bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-γ Exposes a Cryptic Cytotoxic T Lymphocyte Epitope in HIV-1 Reverse Transcriptase
Authors: Sewell Andrew K. , Price David A. , Teisserenc Helene , Booth Bruce L. , Gileadi Uzi , Flavin Fiona M. , Trowsdale John , Phillips Rodney E. , Cerundolo Vincenzo
Published: December 2022

The proteasome, an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells, is responsible for the degradation of most cellular proteins and is believed to be the main source of MHC class I-restricted antigenic peptides for presentation to CTL. Inhibition of the proteasome by lactacystin or various peptide aldehydes can result in defective Ag presentation, and the pivotal role of the proteasome in Ag processing has become generally accepted. However, recent reports have challenged this observation. Here we examine the processing requirements of two HLA A*0201-restricted epitopes from HIV-1 reverse transcriptase and find that they are produced by different degradation pathways. Presentation of the C-terminal ILKEPVHGV epitope is impaired in ME275 melanoma cells by treatment with lactacystin, and is independent of expression of the IFN-γ-inducible proteasome β subunits LMP2 and LMP7. In contrast, both lactacystin treatment and expression of LMP7 induce the presentation of the N-terminal VIYQYMDDL epitope. Consistent with these observations we show that up-regulation of LMP7 by IFN-γ enhances presentation of the VIYQYMDDL epitope. Hence interplay between constitutive and IFN-γ-inducible β-subunits of the proteasome can qualitatively influence Ag presentation. These observations may have relevance to the patterns of immunodominance during the natural course of viral infection.
Human Virus-Specific CD8+ CTL Clones Revert from CD45ROhigh to CD45RAhigh In Vivo: CD45RAhighCD8+ T Cells Comprise Both Naive and Memory Cells
Authors: Wills Mark R. , Carmichael Andrew J. , Weekes Michael P. , Mynard Kim , Okecha Georgina , Hicks Ray , Sissons J. G. Patrick
Published: December 2022

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA− population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.
Therapeutic Protective Effects of IL-12 Combined with Soluble IL-4 Receptor Against Established Infections of Herpes Simplex Virus Type 1 in Thermally Injured Mice
Authors: Kobayashi Hiroyuki , Kobayashi Makiko , Utsunomiya Tokuichiro , Herndon David N. , Pollard Richard B. , Suzuki Fujio
Published: December 2022

The effect of combination therapy between IL-12 and soluble IL-4R (sIL-4R) on the established infection of HSV-1 in thermally injured mice (TI mice) was investigated. All of the TI mice infected with lethal amounts of HSV-1 died when IL-12 was given therapeutically at a dose of 500 U/mouse. However, 80% of these mice treated prophylactically with IL-12 survived compared with 0% survival of the same mice treated with saline. The therapeutic administration of IL-12 to TI mice currently infected with HSV-1 caused an 80% survival of these mice when the treatment was combined with sIL-4R. Although IL-12 did not stimulate IFN-γ production in cultures of splenic T cells from TI mice, IFN-γ was produced by stimulation with IL-12 when the producer cells were prepared from TI mice that had been treated previously with sIL-4R. After stimulation with anti-CD3 mAb, splenic T cells from TI mice with the established infection of HSV-1 produced IL-4 into their culture fluids. However, IL-4 was not produced by splenic T cells that were prepared from the same infected mice treated with IL-12 and sIL-4R in combination. The results obtained herein indicate that the efficacies of the combination therapy against the established infection of HSV-1 may result from the IFN-γ production stimulated by IL-12 in TI mice that are treated with sIL-4R for reducing burn-associated type 2 T cell responses.
The Promoter of the C1 Inhibitor Gene Contains a Polypurine·Polypyrimidine Segment that Enhances Transcriptional Activity
Authors: Zahedi Kamyar , Bissler John J. , Prada Anne E. , Prada Jorge A. , Davis Alvin E.
Published: December 2022

