The cytokine profiles resulting from these signals are unique. These data shed light on the responses of CD91-expressing antigen presenting cells to immunogenic HSPs released from dying cells or introduced by vaccination.
Diphenyl difluoroketone (EF-24) modulates dendritic cell responses. (101.30)
Authors: Vilekar Prachi , Awasthi Vibhudutta , Lagisetty Pallavi , Natarajan Aravindan , Awasthi Shanjana 
Published: January 2023

Abstract
Dendritic cells (DCs) are the most potent antigen-presenting cells that link the antigen-specific innate host responses with adaptive immunity. They recognize pathogens, pathogen-ligands and inflammatory signals via pattern recognition receptors. Inflammatory signals induce DC maturation and release of cytokines, chemokines and chemical mediators. We synthesized EF24, a known more potent analog of curcumin, and studied its immunomodulatory properties using immortalized murine DCs (JAWS II). We hypothesized that pharmacological downregulation of proinflammatory pathway by EF24 may suppress lipopolysaccharide (LPS)-induced phenotypic and functional changes in DCs. Murine bone marrow-derived JAWSII DCs were incubated with 1-10 µM EF24 with or without LPS (100 ng/ml). We investigated cell viability, morphology, expression of DC-specific markers and TLR4, NF-κB activity and cytokines. The short- and long-term treatment with EF24 suppressed the LPS-induced expression of DC-markers (CD80, CD86, MHC class II). Immunohistochemical expression of TLR4 in the cells was significantly reduced. Electrophoretic-mobility shift assay showed inhibition of LPS-induced NF-κB activation. EF24 also reduced the expression of IL-6, TNF-α and MCP-1 in DCs. There was no effect of EF24 on viability of DCs in the concentrations used. Overall, our results suggest that the immunomodulatory properties of EF24 may be of therapeutic value in inflammatory conditions, such as septic shock.
Amplifying TLR-MyD88 signals within tumor-specific T-cells enhances antitumor activity to low concentrations of subdominant tumor-antigens (101.35)
Authors: Davila Eduardo , Geng Degui , Srivastava Ratika , Riker Adam , Markovic Svetomir 
Published: January 2023

Abstract
There is a dire need to understand the cellular signals that can potentiate T-cell responses to weakly immunogenic tumor antigens (TAg) and to tumors expressing suboptimal levels of these antigens. We examined the anti-tumor activity and survival of TLR2-MyD88-stimulated tumor-specific CD8 T-cells derived from melanoma patients and TAg-specific T-cell receptor transgenic pmel mice. TLR2 engagement on pmel CD8 T-cells, but not on TLR2-/-pmel or MyD88-/-pmel T-cells, reduced the activation threshold to a subdominant TAg, resulting in increased production of effector molecules and cytotoxicity. Wild-type or MyD88-/- mice treated with pmel T-cells and TLR2 ligand, but not TLR2-/-pmel or MyD88-/-pmel T-cells, showed significant tumor regression of an established melanoma tumor. Over-expressing TLR2 in pmel T-cells eradicated established tumors and four-times fewer cells were needed to generate anti-tumor responses. The enhanced anti-tumor activity was associated with improved survival and increased effector function of TLR2-MyD88-stimulated T-cells. Activating TLR-MyD88 signals in patient-derived T-cells reduced the activation threshold to several weakly immunogenic TAgs, resulting in increased cytokine production, expansion and cytotoxicity. These data highlight the physiological importance of activating TLR-MyD88 signals within tumor-reactive T-lymphocytes for enhancing their longevity and augmenting effector function against suboptimal levels of weakly-immunogenic antigens.
Vascular Disrupting Agent, DMXAA, Enhances Therapeutic HPV Vaccine-Induced CTL responses and Antitumor Effects (101.37)
Authors: Zeng Qi , Peng Shiwen , Monie Archana , Hung Chien-Fu , Wu T.-c 
Published: January 2023

