bluuebunny/crossref_metadata_2025 huggingface dataset:
Extinction of IL-12 Signaling Promotes Fas-Mediated Apoptosis of Antigen-Specific T Cells
Authors: Marth Thomas , Zeitz Martin , Ludviksson Bjorn R. , Strober Warren , Kelsall Brian L.
Published: December 2022

In previous studies we have shown that peripheral tolerance achieved by high dose feeding of OVA to intact OVA-TCR transgenic mice was enhanced when endogenous IL-12 was neutralized simultaneously. To generalize this phenomenon, in the present study we investigated the tolerogenic mechanisms underlying the blockade of IL-12 signaling following oral and systemic Ag delivery. We found that the numbers of Ag-specific T cells in several lymphoid organs were significantly reduced due to T cell apoptosis following oral OVA or systemic OVA administration when combined with anti-IL-12 injection, but there was no decrease in T cell numbers for OVA-fed, OVA-injected, or anti-IL-12 alone-treated mice compared with those in untreated control mice. In addition, mostly Fas+ T cells were subject to apoptotic deletion in the OVA- plus anti-IL-12-treated groups, and an enhanced cell death of T cells upon OVA restimulation in vitro could be partially reversed by blockade of the Fas/Fas ligand interaction. Finally, in a murine model of superantigen-driven T cell expansion and deletion, we observed no deletional effects of anti-IL-12 treatment on CD4+ cells in Fas-deficient (MRL/lpr) mice, but did find these effects in MRL wild-type mice. In summary, our data suggest that in the course of Ag-induced cell proliferation of Th1 cells, signaling through IL-12 is required to prevent an induction of Fas-mediated apoptosis. Thus, the use of anti-IL-12 may be potentially useful in modulating peripheral immune responses by promotion of Fas-mediated cell death.
Absolute Requirement for an Active Immune Response Involving B Cells and Th Cells in Immunity toPlasmodium yoeliiPassively Acquired with Antibodies to the 19-kDa Carboxyl-Terminal Fragment of Merozoite Surface Protein-1
Authors: Hirunpetcharat Chakrit , Vukovic Peter , Liu Xue Qin , Kaslow David C. , Miller Louis H. , Good Michael F.
Published: December 2022

Vaccination of mice with the leading malaria vaccine candidate homologue, the 19-kDa carboxyl terminus of merozoite surface protein-1 (MSP119), results in sterile immunity to Plasmodium yoelii, with no parasites detected in blood. Although such immunity depends upon high titer Abs at challenge, high doses of immune sera transferred into naive mice reduce parasitemia (and protect from death) but do not result in a similar degree of protection (with most mice experiencing high peak parasitemias); this finding suggests that ongoing parasite-specific immune responses postchallenge are essential. We analyzed this postchallenge response by transferring Abs into manipulated but malaria-naive mice and observed that Abs cannot protect SCID, nude, CD4+ T cell-depleted, or B cell knockout mice, with all mice dying. Thus, in addition to the Abs that develop following MSP119 vaccination, a continuing active immune response postchallenge is required for protection. MSP119-specific Abs can adoptively transfer protection to strains of mice that are not protected following vaccination with MSP119, suggesting that the Ags targeted by the immune response postchallenge include Ags apart from MSP119. These data have important implications for the development of a human malaria vaccine.
Use of a Photoactivatable Taxol Analogue to Identify Unique Cellular Targets in Murine Macrophages: Identification of Murine CD18 as a Major Taxol-Binding Protein and a Role for Mac-1 in Taxol-Induced Gene Expression
Authors: Bhat Nayantara , Perera Pin-Yu , Carboni Joan M. , Blanco Jorge , Golenbock Douglas T. , Mayadas Tanya N. , Vogel Stefanie N.
Published: December 2022

Taxol, a potent antitumor agent that binds β-tubulin and promotes microtubule assembly, results in mitotic arrest at the G2/M phase of the cell cycle. More recently, Taxol was shown to be a potent LPS mimetic in murine, but not in human macrophages, stimulating signaling pathways and gene expression indistinguishably from LPS. Although structurally unrelated to LPS, Taxol’s LPS-mimetic activities are blocked by inactive structural analogues of LPS, indicating that despite the species-restricted effects of Taxol, LPS and Taxol share a common receptor/signaling complex that might be important in LPS-induced human diseases. To identify components of the putatively shared Taxol/LPS receptor, a novel, photoactivatable Taxol analogue was employed to identify unique Taxol-binding proteins in murine macrophage membranes. Seven major Taxol-binding proteins, ranging from ∼50 to 200 kDa, were detected. Although photoactivatable Taxol analogue failed to bind to CD14, the prominent Taxol-binding protein was identified as CD18, the ∼96-kDa common component of the β2 integrin family. This finding was supported by the concomitant failure of macrophage membranes from Mac-1 knockout mice to express immunoreactive CD18 and the major Taxol-binding protein. In addition, Taxol-induced IL-12 p40 mRNA was markedly reduced in Mac-1 knockout macrophages and anti-Mac-1 Ab blocked secretion of IL-12 p70 in Taxol- and LPS-stimulated macrophages. Since CD18 has been described as a participant in LPS-induced binding and signal transduction, these data support the hypothesis that the interaction of murine CD18 with Taxol is involved in its proinflammatory activity.
Alterations in C3 Activation and Binding Caused by Phosphorylation by a Casein Kinase Released from Activated Human Platelets
Authors: Ekdahl Kristina Nilsson , Nilsson Bo
Published: December 2022