The C1 inhibitor (C1INH) promoter is unusual in two respects: 1) It contains no TATA sequence, but instead contains a TdT-like initiator element (Inr) at nucleotides −3 to +5; 2) it contains a polypurine·polypyrimidine tract between nucleotides −17 and −45. Disruption of the Inr by the introduction of point mutations reduced promoter activity by 40%. A TATA element inserted at nucleotide −30 in the wild-type promoter and in promoter constructs containing the mutated Inr led to a 2-fold increase in basal promoter activity. Previous studies suggested that the potential hinged DNA-forming polypurine·polypyrimidine tract might be important in the regulation of C1INH promoter activity. The present studies indicate that this region is capable of such intramolecular triple helix formation. Disruption of the polypurine·polypyrimidine sequence by substitution of 5 of the 23 cytosine residues with adenine prevented triple helix formation. Site-directed mutagenesis experiments demonstrate that the regulation of promoter activity is independent of hinged DNA-forming capacity but requires an intact AC box (ACCCTNNNNNACCCT) or the overlapping PuF binding site (GGGTGGG). The C1INH gene also contains a number of potential regulatory elements, including an Sp-1 and an hepatocyte nuclear factor-1 binding site and a CAAT box. The role of these elements in regulation of the C1INH promoter was examined. Elimination of the hepatocyte nuclear factor-1 site at nucleotides −94 to −81 by truncation reduced the activity of the promoter by ∼50%. Similarly, site-directed mutations that disrupt this site reduce promoter activity by 70%.
Recognition of Class I MHC by NK Receptor Ly-49C: Identification of Critical Residues
Authors: Lian Rebecca H. , Li Yan , Kubota Satoko , Mager Dixie L. , Takei Fumio
Published: December 2022

The Ly-49 family of inhibitory receptors plays a major role in regulating mouse NK cell cytotoxicity. Two of its members, Ly-49C and I, are recognized by the mAb 5E6, which also defines a subset of NK cells involved in the hybrid resistance phenomenon. Previous studies have shown that Ly-49C binds to a broad spectrum of class I MHC molecules, while Ly-49I apparently does not bind to any class I MHC molecules tested. In the present investigation we have defined the amino acid residues of Ly-49C that are critical for determining its ligand specificities. First, using quantitative COS cell adhesion assays, we demonstrated that Ly-49CB6 bound to Dd, Db, Kb, or Kk as well as to murine leukemic cell lines GM979 (H-2s) and IC-21 (H-2b). In contrast, COS cells expressing Ly-49IB6 did not significantly bind to any of the class I MHC tested. To determine which amino acid residues of Ly-49C are critical for their specific binding to class I MHC, a series of chimeric and mutant Ly-49C and I were generated and tested. Exchanging the critical residues between Ly-49C and I significantly affected their binding specificities. Finally, we identified the epitopes on Ly-49C recognized by mAbs 5E6 and 4LO3311 that functionally inhibit Ly-49C recognition of its ligands. These results further define the class I specificities of Ly-49C and provide insight into the structural basis for how class I MHC is recognized by the Ly-49 family of NK receptors.
The Th1/Th2 Balance Does Not Account for the Difference of Susceptibility of Mouse Strains to Theiler’s Virus Persistent Infection
Authors: Monteyne Philippe , Bihl Franck , Levillayer Florence , Brahic Michel , Bureau Jean-François
Published: December 2022

Theiler’s virus causes a persistent infection with demyelination that is studied as a model for multiple sclerosis. Inbred strains of mice differ in their susceptibility to viral persistence due to both H-2 and non-H-2 genes. A locus with a major effect on persistence has been mapped on chromosome 10, close to the Ifng locus, using a cross between susceptible SJL/J and resistant B10.S mice. We now confirm the existence of this locus using two lines of congenic mice bearing the B10.S Ifng locus on an SJL/J background, and we describe a deletion in the promoter of the Ifng gene of the SJL/J mouse. We studied the expression of IFN-γ, IL-2, IL-10, and IL-12 in the brains of SJL/J mice, B10.S mice, and the two lines of congenic mice during the first 2 wk following inoculation. We found a greater expression of IFN-γ and IL-2 mRNA in the brains of B10.S mice compared with those of SJL/J mice. Also, the ratio of IL-12 to IL-10 mRNA levels was higher in B10.S mice. However, the cytokine profiles were the same for the two lines of resistant congenic mice and for susceptible SJL/J mice. Therefore, the difference of Th1/Th2 balance between the B10.S and SJL/J mice is not due to the Ifng locus and does not account for the difference of susceptibility of these mice to persistent infection.
Antibody Prevents Virus Reactivation Within the Central Nervous System
Authors: Lin Mark T. , Hinton David R. , Marten Norman W. , Bergmann Cornelia C. , Stohlman Stephen A.
Published: December 2022