Abstract
Antigen-specific immunotherapy has emerged as an attractive approach for the treatment of cancers. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 peptide-based vaccination for their ability to enhance the E7-specific CD8+ T cell immune responses and antitumor effects in treated mice. We found that the combination of DMXAA treatment with E7 long peptide (aa43-62) vaccination mixed with polyI:C generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes and tumor-infiltrating lymphocytes (TILs) of tumor bearing mice. Furthermore, we found that the DMXAA-mediated enhancement of E7-specific CD8+ T cell immune responses was dependent on the timing of administration of DMXAA and was also applicable to other antigen-specific vaccines. We further tested the combination of DMXAA with E7 peptide vaccination in a vaginal TC-1 tumor growth model and observed a significant enhancement in the antitumor effects in treated mice. Taken together, our data demonstrated that administration of the vascular disrupting agent, DMXAA, enhances therapeutic HPV vaccine-induced CTL responses and antitumor effects against E7-expressing tumors in two different locations. Our study has significant implications for future clinical translation.
Programmed Death Ligand-1 expression on the cell surface is regulated by costimulatory molecule CD80 expressed by cancer vaccines (101.4)
Authors: Haile Samuel , Bosch Jacobus , Agu Nnenna , Ostrand-Rosenberg Suzanne 
Published: January 2023

Abstract
Programmed Death Ligand 1 (PDL1) is a major obstacle in the development of cancer immunotherapies. PDL1 is constitutively expressed or induced by IFNγ on many tumor cells, resulting in the prevention of T cell activation or the apoptotic cell death of activated T cells. Cell-based vaccines previously developed in our lab were made by modifying tumor cells to express Major Histocompatibility Complex Class II (MHCII) and costimulatory B7-1 (CD80) molecules. These cell-based vaccines do not express PDL1, even after IFNγ stimulation. As determined by flow cytometry using three anti-PDL1 monoclonal antibodies that detect distinct PDL1 epitopes, and human lung, uveal and cutaneous melanoma, and breast cancer cells, the over-expression of CD80 blocks constitutive and IFNγ-induced cell surface expression of PDL1. Quantitative analysis of CD80 and PDL1 expression indicates an inverse relationship between PDL1 and CD80. CD80+ tumor cells contain intracellular PDL1, as assessed by flow cytometry, suggesting that CD80 regulates PDL1 at the protein level. Tumor cells transfected with CD80 constructs lacking a cytoplasmic domain or with an irrelevant non-signaling cytoplasmic domain, retain the ability to block PDL1 expression at the cell surface indicating that CD80 does not modulate PDL1 transcription. These results suggest that in addition to serving as a costimulatory molecule, CD80 facilitates T cell activation by blocking coinhibitory signals and preventing T cell apoptosis.
Ovarian tumor infiltrating lymphocytes B have pro angiogenic properties (101.7)
Authors: Nunez-Cruz Selene , Guerra Matthew , Connolly Denise , Coukos George , Gimotty Phyllis , Scholler Nathalie 
Published: January 2023

Abstract
Despite extensive studies of the role of tumor-infiltrating lymphocytes-T and macrophages, the role of tumor-infiltrating lymphocytes-B (TIL-B) during cancer progression remains poorly understood. To address this issue, we characterized the phenotype and function of TIL-B in a transgenic mouse model of epithelial ovarian cancer that resembles human serous ovarian cancer. We first observed that CD45+ CD19+ TIL-B cells are one of first populations to infiltrate ovarian tumors and their percentages decreased over time, from 32±5% at 13 weeks to 16±3% at 26 weeks. Our microarrays results revealed that TIL-B transcripts segregated with normal splenocytes, and significantly up-regulated Angiopoietin2, platelet derived growth factor (PDGF), Midkine, Insulin-like growth factor binding proteins and Transforming growth factors beta. In addition, multiplex cytokine assays showed that TIL-B spontaneously secreted PDGF-bb, CXCL9, Leukemia inhibitory factor and Macrophage-colony stimulating factor. Finally, we found that TIL-B secreted the suppressive cytokine IL-10 and attracted endothelial cells in vitro. Our results demonstrated for the first time that TIL-B beared different functional capabilities than normal B cells, in particular TIL-B synthesized angiogenic factors and attracted endothelial cells. These results suggest that TIL-B could promote angiogenesis and thus represent an attractive therapeutic target for solid tumor therapy.
Exosome-mediated MHCII cross-presentation substantially enhanced by the receptor binding activity of influenza hemeagglutinin (130.10)
Authors: Testa James , Apcher Geraud , Eisenlohr Laurence 
Published: January 2023