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6–4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.
Monoclonal Antibodies to Murine Lipopolysaccharide (LPS)-Binding Protein (LBP) Protect Mice from Lethal Endotoxemia by Blocking Either the Binding of LPS to LBP or the Presentation of LPS/LBP Complexes to CD14
Authors: Le Roy Didier , Di Padova Franco , Tees Reet , Lengacher Sylvain , Landmann Regine , Glauser Michel Pierre , Calandra Thierry , Heumann Didier
Published: December 2022

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.
Progesterone-Induced Inhibition of Chemokine Receptor Expression on Peripheral Blood Mononuclear Cells Correlates with Reduced HIV-1 Infectability In Vitro
Authors: Vassiliadou Neratzoula , Tucker Lynne , Anderson Deborah J.
Published: December 2022

Recent studies have shown that progesterone, a sex steroid hormone, enhances the sexual transmission of various pathogens, including SIV. The goal of this study was to determine whether progesterone affects mechanisms underlying the sexual transmission of HIV-1. We first studied the effects of various physiologic concentrations of progesterone on the expression of chemokines and chemokine receptors by T cells and macrophages. Chemokines are involved in leukocyte recruitment to peripheral sites; in addition, the chemokine receptors CCR5 and CXCR4 are HIV-1 coreceptors, and their ligands can block HIV-1 infection. Progesterone treatment had no effect on constitutive expression of CCR5 and CXCR4 by nonactivated T cells and macrophages, but significantly inhibited IL-2-induced up-regulation of CCR5 and CXCR4 on activated T cells (p < 0.05). Progesterone also inhibited both mitogen-induced proliferation and chemokine secretion (macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, RANTES) by CD8+ T lymphocytes. Control and progesterone-treated PBMC cultures were also tested for susceptibility to infection by T cell-tropic (HIV-1MN) and macrophage-tropic (HIV-1JR-CSF) viral strains in vitro. Infection with low titers of HIV-1MN was consistently inhibited in progesterone-treated cultures; progesterone effects on infection with the HIV-1JR-CSF strain were more variable, but correlated with progesterone-induced reductions in CCR5 levels. These results indicate that progesterone treatment can inhibit mechanisms underlying HIV-1 transmission, including infection of CD4+ target cells via CXCR4/CCR5 coreceptors and effects on chemokine-mediated recruitment of lymphocytes and monocytes to mucosal epithelia.
Induction of HIV-1 Replication by Allogeneic Stimulation
Authors: Moriuchi Hiroyuki , Moriuchi Masako , Fauci Anthony S.
Published: December 2022

Allogeneic stimulation presents an immunologic challenge during pregnancy, blood transfusions, and transplantations, and has been associated with reactivation of latently infected virus such as CMV. Since HIV-1 is transmitted vertically, sexually, or via contaminated blood, we have tested the effects of allostimulation on HIV-1 infection. 1) We show that allostimulated lymphocytes are highly susceptible to acute infection with T cell-tropic or dual-tropic HIV-1. 2) We show that allostimulation has dichotomous effects on replication of macrophage-tropic HIV-1; it activates HIV expression in already infected cells but inhibits HIV entry by secreting HIV-suppressive CC chemokines. 3) We show that allogeneic stimulation of latently infected, resting CD4+ T cells induced replication of HIV-1 in these cells. These observations suggest that allogeneic stimulation may play a role in the transmission, replication, and phenotypic transition of HIV-1.
Human CD8+ and CD4+ T Lymphocyte Memory to Influenza A Viruses of Swine and Avian Species
Authors: Jameson Julie , Cruz John , Terajima Masanori , Ennis Francis A.
Published: December 2022

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.
Cutting Edge: The HLA-A*0101-Restricted HY Minor Histocompatibility Antigen Originates fromDFFRYand Contains a Cysteinylated Cysteine Residue as Identified by a Novel Mass Spectrometric Technique
Authors: Pierce Richard A. , Field Erin D. , den Haan Joke M. M. , Caldwell Jennifer A. , White Forest M. , Marto Jarrod A. , Wang Wei , Frost Leslie M. , Blokland Els , Reinhardus Carla , Shabanowitz Jeffrey , Hunt Donald F. , Goulmy Els , Engelhard Victor H.
Published: December 2022