The neurotropic JHM strain of mouse hepatitis virus (JHMV) produces an acute CNS infection characterized by encephalomyelitis and demyelination. The immune response cannot completely eliminate virus, resulting in persistence associated with chronic ongoing CNS demyelination. The contribution of humoral immunity to viral clearance and persistent infection was investigated in mice homozygous for disruption of the Ig μ gene (IgM−/−). Acute disease developed with equal kinetics and severity in IgM−/− and syngeneic C57BL/6 (wt) mice. However, clinical disease progressed in IgM−/− mice, while wt mice recovered. Viral clearance during acute infection was similar in both groups, supporting a primary role of cell-mediated immunity in viral clearance. In contrast to wt mice, in which infectious virus was reduced to below detection following acute infection, increasing infectious virus was recovered from the CNS of the IgM−/− mice following initial clearance. No evidence was obtained for selection of variant viruses nor was there an apparent loss of cell-mediated immunity in the absence of Ab. Passive transfer of anti-JHMV Ab following initial clearance prevented reactivation of infectious virus within the CNS of IgM−/− mice. These data demonstrate the clearance of infectious virus during acute disease by cell-mediated immunity. However, immunologic control is not maintained in the absence of anti-viral Ab, resulting in recrudescence of infectious virus. These data suggest that humoral immunity plays no role in controlling virus during acute infection, but plays an important role in establishing and maintaining CNS viral persistence.
Antiproliferative Effect of IL-1 Is Mediated by p38 Mitogen-Activated Protein Kinase in Human Melanoma Cell A375
Authors: Itoh Saotomo , Hattori Takayuki , Hayashi Hidetoshi , Mizutani Yukiko , Todo Makoto , Takii Takemasa , Yang De , Lee John C. , Matsufuji Senya , Murakami Yasuko , Chiba Taku , Onozaki Kikuo
Published: December 2022

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.
Targeting of Human Dendritic Cells by Autologous NK Cells
Authors: Wilson Julia L. , Heffler Lena C. , Charo Jehad , Scheynius Annika , Bejarano Maria-Teresa , Ljunggren Hans-Gustaf
Published: December 2022

NK cells have the capacity to spontaneously kill tumor cell lines, in particular cell lines of hemopoietic origin. In contrast, they do not generally kill nontransformed autologous cells. However, here we demonstrate that short-term activated polyclonal human NK cells, as well as human NK cell lines, efficiently lyse autologous dendritic cells (DC) derived from peripheral blood monocytes as well as Langerhans-like cells derived from CD34+ stem cells isolated from umbilical cord blood. Lysis of autologous DC by short-term activated NK cells and NK cell lines was dependent on granule exocytosis, since total abrogation of lysis was observed in the presence of EGTA. Induction of DC maturation by LPS, monocyte conditioned media (MCM), or stimulation through CD40 ligand (CD40L) rendered the DC less susceptible to lysis by NK cells. Infection of DC with influenza virus was likewise associated with a reduced susceptibility to lysis by NK cells. Thus, susceptibility to lysis by autologous NK cells is a particular property of immature DC. The present results are discussed in relation to the ability of DC to interact with NK cells and to the ability of NK cells to regulate development of specific immunity.
Follicular Dendritic Cells Protect Malignant B Cells from Apoptosis Induced by Anti-Fas and Antineoplastic Agents
Authors: Schwarz Ying X. , Yang Ming-yan , Qin Dahui , Wu Jiuhua , Jarvis W. David , Grant Steven , Burton Gregory F. , Szakal Andras K. , Tew John G.
Published: December 2022

The observation that follicular dendritic cells (FDC) reduce apoptosis in B cells prompted the hypothesis that FDC might enhance tumor cell survival by protecting malignant B cells from apoptotic death. To test this notion, apoptosis was induced in B cell lymphomas by anti-Fas or various antineoplastic agents in the presence and absence of FDC. Apoptosis was detected and quantified by TUNEL analysis. Induction of apoptosis with anti-Fas, etoposide, cyclophosphamide, and busulfan was markedly antagonized by FDC at FDC to B cell ratios of ≥1:16. For example, treatment with 10 ng/ml anti-Fas caused 60–90% of A20 cells to undergo apoptosis in 6 h, whereas addition of FDC reduced apoptosis to background levels (3–15%). Similarly, treatment with busulfan induced apoptosis in 55–80% of A20 cells, whereas addition of FDC reduced B cell death to ≤15%; moreover, depletion of FDC abrogated the protective actions. In contrast, the apoptosis-inducing effect of Adriamycin was not reversed by FDC. The ability to block apoptosis induced by anti-Fas or busulfan was not limited to A20 but was observed in four other malignant pre-B cell or B cell lines. The mechanism by which FDC spare malignant B cells from apoptosis did not involve alterations in levels of Bcl-2, Bcl-XL, or Bax. Collectively, these data raise the possibility that FDC may enhance tumor cell survival by protecting malignant B cells against apoptosis induced by anti-Fas and some but not all chemotherapeutic agents.
Ability of a Nondepleting Anti-CD4 Antibody to Inhibit Th2 Responses and Allergic Lung Inflammation Is Independent of Coreceptor Function
Authors: Li Li , Crowley Mary , Nguyen Andrea , Lo David
Published: December 2022