Abstract
T cell responses to viral infection often depend upon cross-presentation, a process in which antigen is transferred from an infected cell to a professional antigen presenting cell. This process has been extensively studied with respect to MHC class I-restricted activation of CD8+ T cells, with several mechanisms having been reported including transfer of peptide-loaded heat shock proteins, apoptotic bodies and exosomes. In contrast, little work has been done on MHCII-restricted cross-presentation. Using infectious influenza virus and its glycoproteins expressed in antigen presenting cells, we readily observed MHCII cross-presentation, but only for one of the three epitopes that we routinely study, the “S1” epitope of the hemagglutinin glycoprotein (HA). This selectivity was explained by subsequent experiments that identified the process to be exosome-mediated. While exosomes are naturally endocytosed at a basal level, blocking antibodies and neuraminidase treatment of acceptor APCs revealed that cross-presentation was substantially enhanced by the sialic acid binding activity of the HA that had been incorporated into exosomes produced by the donor cells. This mechanism could provide an explanation for the skewed CD4+ responses to influenza that have been reported and may be an important component of the immune response to other enveloped viruses.
The effects of HLA-DO on peptide binding by MHC Class II HLA-DR1 (130.14)
Authors: Poluektov Yuri , Kim AeRyon , Khoruzhenko Stanislav , Sadegh-Nasseri Scheherazade 
Published: January 2023

Abstract
Ever since the discovery of the HLA-DOA and HLA-DOB genes in the HLA Class II region, controversy has surrounded their true function. Studies resulting in transcriptional modification of the HLA-DO gene analogs in mice (H-2O) support the general theory that DO decreases the presentation of antigens through the MHC class II system by inhibiting the effects of the MHC class II accessory molecule HLA-DM. The mechanism of such inhibition, however, still remains a mystery. To shed some light on this issue, we have constructed a recombinant form of soluble HLA-DO, without its cytoplasmic anchors, and conducted peptide binding experiments with soluble forms of recombinant HLA-DM and HLA-DR1. Our experiments indicate that DO can have both a negative and a positive effect on peptide presentation by the MHC Class II molecule HLA-DR1, depending on the quality of the anchoring residues of the peptide. In addition, HLA-DO only affects peptide association to the MHC Class II molecules and has virtually no effect on peptide dissociation.
Mapping TCR recognition of a beryllium antigen with implications for chronic beryllium disease (130.17)
Authors: Bowerman Natalie , Falta Michael , Kappler John , Fontenot Andrew 
Published: January 2023

Abstract
Exposure to the metal beryllium in the workplace can result in chronic beryllium disease (CBD), which is associated by the accumulation of large expansions of beryllium-specific CD4+ Th1 cells in the lung. Several T cell clones have been isolated from the lung of a CBD patient that recognize beryllium in the context of HLA-DPB1*0201/peptide. Sequence comparison of the TCR complementarity determining regions (CDRs) has revealed nearly identical Vβ and CDR3 Vα domains, yet different CDR1-2 Vα regions (due to expressing different TCRAV gene segments). Hence, we propose an atypical docking footprint for recognition of the beryllium antigen by these specific TCRs, with CDR1α and CDR2α not significantly contributing to T cell recognition. To test this hypothesis, we have focused on a particular Vα22+/Vβ5+ beryllium-specific TCR derived from a T cell clone designated JK28. Single-site alanine substitutions have been introduced across the CDRs of this TCR in order to delineate amino acids that are critical for recognition of the HLA-DP2/Be2+/peptide complex. TCR variants containing alanine substitutions were expressed on a murine α-β- T cell hybridoma also transduced with human CD4. Assays comparing IL-2 secretion when T cell hybridomas containing wildtype and mutated TCRs are stimulated with beryllium support the notion that the Vβ region contributes most to antigen recognition by this TCR. Supported by grants from the NIH, HL062410 and HL092997.
Quantitative analysis of immune synapse formation using imaging flow cytometry. (130.18)
Authors: George Thaddeus , Spurkland Anne , Sundvold-Gjerstadt Vibeke , Burbach Brandon , Shimizu Yoji , Hall Brian , Friend Sherree 
Published: January 2023