In this report, we describe the use of novel mass spectrometry instrumentation to identify a male-specific minor histocompatibility Ag restricted by HLA-A*0101 (A1-HY). This Ag has the sequence IVDC*LTEMY, where C* represents a cysteine disulfide bonded to a second cysteine residue. The core peptide sequence is found in the protein product of DFFRY, a Y chromosome gene not previously identified as the source of an HY Ag. The male-specific form of the peptide differs from its X chromosomal counterpart by the substitution of serine for the C* residue. Both peptides are expressed on the cell surface at 30 or fewer copies per cell. However, A1-HY-specific CTL recognize the DFFRY-derived peptide at a 1500-fold lower dose than the female homologue. Thus, these studies have identified a new source of HY epitopes and provide additional information about the influence of posttranslational modifications of class I-associated peptides on T cell recognition.
Antigen-Induced Unresponsiveness Results in Altered T Cell Signaling
Authors: McKay Dianne B. , Irie Hanna Y. , Hollander Georg , Ferrara James L. M. F. , Strom Terry B. , Li YongSheng , Burakoff Steven J.
Published: December 2022

Pretransplant exposure to allogeneic lymphocytes can result in donor-specific unresponsiveness and prolonged allograft survival. Intracellular signaling events have been described in anergic T cell clones, but the biochemical events underlying in vivo induced unresponsiveness have not been studied in detail. We employed a TCR transgenic mouse, bearing the 2C TCR, providing adequate numbers of homogenous peripheral T cells to study biochemical aspects of T cell unresponsiveness in vivo. 2C mice exposed to semiallogeneic lymphocytes (H-2b × H-2d) experienced prolonged H-2d cardiac allograft survival, and cells from these mice did not proliferate or make IL-2 in response to alloantigen (H-2d). Importantly, there were marked differences in TCR-associated tyrosine phosphorylation activation patterns. The targets for the unresponsive state appear to be diminished Lck activation and absent ZAP-70 and LAT (linker for activation of T cells) phosphorylation. Our study demonstrates that Ag-induced tolerance in vivo is accompanied by altered early TCR-mediated signaling events.
Protein-Protein and DNA-Protein Interactions Affect the Activity of Lymphoid-Specific IFN Regulatory Factors
Authors: Meraro David , Hashmueli Sharon , Koren Belly , Azriel Aviva , Oumard André , Kirchhoff Sabine , Hauser Hansjörg , Nagulapalli Sujatha , Atchison Michael L. , Levi Ben-Zion
Published: December 2022

IFN regulatory factors (IRFs) constitute a family of transcription factors that are involved in IFN signaling and the development and differentiation of the immune system. Targeted gene disruption studies in mice assigned their primary role to the immune system. Two lymphoid-specific IRF members, IFN consensus sequence binding protein (ICSBP) and IRF-4, bind target DNA with greater efficiency following interaction with two transcription factors, PU.1 and E47, leading to transcriptional synergy. PU.1 and E47 are essential for proper differentiation and maturation of lymphoid cells. In addition, ICSBP interacts with two IRF members, IRF-1 and IRF-2, which also have central roles in the regulation of cell-mediated immunity. Previously, we identified a region in ICSBP, termed the IRF association domain (IAD), that is conserved in all IRFs (excluding IRF-1 and IRF-2) and is essential for its interactions with other IRF proteins. Here we show that the IAD is an independent module used by ICSBP and IRF-4 for protein-protein interactions. In addition, an IAD of IRF-2 (IAD2), necessary for interaction with ICSBP, was identified and found to be conserved in IRF-1. The IAD2 shares similar characteristics with the PEST domain that is essential for the interaction of PU.1 with IRF-4. We also show that the ICSBP DNA binding domain is indispensable for the formation of DNA binding heterocomplexes and transcriptional activity. Therefore, our results shed light on the molecular mechanisms that affect IRF activities in the immune system via discrete functional domains.
Increased p27Kip1 Cyclin-Dependent Kinase Inhibitor Gene Expression Following Anti-IgM Treatment Promotes Apoptosis of WEHI 231 B Cells
Authors: Wu Min , Bellas Robert E. , Shen Jian , Yang William , Sonenshein Gail E.
Published: December 2022

Engagement of the B cell receptor of WEHI 231 immature B cells leads sequentially to a drop in c-Myc, to induction of the cyclin-dependent kinase inhibitor p27Kip1, and finally to apoptosis. Recently we demonstrated that the drop in c-Myc expression promotes cell death, whereas the induction of p27 has been shown to lead to growth arrest. In this paper, we demonstrate that increased p27 expression also promotes apoptosis of WEHI 231 B cells. The rescue of WEHI 231 cells by CD40 ligand engagement of its receptor prevented the increase in p27 induction. Inhibition of p27-ablated apoptosis induced upon expression of antisense c-myc RNA. Furthermore, specific induction of p27 gene expression resulted in apoptosis of WEHI 231 cells. Lastly, inhibition of expression of c-Myc, upon induction of an antisense c-myc RNA vector, was sufficient to induce increased p27 levels and apoptosis. Thus, these findings define a signaling pathway during B cell receptor engagement in which the drop in c-Myc levels leads to an increase in p27 levels that promotes apoptosis.
Bacterial/CpG DNA Down-Modulates Colony Stimulating Factor-1 Receptor Surface Expression on Murine Bone Marrow-Derived Macrophages with Concomitant Growth Arrest and Factor-Independent Survival
Authors: Sester David P. , Beasley Shannon J. , Sweet Matthew J. , Fowles Lindsay F. , Cronau Stephen L. , Stacey Katryn J. , Hume David A.
Published: December 2022

