bluuebunny/crossref_metadata_2025_split huggingface dataset:
PD-1-mediated deletion induces tolerance of CD8 T cell response specific for H60 under helpless condition (P2225)
Authors: Choi Eun Young , Ryu Su Jeong , Kim Juhyun 
Published: January 2023

Abstract
H60 is a dominant minor H antigen, for which specific CD8 T cell response dominates over those for other minor H antigens in B6 anti-BALB.B response. The specific CD8 T cell response for H60 is CD4 help-dependent, as burst expansion of the specific cells during the primary and memory responses occurs only when CD4 T cell activation accompanies. When the CD4 help is not provided, primary expansion was limited and, then, recall expansion of the specific CD8 T cells is impaired even when the CD4 help is provided during the memory response. This tolerance phenomenon was ascribed to intrinsic property of H60-specific CD8 T cells activated under the helpless condition. The H60-specific CD8 T cells activated under helpless condition could develop into functionally active effector cells, migrating to inflamed sites. However, they were more prone to cell death with up-regulation of PD-1 on the H60-specific CD8 T cells activated under helpless condition. Recall expansion of H60-specific CD8 T was restored in the PD-1 deficient mice that had been primed under helpless condition, suggesting the significance of negative regulation by PD-1 in the tolerance induction of H60-specific CD8 T cell response after the helpless priming. These results will provide useful information for induction of tolerance after allogeneic transplantation.
Depletion of activated lymphocytes during the emergence of ovarian cancer (P2004)
Authors: Riggs James , Suarez Joyce 
Published: January 2023

Abstract
We are modeling the myeloid suppression found in tumors by characterizing how peritoneal cavity (PerC) macrophages (Mϕs) suppress lymphocyte activation. Cultured PerC Mϕs suppress the proliferation of C57BL/6J T cells by iNOS catabolism of arginine. To make our model more relevant to in vivo biology we initiated studies of ovarian cancer in the PerC. The ID8 cell line (mouse ovarian surface epithelial cell carcinoma) forms ovarian tumors and hemorrhagic ascites 5 weeks following i.p. injection into intact, syngeneic C57BL/6J mice. We found cancer development was accelerated in IL10-/- mice and delayed in iNOS-/- mice indicative that inflammation plays a role in cancer progression. Assessing mice before the ovarian cancer was fully developed we observed a depletion of CD44hi T and CD11b+IgMhi B cells in the PerC. With disease progression B and T cell depletion also became evident in the spleen. Lymphocyte depletion in the PerC correlated with ID8 expansion and the emergence of CD11b+F4/80+Gr-1+CD80+ myeloid suppressor cells (MSCs); splenic MSCs were CD80-. It was interesting to observe that GITR+ T cells, naturally enriched in the PerC of healthy, normal mice, persisted during MSC emergence in both the spleen and PerC. We are investigating what triggers the selective death of these important lymphocyte subsets. These results reveal an additional, challenging aspect of the immune suppression characteristic of myeloid cell emergence during cancer development.
Neutrophils inhibit tumor growth at early stages of tumor formation. (P2006)
Authors: Liu Yuhong , O’Leary Claire , Wang Liang-Chuan , Dai Ning , Kapoor Veena , Liu Peihui , Mei Junjie , Oliver Paula , Albelda Steven , Worthen George 
Published: January 2023

Abstract
Neutrophils are important innate immune cells involved in microbial clearance at sites of infection. However, the roles of neutrophils in cancer development are not clear. We hypothesized that neutrophils might mediate antitumor effects at the initial tumor formation stage. To test this hypothesis, we injected 2x106 mouse lung Lewis cancer cells into the flanks of C57BL/6 and Cxcr2-/- mice, the latter lack the receptor for ELR+ CXC neutrophil chemokines. We observed increased tumor growth in Cxcr2-/- mice as compared with controls within 3 weeks of injection. FACS analysis indicated fewer neutrophils and more IL-17-producing cells in the tumors of Cxcr2-/- mice than those of C57BL/6 mice. We further tested the roles of neutrophils using anti-Ly6G antibody depletion at the time of tumor cell injection. We demonstrated that tumors grew faster in the neutrophil depleted group, while the percentages of IL-17-producing cells in the tumors and the levels of G-CSF in the plasma were also increased in the absence of neutrophils. To test whether neutrophils have a direct cytotoxic effect on LLC cells, we performed in vitro cell killing assays. We demonstrated neutrophils isolated from both circulation and bone marrow exerted cytotoxic effects. In conclusion, by using Cxcr2-/- and neutrophil antibody-depleted C57BL/6 mice, we have demonstrated that initial tumor growth of LLC cells is enhanced in association with inaccessibility of neutrophils and activation of the IL-17/G-CSF axis.
Preclinical modelling of immune checkpoint blockade (P2012)
Authors: Stewart Ross , Mullins Stefanie , Watkins Amanda , Morrow Michelle , Sainson Richard , Robbins Paul , La Vallee Theresa , Wilkinson Robert 
Published: January 2023