Nondepleting anti-CD4 Abs have been used in vivo to induce Ag-specific immunological tolerance in Th1 responses, including tissue allograft rejection and autoimmune diabetes. To examine whether this Ab (YTS177.9) acts by provoking a Th2 shift, we tested the effect in a mouse model of allergic lung inflammation. Interestingly, nondepleting anti-CD4 treatment induces tolerance to allergens as well, especially when given during initial priming. In vitro studies indicate that the effect of the Ab is independent of CD4 coreceptor function, as Ab treatment also inhibits proliferation and induces a persistent anergy in naive CD4 T cells stimulated by anti-CD3/CD28. Moreover, the Ab stimulated a distinct pattern of tyrosine phosphorylation in T cells even in the absence of TCR triggering, suggesting that signaling through CD4 alone induces significant physiological changes in T cell function. These results show that tolerance induced by anti-CD4 triggering is not a simple shift in Th1/Th2 effector function or depletion of Ag-specific cells, but may instead induce a persistent clonal anergy capable of blocking subsequent immunity.
Selective Pharmacologic Inhibition of Murine and Human IL-12-Dependent Th1 Differentiation and IL-12 Signaling
Authors: Coon Michael E. , Diegel Michael , Leshinsky Natasha , Klaus Stephen J.
Published: December 2022

We have previously shown that lisofylline (LSF) inhibits murine Th1-mediated disease in vivo by blocking IL-12-induced differentiation of Th1 cells. The cellular and molecular mechanisms underlying this inhibition were further explored by testing LSF in several IL-12-responsive model systems in vitro. IL-12-dependent Th1 differentiation was abrogated by LSF and yielded effector T cells that were deficient in proinflammatory cytokine secretion, including IFN-γ, IL-2, and TNF-α. The diminished Th1 phenotype resulted from both a lower frequency of IL-12-derived Th1 clones and a reduced capacity of individual clones to secrete IFN-γ due to lower levels of IFN-γ mRNA. The arrest in Th1 development resulted from a blockade of IL-12 signaling that preceded the Th0 to Th1 transition. Thus, LSF blocked IL-12-enhanced IFN-γ production in anti-CD3-stimulated T cells and prevented IL-12-mediated repression of the transcription factor GATA-3. Lisofylline also inhibited IL-12-induced increases in STAT4 tyrosine phosphorylation, but did not block TCR signaling or inhibit acquisition of IL-12 responsiveness. These findings were extended to show that LSF also inhibits IL-12-dependent responses in human T cells. LSF, which has one asymmetric chiral center, was selectively inhibitory for IL-12 signaling compared with its S-enantiomer (1501-S) and the oxidized side chain analog, pentoxifylline. The results suggest that LSF may be useful as a modulator of Th1-mediated disease in humans.
The −180 Site of the IL-2 Promoter Is the Target of CREB/CREM Binding in T Cell Anergy
Authors: Powell Jonathan D. , Lerner Cara G. , Ewoldt Gerald R. , Schwartz Ronald H.
Published: December 2022

Anergic T cells display a marked decrease in their ability to produce IL-2 even in the presence of optimal TCR and costimulatory signals. Using IL-2 enhancer/promoter-driven reporter constructs, we have previously identified a region that appears to be a target for cis transcriptional repression in anergy. This region of the promoter, which shares partial homology with a consensus AP-1-binding sequence, is located about −180 bp from the transcriptional start site. In the present study, we demonstrate that cAMP response element-binding protein/cAMP response element modulator (CREB/CREM), activating transcription factor-2/c-Jun, and Jun-Jun/Oct complexes bind to this site. However, the induction of anergy by prolonged stimulation through the TCR led to an increase in binding of only the CREB/CREM complex. Furthermore, the level of binding of this complex appeared to be up-regulated in both resting and restimulated anergic T cells. Finally, an IL-2 promoter-driven reporter construct that contained a mutation that specifically reduced the binding of the CREB/CREM complex displayed a decreased ability to be affected by anergy, while a construct that contained a mutation that decreased the binding of the Jun-Jun/Oct complex was still susceptible to anergy. These findings suggest that the −180 region of the IL-2 promoter is the target of a CREB/CREM transcriptional inhibitor that contributes to the repression of IL-2 production in T cell anergy.
Contributions of Leukemia Inhibitory Factor Receptor and Oncostatin M Receptor to Signal Transduction in Heterodimeric Complexes with Glycoprotein 130
Authors: Hermanns Heike M. , Radtke Simone , Haan Claude , Schmitz-Van de Leur Hildegard , Tavernier Jan , Heinrich Peter C. , Behrmann Iris
Published: December 2022

Leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M (OSM) lead to heterodimerization of LIF receptor (LIFR) or the OSM-specific receptor (OSMR) with glycoprotein (gp) 130, the common receptor subunit for IL-6-type cytokines. Thereby intracellular signaling via Janus kinases (Jaks) and STAT transcription factors is initiated. We investigated the contributions of LIFR and OSMR to signal transduction in the context of heterodimers with gp130. Chimeric receptors based on the extracellular parts of the IL-5R α- and β-chains were generated, allowing the induced heterodimerization of two different cytoplasmic tails. Our studies demonstrate that upon heterodimerization with the gp130 cytoplasmic region, the cytoplasmic parts of both LIFR and OSMR were critical for activation of an acute phase protein promoter in HepG2 hepatoma cells. The membrane-proximal region of LIFR or OSMR was crucial for the ability of such receptor complexes to induce DNA binding of STAT1 and STAT3 in COS-7 cells. Membrane-distal regions of LIFR and OSMR contributed to STAT activation even in the absence of gp130 STAT recruitment sites. We further show that the Janus kinases Jak1 and Jak2 constitutively associated with receptor constructs containing the cytoplasmic part of LIFR, OSMR, or gp130, respectively. Homodimers of the LIFR or OSMR cytoplasmic regions did not elicit responses in COS-7 cells but did in HepG2 cells and in MCF-7 breast carcinoma cells. Thus, in spite of extensive functional similarities, differential signaling abilities of gp130, LIFR, and OSMR may become evident in a cell-type-specific manner.
Nitric Oxide Plays a Critical Role in the Recovery of Lewis Rats from Experimental Autoimmune Encephalomyelitis and the Maintenance of Resistance to Reinduction
Authors: O’Brien Nikki C. , Charlton Brett , Cowden William B. , Willenborg David O.
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS and an animal model for the human demyelinating disease, multiple sclerosis. In the Lewis rat, myelin basic protein (MBP)-CFA-induced EAE is an acute monophasic disease from which animals recover fully, do not relapse, and develop a robust long-term resistance to further active reinduction of disease. In this paper, we report that rats recovering from MBP-CFA-induced EAE have significantly increased serum levels of reactive nitrogen intermediates indicative of increased NO production. These levels remain elevated after the recovery period and increase even further early after a rechallenge with MBP-CFA, and all animals are totally refractory to a second episode of disease. Oral treatment of rats with N-methyl-l-arginine acetate (l-NMA), beginning at peak disease on day 11 postimmunization, results in significant prolongation of disease and an alteration in the presentation of clinical symptoms from that of solely hind limb paresis/paralysis to severe fore limb involvement as well. Treatment of fully recovered rats with l-NMA 24 h before a rechallenge with MBP-CFA leads to decreased serum reactive nitrogen intermediate levels and results in a second episode of EAE in 100% of animals. Furthermore, l-NMA treatment of fully recovered rats in the absence of a rechallenge immunization leads to spontaneous relapse of disease.
The soy peptide lunasin enhances cross-presentation by dendritic cells (APP3P.109)
Authors: Tung Chun-Yu , Casiano-Rivera Félix , Yao Shuyu , Sun Jie , Chang Hua-Chen
Published: December 2022