Unmethylated CpG motifs within bacterial DNA constitute a pathogen-associated molecular pattern recognized by the innate immune system. Many of the immunomodulatory functions of bacterial DNA can be ascribed to the ability to activate macrophages and dendritic cells. Here we show stimulatory DNA, like LPS, caused growth arrest of murine bone marrow-derived macrophages proliferating in CSF-1. Stimulatory DNA caused selective down-modulation of CSF-1 receptor surface expression. Flow cytometric analysis of CSF-1-deprived bone marrow-derived macrophages revealed that in contrast to the synchronous reduction of CSF-1 receptor upon CSF-1 addition, activating DNA (both bacterial DNA and CpG-containing oligonucleotide) caused rapid removal of receptor from individual cells leading to a bimodal distribution of surface expression at intermediate times or submaximal doses of stimulus. Despite causing growth arrest, both stimulatory DNA and LPS promoted factor-independent survival of bone marrow-derived macrophages, which was associated with phosphorylation of the mitogen-activated protein kinase family members, extracellular-regulated kinase 1 and 2. CSF-1 receptor down-modulation may polarize the professional APC compartment to the more immunostimulatory dendritic cell-like phenotype by suppressing terminal macrophage differentiation mediated by CSF-1.
Membrane Topology and Dimerization of the Two Subunits of the Transporter Associated with Antigen Processing Reveal a Three-Domain Structure
Authors: Vos Jan C. , Spee Pieter , Momburg Frank , Neefjes Jacques
Published: December 2022

Presentation of peptides derived from cytosolic and nuclear proteins by MHC class I molecules requires their translocation across the membrane of the endoplasmic reticulum (ER) by a specialized ABC (ATP-binding cassette) transporter, TAP. To investigate the topology of the heterodimeric TAP complex, we constructed a set of C-terminal deletions for the TAP1 and TAP2 subunits. We identified eight and seven transmembrane (TM) segments for TAP1 and TAP2, respectively. TAP1 has both its N and C terminus in the cytoplasm, whereas TAP2 has its N terminus in the lumen of the ER. A putative TM pore consists of TM1–6 of TAP1 and, by analogy, TM1–5 of TAP2. Multiple ER-retention signals are present within this region, of which we positively identified TM1 of both TAP subunits. The N-terminal domain containing TM1–6 of TAP1 is sufficient for dimerization with TAP2. A second, independent dimerization domain, located between the putative pore and the nucleotide-binding cassette, lies within the cytoplasmic peptide-binding domains, which are anchored to the membrane via TM doublets 7/8 and 6/7 of TAP1 and TAP2, respectively. We present a model in which TAP is composed of three subdomains: a TM pore, a cytoplasmic peptide-binding pocket, and a nucleotide-binding domain.
NADPH Oxidase Activation and Assembly During Phagocytosis
Authors: DeLeo Frank R. , Allen Lee-Ann H. , Apicella Michael , Nauseef William M.
Published: December 2022

Generation of superoxide (O2−) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.
Silymarin Suppresses TNF-Induced Activation of NF-κB, c-Jun N-Terminal Kinase, and Apoptosis
Authors: Manna Sunil K. , Mukhopadhyay Asok , Van Nguyen T. , Aggarwal Bharat B.
Published: December 2022

Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-inflammatory, cytoprotective, and anticarcinogenic effects. How silymarin produces these effects is not understood, but it may involve suppression of NF-κB, a nuclear transcription factor, which regulates the expression of various genes involved in inflammation, cytoprotection, and carcinogenesis. In this report, we investigated the effect of silymarin on NF-κB activation induced by various inflammatory agents. Silymarin blocked TNF-induced activation of NF-κB in a dose- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of ΙκBα, an inhibitor of NF-κB. Silymarin blocked the translocation of p65 to the nucleus without affecting its ability to bind to the DNA. NF-κB-dependent reporter gene transcription was also suppressed by silymarin. Silymarin also blocked NF-κB activation induced by phorbol ester, LPS, okadaic acid, and ceramide, whereas H2O2-induced NF-κB activation was not significantly affected. The effects of silymarin on NF-κB activation were specific, as AP-1 activation was unaffected. Silymarin also inhibited the TNF-induced activation of mitogen-activated protein kinase kinase and c-Jun N-terminal kinase and abrogated TNF-induced cytotoxicity and caspase activation. Silymarin suppressed the TNF-induced production of reactive oxygen intermediates and lipid peroxidation. Overall, the inhibition of activation of NF-κB and the kinases may provide in part the molecular basis for the anticarcinogenic and anti-inflammatory effects of silymarin, and its effects on caspases may explain its role in cytoprotection.
IL-12 Drives IFN-γ-Dependent Autoimmune Kidney Disease in MRL-FaslprMice
Authors: Schwarting A. , Tesch G. , Kinoshita K. , Maron R. , Weiner H. L. , Kelley V. Rubin
Published: December 2022

IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Faslpr mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-γ, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-γ-secreting T cells within the kidney, which, in turn, incites injury in MRL-Faslpr mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Faslpr tubular epithelial cells (IL-12 “carrier cells”), which were implanted under the kidney capsule of MRL-Faslpr mice before renal disease for a sustained period (28 days). IL-12 “carrier cells” generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4−CD8− B220+) T cells adjacent to the implant site. We detected more IFN-γ-producing T cells (CD4 > CD8 > CD4−CD8− B220+) at 28 days (73 ± 14%) as compared with 7 days (20 ± 8%) after implanting the IL-12 “carrier cells;” the majority of these cells were proliferating (60–70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-γ was required for IL-12-incited renal injury, because IL-12 “carrier cells” failed to elicit injury in MRL-Faslpr kidneys genetically deficient in IFN-γ receptors. Furthermore, IFN-γ “carrier cells” elicited kidney injury in wild-type MRL-Faslpr mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-γ-secreting CD4, CD8, and CD4−CD8− B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Faslpr mice.
The MHC class II-associated invariant chain regulates TLR7 trafficking and signaling in B cells (APP3P.100)
Authors: Manoury Benedicte , Maschalidi Sophia , Achour Karim , Lennon-Duménil Ana-Maria , Tohmé Mira
Published: December 2022

Intracellular Toll-like receptors localise in endosomes and sense nucleotides from viruses and bacteria. This recognition induces their conformational changes resulting in the production of proinflammatory cytokines and MHC class II (MHCII) antigenic presentation. TLR9 trafficking and signalling has been well described, however, regulation of Toll-like receptor 7 which recognizes single-stranded RNA from viruses in endosomes is still largely unknown. Increasing evidences describe a cross-talk between proteins that regulate both innate and adaptive responses. For example, UNC93B1, a chaperone essential for intracellular TLRs trafficking also interferes with the MHCII antigen presentation pathway and MHCII molecules were recently described to regulate TLR9 signaling. Here, we show, that in B cells, in contrast to dendritic cells, at the steady state and after stimulation, TLR7 and TLR9 reside in the lysosomes together with the MHCII molecule, the invariant chain (Ii) and H-2 DM. Following stimulation by TLR7 ligand, Ii specifically interacts with TLR7 but not with TLR9 and its adaptor molecule MyD88. Surprisingly, in the absence of Ii chain or when its expression is downregulated upon viral infection, TLR7 but not TLR9 is localized in the endoplasmic reticulum leading to an exacerbation of TLR7 innate but not adaptive function. This suggests a new role for Ii chain by acting as a negative regulator of TLR7 signaling in B cells
Targeting antigen to CD40 reinforces dendritic cells to cross-prime antigen-specific CD8+ T cells (APP3P.110)
Authors: Yin Wenjie , Gorvel Laurent , Li Dapeng , Joo HyeMee , Duluc Dorothée , Upchurch Katherine , Gu Chao , Ouedraogo Richard , Xue Yaming , Wang Zhiqing , Zurawski Sandra , Gorvel Jean-Pierre , Zurawski Gerard , Oh SangKon
Published: December 2022

Dendritic cells are major antigen-presenting cells that can efficiently cross-prime antigen-specific CD8+ T cells. Thus, targeting antigen to dendritic cells via surface receptors is an appealing strategy to mount CD8+ T cell-mediated immunity against intracellular pathogens and cancers. Nonetheless, which targeted receptor is the most efficient at priming CD8+ T cells remains elusive. Herein, we report the superior function of CD40 over nine different lectins and scavenger receptors at priming antigen-specific CD8+ T cells. A quantitative analysis of intracellular trafficking of antibody-bound receptors revealed that αCD40 monoclonal antibody localized mainly at the plasma membrane and subsequently accumulated at early endocytic compartments whereas αLOX-1 and αDectin-1 monoclonal antibodies localized at both early and late endocytic compartments in dendritic cells. Regardless of the differences in their subcellular localizations, targeting antigen to CD40 and lectins resulted in the same pattern of peptide epitope-specific IFNg+CD8+ T cell responses. We also report that poly(I:C) can significantly enhance both CD8+ and CD4+ T cell responses elicited by targeting antigen to CD40 in both human in vitro and in human CD40 transgenic mouse in vivo settings. This study provides key information for the rational design of DC-targeting vaccines against cancers and intracellular pathogens.
Implications of T cell receptor cross-reactivity on the CD4+ T cell repertoire (APP3P.111)
Authors: Nelson Ryan , Jenkins Marc
Published: December 2022