Abstract
Harnessing the power of the immune system to identify and eliminate cancer is a promising area of development in cancer therapy. Increasing T-cell activation, in particular, is now a clinically validated approach to the treatment of cancer. Cytotoxic T lymphocyte antigen 4 (CTLA-4, CD152) and PD-L1 (B7-H1, CD274) are part of a complex system of receptors and ligands that are involved in limiting T-cell activation through the delivery of inhibitory signals. MedImmune is currently developing tremelimumab (anti-CTLA4) and MEDI4736 (anti-PD-L1), for the treatment of advanced or metastatic solid tumours. Both of these antibodies antagonise T-cell inhibition signals with the aim of enhancing anti-tumor T cell activity. We have established and characterised a panel of syngeneic mouse tumor models and employed these to explore the activity of anti-mouse CTLA-4 and PD-L1 antibodies using pharmacodynamic (PD) and tumor growth inhibition (TGI) endpoints. Published studies characterising the immunogenic nature of some existing cancer therapeutics provide reason to believe that a combination strategy, utilising checkpoint blockade together with current standard of care, or novel therapeutics, could result in increased activity. We have gone on to explore the value of such combinations in preclinical mouse models.
Hepatic T cell suppressors and their inhibition roles in hepatic viral clearance (P6353)
Authors: Tang Li , Liu Biao , Wang Min , Wang Xiaowen , He Fuchu 
Published: January 2023

Abstract
The unique microenvironment of the liver favors T cell inactivation, tolerance, and apoptosis. As such, it is a challenging environment in which to efficiently clear viruses, which may explain why HBV and HCV often establish a chronic viral presence. The principal mechanisms that account for hepatic T cell failure and thus influence the outcome of liver infections, including the identification of inhibitory molecules and their ligands, are beginning to be understood. We recently identified LSECtin from the liver and reported that LSECtin negatively regulates T cell receptor-mediated signaling and T cell Immunity. We then investigate whether LSECtin regulates anti-viral immunity and virus invasiveness in the liver. Using a mouse model infected with a hepatotropic adenovirus, we found that the absence of LSECtin led to a higher frequency of CTL, which produced higher levels of antiviral cytokines and cytotoxic factors and showed increased expression of T-bet and Runx3, thus mediated more efficient virus-specific cytotoxity than WT cells. As a consequence, LSECtin deficiency significantly accelerated liver adenovirus clearance. We further constructed an immunocompetent mouse model of acute hepatitis B viral infection to demonstrate that LSECtin significantly delayed clearance of HBV from blood and infected hepatocytes through limiting the frequency of HBV-specific IFN-γ-producing cells.
MMP-8 mediates vascular breakdown during viral infection of the nervous system (P6368)
Authors: McGavern Dorian , Gossa Selamawit 
Published: January 2023