Lunasin, a natural seed peptide containing 43 amino acids, was originally isolated from soybeans. We recently discovered a novel function of lunasin as an immune modulating agent. Lunasin stimulation resulted in dendritic cell (DC) maturation and activation by increasing the expression of co-stimulatory molecules and the production of type I interferons, pro-inflammatory cytokines and chemokines. Lunasin acts as an adjuvant in vivo was demonstrated in a prophylactic model against OVA-expressing A20 B-lymphomas. The objective is to determine the mechanisms by which lunasin promotes antigen-specific antitumor immune responses. We hypothesize that lunasin enhances cross-presentation of tumor antigens by DCs, resulting in priming of tumor specific cytotoxic T lymphocytes (CTLs). To test our hypothesis, in vitro cross-presentation of soluble OVA by isolated DCs (CD11c+) was performed. Lunasin-treated DCs stimulated greater proliferation of OVA specific CD8+ T cells (OT-I) cells than DCs cultured in medium only. To further define the molecular mechanisms, we determined the activation of transcription factors involved in DC maturation by lunasin. Increases in phosphorylation of STAT1 and NF-kB were found in DCs treated with lunasin. The data may delineate the signaling pathways involved in gene expression induced by lunasin in DCs. These results show that lunasin enhances cross-presentation of tumor antigens by DCs, resulting in effective priming of antigen specific CD8+ CTLs.
Macrophage anti-viral responses to diabetogenic Coxsackie virus infections are mediated by free radicals (BA6P.134)
Authors: Das Shaonli , Anderson Brian , Burg Ashley , Tse Hubert
Published: December 2022

Reactive oxygen species (ROS) induces Type-1 diabetes (T1D) by directly killing pancreatic β-cells, triggering pro-inflammatory cytokine production, and promoting autoreactive T cell maturation. We hypothesize that free radical synthesis in T1D can synergize and enhance autoimmune responses in T1D onset and progression. Free radicals can be identified by immuno-spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and utilizing a DMPO-specific antibody to detect DMPO-radical adducts. We previously reported a delay in spontaneous T1D onset in Non-obese diabetic (NOD) mice impaired in NADPH Oxidase (NOX)-derived superoxide synthesis (NOD.Ncf1m1J). Bone marrow-derived macrophages (BM-Mϕ) from NOD.Ncf1m1J mice infected with Coxsackie virus CB3 strain displayed a significant decrease (p<0.0001) in fluorescence intensity of DMPO adducts at 4, 6,12 and 24 hours post-infection concomitant with a decrease (p=0.0027) in STAT1 phosphorylation compared to NOD BM-Mϕ. CB3 infected-NOD.Ncf1m1J BM-Mϕ also exhibited a decrease in Tnfa (p=0.0037) and Ifih1 (p=0.002) mRNA accumulation in contrast with NOD BM-Mϕ. These findings suggest that anti-viral responses in NOD.Ncf1m1J BM-Mϕ are redox-regulated and were compromised by ROS-deficiency. Our results further illustrate the importance of NOX-derived superoxide in modulating autoimmune responses in T1D. Future studies will utilize immuno-spin trapping to visualize the interface between free radicals and immune cells during T1D progression.
Understanding neuronal gene expression changes induced by distal demyelination or inflammatory insult (BA6P.137)
Authors: Clarkson Ben , Mirchia Kanish , Kim Hyo , Sauer Brian , LaFrance-Corey Reghann , Howe Charles
Published: December 2022

MR imaging suggests an anatomic correspondence between WM and GM lesions in progressing MS patients, suggesting an underlying neuronal-signaling or neurodegenerative element to the progressive disease state. While retrograde signals evolving from stressed axons that alter soma gene expression have been previously described in models of axonal transection or crush injury, those resulting from inflammation and demyelination are unknown. Using microfluidic chambers that segregate neuronal soma and axons, we have previously demonstrated increased axonal MHC I following treatment with IFNg, a cytokine prominent in demyelinating MS lesions. Furthermore, expression of antigen-loaded MHC I predisposed axons to injury by antigen-specific CD8+ T cells in vitro. Here we report that neurons also upregulate expression of MHC class Ib mRNAs in response to axonal stimulation with IFNg. Genes most highly upregulated following cytokine treatment included H2-M3, H2-Q5, H2-Q6, H2-Q7, H2-Q8, H2-T10 , and H2-T23. We further report the development of an in vivo model system for measuring axonal vulnerability to antigen-specific immune attack following demyelination using an AAV vector to induce neuron-specific expression of axonally-targeted neoantigen (ovalbumin) tagged with cleavable eGFP. We also present optimized methods using AAV vectors in mice for infecting retinal, motor cortex, and hippocampal neurons to induce target gene expression in axons projecting to optic nerve and corpus collosum.
Neutrophils regulate humoral autoimmunity by restricting interferon gamma production via the generation of reactive oxygen species (BA7P.142)
Authors: Cao Wei , Li Jingjing , Huang Xinfang , Dorta-Estremera Stephanie , Di Domizio Jeremy , Anthony Scott , Schluns Kimberly , Lanier Lewis
Published: December 2022