Naïve CD4+ T cell populations with T cell receptors (TCR) specific for different major histocompatibility complex II (MHCII)-bound foreign peptides vary in size for unknown reasons. Negative selection on self peptides could play a role. We found that MHCII-binding nonamer peptides only had to have the same residues at five positions to be recognized by the same TCR. This degree of TCR cross-reactivity between self and foreign peptides reduced the sizes of foreign peptide-specific T cell populations via clonal deletion. Small foreign p:MHCII-specific populations that underwent a greater degree of cross-reactive negative selection were on average lower affinity and produced more variable effector cell responses to immunization. In contrast, large foreign p:MHCII-specific T cell populations with minimal evidence of negative selection were higher affinity and produced larger responses to immunization. Reciprocally, an incompletely deleted naïve T cell population specific for a tissue-restricted self peptide could be triggered by similar microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides influences the size of certain foreign peptide-specific T cell populations, and may allow tissue-restricted self peptide-specific populations to cause autoimmunity after infection.
T cell-dependent and -independent regulatory roles of a new inhibitory molecule VISTA (APP3P.115)
Authors: Ohno Tatsukuni , Kondo Yuta , Azuma Miyuki
Published: December 2022

[Objective] VISTA is a new member of the CD28-B7 family co-inhibitory molecules. VISTA is expressed on T cells and myeloid cells, but not on B cells. It seems that VISTA functions as a co-inhibitory molecule, but the actual contribution as a receptor or ligand has not been clear. Here, we generated a new mAb against VISTA and examined the effects on T-dependent and -independent immune responses. [Results and Discussion] We generated a new anti-VISTA mAb (MIH63). In a T-cell-mediated DNFB-induced contact hypersensitivity model, treatment with MIH63 at sensitization, but not at challenge, enhanced ear swelling. In the MIH63-treated mice, the ratio of CD8+ T cells was markedly reduced, whereas the ratio of IFN-g+ effector CD8+ T cells was enhanced. Blockade of FcgR by 2.4G2 cancelled the reduction of CD8+ T cells by MIH63 without affecting ear swelling and CD8+ T cell activation. These results suggest that MIH63 possesses both antagonistic and ADCC abilities. Adaptive transfer of MIH63-treated sensitized T cells enhanced ear swelling after ear challenge. In a T cell-independent LPS-induced shock model, treatment with MIH63 markedly exacerbated the mortality and increased plasma TNFα and IL-6 levels and liver transaminases (AST/ALT). Our results demonstrated that treatment with anti-VISTA mAb enhanced both T cell-dependent and -independent immune responses, suggesting regulatory roles of VISTA. We will further report the actual contributing cells that express VISTA.
B cell directed tolerogenic fusion proteins (APP3P.117)
Authors: Zhang Ai-Hong , Wang Hong , Kim Yongchan , Scott David
Published: December 2022

We previously demonstrated that retroviral delivery of antigen-IgG fusion proteins in B cells ex vivo rendered these cells tolerogenic. Such B cells, upon adoptive transfer, could block or reverse the adverse immune responses in rodent models of multiple sclerosis, type 1 diabetes and hemophilia. In order to create a non-viral­ delivery system taking advantage of the tolerogenicity of IgG fusion proteins directed into B cells, we have prepared a fusion comprising anti-human CD20 single chain variable fragment and with target T-cell epitope on a human IgG4 scaffold. We term this fusion a B-cell-specific antigen IgG tolerogen (BAIT). As an example, the BAIT fusion containing a DR1 restricted T cell epitope FVIII2191-2210 (BAIT-FVIII2191) was prepared in a CHO cell expression system via stable transfection. Ex vivo analyses demonstrated that biotinylated BAIT-FVIII2191 could be specifically taken up by CD20+ve B cells from human PBMC. In this report, we will evaluate the presentation of B-cell directed BAIT-FVIII2191 to FVIII2191-2210 specific human T cells, and then test in vivo using DR1-transgenic FVIII KO mice. The results obtained from these studies will facilitate the translation of our successful B-cell gene therapy approach into a novel protein therapeutic for adverse immune responses, such as anti-FVIII inhibitor development in hemophilia A.
NLRC5 regulates MHC-I expression and immunity during viral infection (APP3P.101)
Authors: Lupfer Christopher , Stokes Kate , Kanneganti Thirumala-Devi
Published: December 2022

NLRC5 is a member of the NOD-Like receptor family and reportedly functions in the regulation of type I interferons and inflammasome activation. In addition, NLRC5 functions as a transcriptional transactivator of MHC-I gene expression. We have observed that Nlrc5-deficient mice infected with influenza A virus have normal type I interferon responses and inflammasome activation but have dramatically reduced H-2Db and H-2Kb major histocompatibility complex (MHC)-I allele expression on virtually all myeloid and lymphoid cells. Furthermore, Nlrc5-/- mice have diminished capacity for CD8+ T cell responses directed toward influenza A virus following infection. These data suggest that during influenza A virus infection, NLRC5 plays an important function in the generation of adaptive immunity but has no detectable role in the innate immune response.
Retina-specific T cells provide neuroprotection in a mouse model of glaucoma (BA6P.140)
Authors: Kielczewski Jennifer , Horai Reiko , Caspi Rachel
Published: December 2022