Abstract
Viral infection of the central nervous system (CNS) routinely induces immune-mediated vascular damage that compromises the integrity of the blood brain barrier (BBB) and induces neurological complications. We have shown previously that cytotoxic lymphocytes massively recruit monocytes and neutrophils to the brain following infection with lymphocytic choriomeningitis virus (LCMV). These cells promote vascular leakage through synchronous extravasation. Here, we focus on identifying the mechanism by which innate myelomonocytic cells injure brain vasculature. Quantitative analyses revealed that 6 metalloproteinases (MMP-2, 3, 8, 12, 13, and 19) and 1 disintegrin (ADAM-8) were highly expressed in symptomatic mice with LCMV meningitis. Among these, the collagenase, MMP-8, was expressed greater than 2000-fold in neutrophils and 200-fold in monocytes. We therefore postulated that vascular collagenase activity during viral meningitis promotes myelomonocytic cell extravasation and vessel leakage. This theory was tested experimentally by administering a specific MMP-8 antagonist to LCMV-infected mice prior to the development of symptoms. Our results revealed that MMP-8 antagonism significantly reduced vascular damage, BBB breakdown, and the development of symptoms in infected mice. These data demonstrate that inhibition of collagenases might be a viable therapeutic approach to lessen the pathogenic effects of innate inflammatory cells following CNS viral infection.
A case for research consortia to accelerate vaccine candidate discovery: identification of a stable antigenic HIV-1 Clade A Env candidate (P6370)
Authors: Fliing Steven , Hoffenberg Simon , Caulfield Michael , Parks Christopher , King C. , Burton Dennis , Ward Andrew , Wilson Ian , Moore John , Koff Wayne 
Published: January 2023

Abstract
IAVI’s NAC provides a multi-disciplinary platform to decipher the interaction of bNAbs with HIV Env and accelerate translation to development of vaccines capable of eliciting potent neutralizing abs. Recent successes highlight the value of a consortium to expedite progress. IAVI initiated Protocol G to identify neutralizing sera from HIV-1 infected patients. Using these samples, a cross-disciplinary team discovered abs with unprecedented potency against HIV. PG9 was isolated from a subject infected with HIV-1 clade A. Consortium members reasoned that Env sequences from the clade A infected donor could lead to immunogen discovery. Using bioinformatics, MRCA sequence was aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database and BG505 was identified with the highest homology. Soluble BG505 retains features of native trimer. While native Env trimer is the target for bnAbs, its instability impedes efforts to develop recombinant Env. Because BG505 retains features of native trimer, it might be optimal for immunogen design. NAC partners report construction of a soluble trimer from BG505 that binds PG9 with high affinity and show it is uniquely stable. Formulations of gp120 and trimers are under analysis for immunogenicity. Challenges remain in development including analysis of structure, immunogenicity, ab responses elicited; and production for human testing. A consortium approach of multidisciplinary experience will be required to expedite advancement of vaccine concepts.
Measuring immunological synapse and actin organization using the FlowSight imaging flow cytometer (P5018)
Authors: Pugsley Haley , Kong Raymond , Hall Brian , Vaidyanathan Shobana , Basiji David , Friend Sherree 
Published: January 2023

Abstract
Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse (IS) after T cell receptor stimulation are required for the antigen-specific response. One way to measure an IS is by fluorescently labeling the molecules that have been recruited to the synapse and imaging via microscopy. However, IS are often rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis FlowSight imaging flow cytometry platform to objectively collect imagery of large numbers of cells to assess the percentage of T cells involved in an organized IS. In this study, Raji B cells were loaded with Staphylococcal enterotoxin B (SEB) to make APCs. The SEB-loaded APCs were incubated with human T cells to create T cell-APC conjugates. In addition, we investigated the inhibition of actin by cytochalasin D and how that affects the T cell-APC conjugates. The T cells, APCs and conjugates were fluorescently labeled. Using the FlowSight imaging flow cytometry platform we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized IS in an objective and statistically significant manner.
Plasma membrane interactions that determine presentation of antigens by Antigen-presenting cells. (P5022)
Authors: Hari Aswin , Shi Yan 
Published: January 2023

Abstract
Antigen presentation is a critical process in host defense for initiating adaptive immune responses. The APC loads peptides derived from antigens onto major histocompatibility complex (MHC) molecules and exports them to the surface for T cells sampling. If the primary interaction between an APC and T cell is successful, i.e. the TCR binds an appropriate MHC-peptide complex, this leads to an adaptive immune response. Peptide antigens are presented on two types of MHC molecules, MHC I and MHC II via several distinct processing pathways. In turn, the MHC molecule dictates which type of T cell can be activated and thereby the nature of the adaptive immune response. Therefore, antigen loading onto MHC molecules is a critical determinant of the adaptive immune responses. Exogenous antigens can be presented on MHC I and II molecules. Antigen processing mechanisms have been elucidated in great detail, but the fundamental reason behind why an antigen is directed into a specific pathway is not known. One of the foremost aims for this project is to determine why particulate antigens are presented more often on MHC I and why soluble antigens are more efficiently presented on MHC II, by deciphering the interactions of plasma membrane with particulate versus soluble antigen. This fundamental sensing mechanism of the human body when elucidated can help in generating an antiviral response in diseases like HIV and in field of preventative medicine.
X-ray crystal structure of HLA-DQ2 in complex with CLIP1 and CLIP2 (P5026)
Authors: Nguyen Thanh Binh , Jayaraman Priya , Bergseng Elin , Madhusudhan Mallur Srivatsan , Sollid Ludvig M. , Kim Chu-Young 
Published: January 2023