The mechanism by which plasmacytoid dendritic cells (pDCs) promote humoral autoimmunity is unclear. In amyloid-induced experimental autoimmune model, pDC activation is accompanied by prominent neutrophilia. We investigated the contribution of neutrophils since neutrophils have been critically implicated in the pathogenesis of systemic autoimmunity. Unexpectedly, neutrophil depletion enhanced anti-nuclear antibody development, which was correlated with heightened IFN-γ production by natural killer (NK) cells. IFN-α/β produced by pDCs activated NK cells via the induction of IL-15. Neutrophils released reactive oxygen species (ROS), which negatively modulated the levels of IL-15, thereby inhibiting IFN-γ production. Consistently, mice deficient of NADPH oxidase 2 (Nox2) produced increased amounts of IFN-γ and developed augmented titers of autoantibodies. Both the pDC-IFN-α/β pathway and IFN-γ were indispensable in stimulating humoral autoimmunity. Male NZB/W mice had higher levels of superoxide than their female lupus-prone siblings and depletion of neutrophils resulted in spontaneous NK cell and autoimmune B cell activation. Our findings reveal a regulatory role of neutrophils in vivo and highlight the importance of IFN-γ downstream of the pDC-IFN-α/β pathway in systemic autoimmunity.
Role of dendritic cells in immunopathogenesis of Pemphigus vulgarism (BA7P.149)
Authors: Antil Parul , Das Dayasagar , Khandpur Sujay , Sharma Vinod , Singh Manoj , Sharma Alpana
Published: December 2022

Pemphigus vulgaris (PV) is an autoimmune blistering disease that affects skin and mucous membranes characterized by autoantibodies against Dsg3. The breach in immune tolerance, aberrant T cell function and antigen presentation are key immunological events in autoimmunity. Given the central role in induction of immune response and tolerance, dendritic cells (DCs) are most important cells in inciting and sustaining autoimmunity, but it has not yet been explored in PV. We have previously identified potential role of Th cells in pathogenesis of PV. In this study, DC frequency was estimated using HLA-DR, Lin1, CD11c and CD123 by flowcytometry. A significant decline in mDC & pDC frequency in circulation of PV patients (n=20) vs. controls (n=15) was observed. Further, molecular functional characteristics were assessed in cultured monocyte derived differentiated DCs and sorted DCs (FACS AriaIII) directly from circulation of patients and controls. The mRNA levels of their co-stimulatory molecules were estimated. Expression levels of stimulatory molecules (CD40 & CD80) was found to be significantly increased, while a significant decline in inhibitory markers (PSGL1 & ILT3) was observed in sorted and cultured DCs from patients vs. controls. Increased expression level of stimulatory molecules and decreased level of inhibitory molecules further strengthens the defect in functional status of DC. Thus, these co-stimulatory molecules might be a promising avenue for novel therapeutics in PV.
Antibody-mediated regulation of the response to a protective cryptic epitope in rMOG-induced EAE (BA8P.166)
Authors: Riter Melissa , Almatrook Alaa , Rambottom Michael , Cross Anne , Lyons Jeri-Annette
Published: December 2022

Multiple sclerosis is a Th1/Th17-mediated autoimmune disease of the central nervous system, in which B cells are involved. Experimental autoimmune encephalomyelitis (EAE) is the main animal model for MS. Previously we showed that B cell-deficient (B-/-) mice were resistant to rMOG-induced EAE. Further investigation revealed response to a cryptic epitope (MOG61-85) by lymph node (LN) T cells from rMOG-immunized B-/- mice but not WT mice. We investigated the immune response to MOG61-85 and the role of antibody in altering this response. EAE was ameliorated in B-/- mice when coimmunized with MOG35-55 and MOG61-85 compared with MOG35-55 alone. B-/- mice immunized with both peptides and injected with rabbit serum primed to MOG35-55 and MOG61-85 developed more severe EAE with earlier onset than mice receiving preimmune rabbit serum. Purified IgG from rMOG primed WT mice added to cultured LN T cells from co-immunized B-/- mice decreased T cell proliferation to MOG61-85, indicating that antibody inhibited the T cell response to MOG61-85. ELISA of culture supernatants from rMOG-primed WT LN cells demonstrated presence of TGF-β but not IL6 in response to MOG61-85; both cytokines were present in response to MOG35-55. B cell-/- LN cells cultured with MOG61-85 vs. MOG35-55 showed similar percentages of CD4+CD25+FoxP3+ T cells, whereas CD8+CD25+ T cells were slightly increased in those cultured with MOG61-85. The characterization of this CD8+ population is still under investigation.
IL-27 is negatively regulated by PGE2 in bone marrow-derived dendritic cells and macrophages (CCR4P.205)
Authors: Hooper Kirsten , Yen Jui-Hung , Kong Weimin , Ganea Doina
Published: December 2022