Abstract
Antigen-specific immune responses are initiated via direct cellular contact between an antigen presenting cell (APC) and an effector cell. Early events in the interaction between these two cells involve reorganization of the actin cytoskeleton and recruitment of adhesive and signaling molecules to the immunological synapse (IS), which ultimately results in effector cell activation. Because manual image acquisition and quantitative image analysis are time consuming processes, microscopic analyses of protein recruitment to the IS have remained either qualitative or statistically limited, despite the rich information content inherent in high resolution digital images. Here we describe an objective, statistically robust microscopy-based method for quantifying molecular recruitment to the IS using multispectral imaging flow cytometry, which enables quantitative image analysis of large populations of automatically collected images. The analysis technique uses morphology-based features to identify conjugates, followed by measurement of fluorescence specifically at the conjugate junction. Two models are described, including: 1) actin recruitment to the IS following T cell activation by artificial APC coated with anti-CD3; 2) antigen-specific recruitment of ADAP to the IS within TCR transgenic T cells. These data outline an objective and statistically robust method to rapidly quantify molecular recruitment to the immune synapse using automated high speed microscopy.
Endogenous MR1 is transiently expressed on the cell surface and can be stabilized with a unique mAb for activation of mucosal-associated invariant T cells (130.19)
Authors: Chua Weijen , Yu Lawrence , Myers Nancy , Kim Sojung , Huang Shouxioung , Hansen Ted 
Published: January 2023

Abstract
MHC-related protein 1 (MR1) is highly conserved among mammals and restricts the development of mucosal-associated invariant T (MAIT) cells. MAIT cells bear an invariant TCRα chain preferentially paired with limited β chains. MAIT cells reside predominantly in the mucosal tissues but are absent in mice lacking commensal bacteria or B cells. Upon activation, MAIT cells rapidly secret cytokines including IFN-γ, IL-4, and IL-10. Surprisingly, endogenous MR1 has not been detected at the cell surface; but, surface MR1 is detected on cells overexpressing MR1 by transfection or transduction. To better characterize endogenous MR1 expression, we generated mAbs to mouse MR1. Using these mAbs, we show that select B cell lines express low levels of endogenous MR1 at the cell surface. Interestingly, a unique mAb to MR1 was found to enhance MAIT cell activation, whereas other mAbs to MR1 blocked MAIT cell activation. The enhancing and the blocking mAbs recognize different MR1 epitopes mapped by a panel of MR1 mutants and by mAb competition assays. Mechanistically, the enhancing mAb stabilizes MR1 at the cell surface suggesting it either promotes assembly of the MR1 complex or allosterically induces a more favorable conformation. These findings demonstrate that endogenous MR1 transiently visits the cell surface of APCs, and that induced levels of MR1/ligand complexes are required for MAIT cell activation.
Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphinogolipids presented by CD1d (130.22)
Authors: Muindi Karen , Cernadas Manuela , Watts Gerald , Royle Louise , Neville David , Dwek Raymond , Besra Gurdyal , Rudd Pauline , Butters Terry , Brenner Michael 
Published: January 2023