Abstract
The class II MHC protein HLA-DQ2 is strongly associated with type 1 diabetes and celiac disease. CLIP (MHC class II-associated invariant chain peptide) occupies the peptide binding groove of all newly synthesized class II MHC proteins until HLA-DM replaces it with an antigen peptide. We have determined the atomic structure of HLA-DQ2-CLIP1 and HLA-DQ2-CLIP2 complex at 2.8 Ang and 2.2 Ang, respectively. Our crystal structure shows that MRMATPLLM of CLIP1 occupies the P1 to P9 pockets of HLA-DQ2. For CLIP2, PLLMQALPM occupies the P1 to P9 pockets of HLA-DQ2. We present our analysis of the MHC-peptide interaction in these two structures, and we highlight the structural similarities and differences compared to other class II MHC-CLIP complex structures reported to date.
Unexpected side reaction of lysine and arginine side chains preceding a photocleavable group in MHC-II UV-cleavable peptides. (P5028)
Authors: Negroni Maria , Stern Lawrence 
Published: January 2023

Abstract
MHC class II molecules (MHCII) present peptides to CD4+ T cells, and HLA-DM (DM) catalyzes peptide exchange favoring binding of high affinity peptides. The effects of DM on peptide association and dissociation kinetics are not yet clear. In the absence of peptide, DR1 (a MHCII) is in equilibrium between inactive and active conformations. In order to understand the kinetics of this process and how it is affected by DM, we wanted to generate DR1 in the active conformation using a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII-binding peptides. We used well-characterized variants of a peptide derived from influenza hemagglutinin (HA). The variants bind to DR1 with an affinity similar to the parent HA peptide. Surprisingly, photolysis of peptides did not result in peptide release. Mass spectrometry showed that the main photoproduct had a mass decrease of 2Da, resulting from photocleavage followed by unexpected religation to a rearranged peptide that can still bind DR1. Replacement of peptide lysine (or arginine) residues by methionine yielded the expected cleavage products. The methionine substituted peptides can be used for further studies on DR1-peptide binding kinetics and the effect of DM on the different rates of the reaction. These studies provide a caveat to routine use of photocleavable peptides in MHC-peptide exchange studies.
Substrate specificity of human ERAP1 haplotypes in vivo identify three functional classes and multiple fine specificity (P5034)
Authors: Reeves Emma , Edwards Christopher , Elliott Tim , James Edward 
Published: January 2023

Abstract
Recent genome wide association studies have identified single nucleotide polymorphisms within Endoplasmic Reticulum Aminopeptidase1 (ERAP1) as being associated with autoimmune diseases, cervical carcinoma and hypertension. ERAP1 performs a key final step in antigen processing, trimming N-terminally extended peptides to the final epitope for presentation by MHC I molecules. Here we show that ERAP1 sequenced from individuals is polymorphic, with multiple haplotypes identified. Characterization of the amino acid trimming capacity of the identified haplotypes reveal distinct profiles that correlate with the SNPs they possess. In addition, the properties of the amino acids preferentially trimmed vary between haplotypes. RP-HPLC and mass spectrometry analysis allows classification of the ERAP1 haplotypes into three functional groups; those that have a predominantly i) Normal, ii) Hypo or iii) Hyper-functional activity. These results indicate that the repertoire of peptides presented at the cell surface depends, not only on the MHC I, but also on the ERAP1 expressed within an individual. This therefore has important implications for predisposition to disease, the ability to combat cancer and infection, and also vaccine efficacy.
The impact of conformational entropy on epitope selection by MHC class II (P5010)
Authors: Ferrante Andrea 
Published: January 2023