Glaucoma is a disease that leads to degeneration of retinal ganglion cells (RGC) and is potentially blinding if left untreated. Based on the concept of “beneficial autoimmunity”, we examined whether naïve, retina-specific T lymphocytes can provide neuroprotection to retinal ganglion cells in an optic nerve crush injury glaucoma model. Purified T cells from mice that express the transgenic R161H TCR for the retinal antigen IRBP, as well as eGFP (R161H-eGFP), were adoptively transferred into wild type (WT) mice. Also, irradiated bone marrow (BM) chimeras were stably engrafted with <10% circulating R161H-eGFP cells. Recipient mice were subjected to retrograde fluorogold labeling for quantification of RGC prior to an optic nerve crush injury. Retinas were imaged 4 or 7 days later for quantification of RGCs and donor R161H-eGFP T cells were detected by confocal microscopy. In both types of recipients, T cell influx into the eye and neuroprotection were apparent, but differed in kinetics. In adoptively transferred mice, R161H T cells were detected in the eye 4 days post injury and were associated with 27% less RGC loss compared to controls infused with normal polyclonal eGFP+ T cells (p<0.05). In the BM chimeric mice, major influx of R161H-eGFP cells occurred at 7 days post injury and was associated with a 16% statistically significant reduction in RGC loss. We conclude that naïve retina-specific T cells can mediate neuroprotection of retinal ganglion cells.
A novel rodent model for autoimmune pulmonary alveolar proteinosis (BA6P.123)
Authors: Ferretti Andrew , Fortwendel Jarrod , Gebb Sarah , Barrington Robert
Published: December 2022

Pulmonary alveolar proteinosis (PAP) is a rare lung syndrome caused by the accumulation of surfactants in the alveoli. The most prevalent clinical form of PAP is autoimmune (aPAP) whereby IgG autoantibodies neutralize the cytokine colony stimulating factor 2 (Csf2, formerly GM-CSF). Csf2 is a pleiotropic cytokine that promotes the differentiation, survival, and activation of alveolar macrophages (AM), the cells responsible for surfactant catabolism. IgG-mediated neutralization of Csf2 thereby inhibits AM homeostasis and function, leading to an innate immunodeficiency with increased susceptibility to opportunistic infections. Importantly, there are no rodent models for this disease, and therefore underlying immune mechanisms regulating Csf2-specific IgG in aPAP are not well understood. Herein, we identify autoimmune-prone Rasgrp1-deficient (KO) mice as the first rodent model of aPAP: 1) analysis of lung sections revealed an eosinophilic, periodic acid Schiff positive proteinaceous accumulation in the alveoli; 2) KO mice exhibit reduced pulmonary compliance; 3) AM from KO mice are enlarged, exhibit impaired protein levels and have reduced surfactant degradation; and 4) serum Csf2-specific IgG are increased (ELISA) in KO mice. Finally, while control mice infected intranasally with Aspergillus fumigatus clear conidia readily, KO mice failed to clear conidia from the lungs. Collectively these data suggest that KO mice represent the first rodent model for aPAP.
Ex vivo killing of primary melanocytes by syngeneic mononuclear cells from active, autoimmune vitiligo lesions in Smyth chickens (BA7P.152)
Authors: Falcon Daniel , Dienglewicz Robert , Erf Gisela
Published: December 2022

In humans autoimmune vitiligo is characterized by the loss of pigment-producing melanocytes in the skin. The vitiligo-prone Smyth line (SL) chicken together with the MHC-matched parental Brown line (BL) and vitiligo-resistant Light Brown Leghorn line of chickens constitute a spontaneous, non-transgenic animal model for this disorder. Cell-mediated immunity (CMI) is known to contribute to the etiopathology of vitiligo. Furthermore, CMI was demonstrated in vivo in vitiliginous SL chickens based on the delayed wattle response (DWR) to melanocyte lysates. Immunohistochemical analysis of DWR-positive tissue revealed extensive T cell infiltration indicative of a CMI response. In order to examine CMI in SL vitiligo (SLV) ex vivo we developed a co-culture assay using primary effector and target cells from growing feathers (the target tissue). Mononuclear cells (MNCs) were harvested from feathers of chickens with active SLV and BL controls. Target cells (melanocytes) were isolated from pigmented feathers prior to development of SLV. To detect melanocyte-specific death a modified flow cytometric method of Lee-MacAry et al (2001) was used. Regardless of whether melanocytes were isolated from chickens that did or did not eventually develop SLV, co-culture with MNCs from SLV chickens resulted in greater melanocyte death compared to controls. Use of this ex vivo system with sorted MNCs from active, autoimmune SLV lesions will help identify roles of individual subsets in melanocyte death.
Differential effects of IFNβ on IL-12, IL-23, and IL-10 expression in TLR-stimulated dendritic cells (BA7P.157)
Authors: Yen Jui-Hung , Kong Weimin , Hooper Kirsten , Emig Fran , Rahbari Kate , Kuo Ping-Chang , Scofield Barbara , Ganea Doina
Published: December 2022