Abstract
CD1d molecules present lipid antigens that activate CD1d-restricted natural killer T (NKT) cells. NKT cell activation leads to potent downstream activation of other immune cells through cell-cell interactions and rapid production of large quantities of cytokines. Endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of TLR induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The repertoire of GSL bound to CD1d expressed in myeloid antigen presenting cells at steady state and in the setting of activation has not been delineated. We have identified the range of GSL bound to soluble CD1d molecules that sample the ER/secretory routes and cell surface cleaved CD1d that also samples the endocytic system. CD1d bound specific GSL preferentially as the CD1d GSL profile did not solely reflect cellular GSL abundance. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, while GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation the quantities of soluble CD1d bound GM3 and GM1a markedly increased. A unique a-galactose-terminating GSL was also found to be preferentially bound to CD1d at steady state and increased with APC activation. Our studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation. In press PNAS
Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC I in the endoplasmic reticulum (130.26)
Authors: Kropp Laura , Garg Manish , Binder Robert 
Published: January 2023

Abstract
Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC I are associated with several chaperones. Hsp70, hsp90, and the TCP1-ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC I and hence presentation of the MHC 1-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC I heavy chains and beta2-microglobulin as a trimolecular complex.
Surface peptide-MHC generation from Listeria-derived recombinant proteins is independent of protein half-life (130.30)
Authors: Wolf Benjamin , Kumagai Chiharu , Guterman Jacqueline , Princiotta Michael 
Published: January 2023

Abstract
The generation of surface peptide-MHC (pMHC) complexes from virally infected cells is dependant on the degradation of newly synthesized proteins, with the amount of pMHC complexes generated directly correlating with the amount of protein degraded. pMHC complex generation from cells infected with the bacteria Listeria monocytogenes, on the other hand, is not as well understood. While it is known that the generation of pMHC complexes from Listeria-infected cells is dependant on newly synthesized bacterial proteins, it is not known whether the amount of pMHC on the cell surface correlates with the amount of protein degraded. To determine whether there is such a correlation, we generated Listeria strains that produce model recombinant proteins containing the minimal Kb binding peptide SIINFEKL. These recombinant proteins are nearly identical (1 amino acid difference) yet differ in half-life once in the eukaryotic cell cytosol. By changing the half-life of the recombinant protein and then comparing Kb-SIINFEKL generation, we have found that full-length proteins secreted into the host cell generate similar amounts of Kb-SIINFEKL regardless of their protein half-life. These data suggest that full-length proteins secreted into the host cell by Listeria follow distinct processing pathways for the production of pMHC complexes.
Direct presentation regulates the magnitude of the CD8+ T cell response to cell-associated antigen through prolonged T cell proliferation (130.32)
Authors: Tatum Angela , Schell Todd 
Published: January 2023

Abstract
The magnitude and complexity of antigen-specific CD8+ T cell responses is determined by both intrinsic properties of the immune system and extrinsic factors such as vaccination. We evaluated mechanisms that regulate the CD8+ T cell response to two distinct determinants derived from the same protein antigen, SV40 T antigen (T Ag), in C57BL/6 mice following immunization with T Ag transformed cells. We found that the immunodominance hierarchy was reversed in a dose-dependent manner by addition of excess naïve T cells targeting the subdominant epitope. However, competition by T cells targeting distinct determinants played only a minor role in limiting T cell accumulation under physiological conditions. In contrast, the magnitude of the T cell response was regulated by the ability of T Ag transformed cells to directly present the T Ag determinants. The hierarchy of the CD8+ T cell response was maintained when antigen presentation in vivo was restricted to cross-presentation, but immunization with T Ag transformed cells capable of direct presentation dramatically enhanced T cell accumulation at the peak of the response. Enhanced accumulation was due to a prolonged period of T cell proliferation, resulting in a delay in T cell contraction. Our findings reveal that direct presentation by non-professional antigen presenting cells can dramatically enhance accumulation of CD8+ T cells during the primary response, revealing a potential strategy to enhance vaccination approaches.