Abstract
The interaction between pathogen-derived peptides and molecules of the Class II Major Histocompatibility Complex (MHCII) is a thermodynamically cooperative process, characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to epitope selection by MHCII needs to be elucidated. Here, cycle-mutated peptides derived from influenza H3 HA306-319 are analyzed as they interact with the human MHCII allele HLA-DR1 (DR1) by isothermal titration calorimetry, fluorescence polarization, circular dichroism (CD) and molecular dynamics (MD) simulation. Peptide ligand to DR1 displays enthalpy/entropy compensation that results in similar free energy of binding, even though individual enthalpy and entropy terms change significantly across the series. CD analysis indicates that complexes, whose entropic contribution to the binding is the greatest, feature spectra that are consistent with an incomplete conversion to the compact form. These distinct binding modes correlate with the stability of the complex during DM-mediated peptide release and are supported by MD simulations. These results indicate that, at the molecular level, binding modes and thermodynamic binding signatures can be very different even for closely related peptides, and these signatures have a significant impact on the outcome of the epitope selection process.
Immunodeficiency and autoimmunity in H2-O deficient mice (P5016)
Authors: Chen Xinjian , Jensen Peter , Gu Yapeng 
Published: January 2023

Abstract
HLA-DO/H2-O is a highly conserved non-polymorphic MHC class II-like molecule whose physiological function remains unknown. The cell maturation-dependent DO expression in B-lymphocytes and DCs suggests a role H2-O in promoting presentation of exogenous Ag by attenuating presentation of endogenous self-peptides. We report herein that H2-O-/- mice spontaneously develop high titers of IgG2a/c antinuclear antibodies (ANA) with specificity for dsDNA, ssDNA, and histones. Reconstitution of RAG1-/- mice with T and B cells from H2-O-/- or WT mice demonstrated that production of ANAs requires participation of CD4+ T cells from H2-O-/- mice. Bone marrow chimeras demonstrated that loss of H2-O expression in thymic epithelial cells did not induce ANAs, and that lack of H2-O expression in bone marrow-derived cells was sufficient to induce the autoimmune phenotype. Despite production of high titers of autoantibodies, H2-O-/- mice exhibit a delayed generation of humoral immunity to model antigens (OVA and KLH), affecting all major T-dependent Ig classes including IgG2a/c. Consistently, presentation of all the natural Ag epitopes by H2-O-/- APC was inefficient as compared to WT APC. Thus, H2-O promotes immunity towards exogenous Ag while preventing autoimmunity. We propose that H2-O, through spatially and temporally inhibiting H2-M, enhances presentation of exogenous antigen by preventing nascent/newly-synthesized class II molecules from forming stable complexes with endogenous self-peptides.
The constant region affects antigen binding of anti-DNA antibodies by altering secondary structure (P4002)
Authors: Putterman Chaim , Xia Yumin , Janda Alena , Eryilmaz Ertan , Casadevall Arturo 
Published: January 2023

Abstract
We previously demonstrated an important role of the constant (C) region in the pathogenicity of anti-DNA antibodies (Ab) through modulation of immunoglobulin affinity and specificity. However, the mechanisms by which the C region affects the binding of autoantibodies to nuclear antigens (Ag) have yet to be elucidated. From a parent IgG3 anti-nuclear Ab, PL9-11, we generated IgG1, IgG2b, and IgG2a variants that share identical variable regions but that differ in their C region. The affinity of these Abs for histone antigens was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine the wavelength shift of the Ab panel upon the addition of DNA and histone Ags. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure of the PL9-11 panel corresponding to Ab-Ag interactions. SPR analysis revealed significant differences of histone 2A/2B binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the Ab isotype, while increasing salt concentrations lowered KD values. Circular dichroism analysis determined that changes in Ab secondary structure content differed between the IgG isotypes upon Ag binding. In conclusion, the Ag-binding affinity of anti-nuclear Abs is dependent on the particular C region. Ag binding effects on Trp fluorescence, as well as secondary structure shifts, were also C region dependent. Alteration of secondary structures influenced by C regions may explain differences in fine specificity of anti-DNA Abs between Abs with similar variable regions, as well as cross-reactivity of anti-DNA Abs with non-DNA Ags.