Autoimmune disease multiple sclerosis (MS) is characterized by immune cell infiltration in the CNS leading to cumulative disability. IFNβ, used clinically in remitting-relapsing MS, reduces lesion formation and rate of relapses. Although the molecular mechanisms are not entirely elucidated, myeloid cells appear to be a major target for the therapeutic effects of IFNβ. Dendritic cells (DC) play a critical role in experimental models of MS, through their effect on Th1/Th17 cell differentiation and expansion. Here we studied the effects of IFNβ on DC expression of cytokines involved in Th1/Th17 differentiation. Administration of IFNβ to EAE mice inhibited IL-12 and IL-23 expression in splenic DC and reduced in vivo differentiation of Th1/Th17 cells. IFNβ affected cytokine expression in TLR-stimulated DC in a similar manner in vitro, inhibiting IL-12 and IL-23 and stimulating IL-10 at both mRNA and protein levels, by signaling through IFNAR. We investigated the role of the signaling molecules STAT1/STAT2, IRF1 and IRF7, and of the PI3KàGSK3 pathway. Inhibition of IL-12 p40 and p35 by IFNβ was mediated through STAT1/STAT2, whereas inhibition of IL-23 was STAT1-depedent, and the stimulatory effect on IL-10 expression was mediated through STAT2. Although IFNβ induces IRF-7 and IRF-1, neither IRF mediated the effects of IFNβ on IL-12, IL-23, or IL-10. We found that the PI3K pathway mediated IL-12 inhibition but did not interfere with the inhibition of IL-23 or stimulation of IL-10.
Natural killer cells protect against cardiac inflammation and fibrosis by directly limiting eosinophilic accumulation (BA7P.144)
Authors: Ong SuFey , Ligons Davinna , Barin Jobert , Wu Lei , Talor Monica , Diny Nicola , Fontes Jillian , Gebremariam Elizabeth , Rose Noel , Cihakova Daniela
Published: December 2022

Myocarditis is the inflammation of the cardiac muscle and is among the leading causes of sudden cardiac failure in young adults. We demonstrate here that natural killer (NK) cells, a subset of the Type I innate lymphoid cell (ILC) group, suppress experimental autoimmune myocarditis (EAM). EAM was initiated in BALB/c mice by immunization with myocarditogenic peptide emulsified in complete Freund's adjuvant. During disease, activated cardiac NK cells accumulated in the heart, secreted interferonγ, perforin, and granzyme-B, and expressed activation markers CD69, TRAIL, and CD27. The depletion of NK cells during EAM with anti-asialo GM1 antibody resulted in a 10-fold increase from 0.12 - 12.0% in the percentage of eosinophils found in the cardiac tissue at the peak of disease. This was accompanied by significantly increased myocarditis severity and elevated fibrosis. Eosinophils were required for the change in disease severity in the absence of NK cells, as treatment with anti-asialo GM1 did not increase myocarditis severity in eosinophil-deficient ΔdoubleGATA1 mice. We show that NK cells limited eosinophilic infiltration during EAM indirectly through altering eosinophil-related chemokine production by resident cardiac fibroblasts both in vitro and in vivo. Additionally, the co-culture of NK cells and eosinophils directly induced eosinophil apoptosis in vitro. Altogether, we define a new pathway of eosinophilic regulation through interactions with NK cells.
Identification of genes/pathways responsible for resistance vs susceptibility to EAE utilizing HLA class-II transgenic mice (BA8P.163)
Authors: Mangalam Ashutosh , Luckey David , Smart Michele , Sarangi Vivekananda , Giri Shailendra , Rodriguez Moses , David Chella
Published: December 2022

MS is an autoimmune disease of the CNS and among all the genetic factors; MHC class-II molecules show the strongest association. To better understand the role of MHC class-II (HLA-DR/DQ) genes in susceptibility and resistance to MS, we utilized transgenic (Tg) mice expressing HLA class-II gene lacking endogenous mouse class II genes. HLA-DR3 Tg mice are susceptible to PLP91-110 induced experimental autoimmune encephalomyelitis (EAE), whereas DQ8 (DQB1*0302) transgenic mice are resistant. To identify the gene expression changes and pathways required for disease susceptibility vs resistance to EAE, we performed mRNA expression profiling of CD4+ T cells from HLA-DR3 (susceptible) and HLA-DQ8 (resistant) Tg mice. Whole genome expression analysis identified 2945 genes in HLA-DR3 Tg mice (PLP91-110 immunized vs. naïve) and 1053 genes in HLA-DQ8 Tg mice (PLP91-110 immunized vs. naïve). Comparison of modulated genes between DR3 and DQ8 identified 772 genes common to both strains, 2173 genes unique to DR3 whereas 281 genes were unique to DQ8 mice. Ingenuity pathway analysis showed up regulation of pathways involved in T cell signaling in inflammation, NF-kB signaling and those responsible for communication between innate and adoptive immunity. Majority of these pathways have IL1β as a major upstream regulator in disease susceptible HLA-DR3 Tg mice. Thus microarray data indicates an important role of IL1β induced encephalitogenic T helper cell differentiation in disease susceptibility.