Interleukin-27 (p28/EBI3) is expressed by activated antigen presenting cells in response to TLR ligands and pro-inflammatory cytokines. The immunosuppressive functions of IL-27 have previously been demonstrated to occur through the induction of type 1 regulatory T cells, inhibition of Th17 cell development, and anti-inflammatory effects in murine models of collagen-induced arthritis (CIA), inflammatory bowel disease (IBD), and experimental autoimmune encephalomyelitis. We previously showed the pro-inflammatory effects of PGE2 in CIA and IBD through stimulation of IL-23 production leading to development of Th17 cells. Here we report on a novel pro-inflammatory function of PGE2 through the inhibition of IL-27 production by LPS-stimulated and poly I:C-stimulated murine bone marrow-derived dendritic cells (BMDC) and macrophages. We determined the effect of PGE2 on LPS-stimulated BMDC was mediated through EP2/EP4 receptors and induction of cAMP. Previous studies detailed the involvement of NF-κB (c-Rel) and interferon regulatory factors in IL-27p28 gene activation. The current project examines the effect of PGE2 on c-Rel, IRF1 and IRF3 utilizing luciferase reporter assays, gene expression and protein analysis. These studies further characterize the inflammatory mechanisms of PGE2, and might identify novel therapeutic targets for treating inflammatory diseases.
Regulation of virus-specific natural killer cell memory by IL-10 and TGF-β (CCR4P.207)
Authors: Geiger Theresa , Beaulieu Aimee , Vivier Eric , Li Ming , Sun Joseph
Published: December 2022

Natural killer (NK) cells are critical for host control of viral infections and are rapidly activated by pro-inflammatory cytokines. These activating cytokines are thought to be counter-balanced by regulatory cytokines to prevent excessive cytotoxicity or inflammation. This study investigates the role of the immunosuppressive cytokines transforming growth factor (TGF)-β and interleukin (IL)-10 in NK cell development and function, particularly in the generation of effector and memory responses against viral infection. We compared TGF-β-insensitive NK cells (using TGF-βRIIfl/fl x NKp46iCre mice) to NK cells from littermate control mice directly, as well as in mixed bone marrow chimera and adoptive transfer settings. Although they exhibited no obvious defect at steady state, TGF-β-insensitive NK cells revealed greater activation but decreased memory cell formation during mouse cytomegalovirus (MCMV) infection. Conversely, IL-10R-deficient mice at steady state contained greater percentages of mature NK cells in all organs. Similar to TGF-β-insensitive NK cells, IL-10R-deficient NK cells were also defective in generation of memory following MCMV infection. These studies reveal a novel and important role for immunosuppressive cytokines in regulating the magnitude of the effector response and in the optimal generation of long-lived NK cells following viral infection.
Innate lymphoid cell development requires TOX-dependent generation of a common ILC progenitor (HEM5P.221)
Authors: Seehus Corey , Aliahmad Parinaz , de la Torre Brian , Iliev Iliyan , Spurka Lindsay , Funari Vincent , Kaye Jonathan
Published: December 2022

Subtypes of innate lymphoid cells (ILC), defined by effector function and transcription factor expression, have recently been identified in both mice and humans. ILC are critical in maintaining barrier homeostasis and orchestrating cross-talk between the innate and adaptive arms of the immune system. In the adult, ILC derive from common lymphoid progenitors in bone marrow by passing through multiple ILC-specific progenitor stages, although transcriptional regulation of the developmental pathways involved in this process remains poorly defined. Previously, we showed that the nuclear DNA-binding protein TOX is required for development of lymphoid tissue inducer cells, a type of ILC3 required for lymph node organogenesis, and conventional natural killer cells, a type of ILC1. Here, we demonstrate that the production of multiple ILC lineages requires TOX, as a result of TOX-dependent development of common ILC progenitors. In vitro and ex vivo studies indicate that this deficit is cell intrinsic. Furthermore, using low-input RNA sequencing, we obtained robust whole transcriptome data from wild-type and TOX-deficient ILC progenitors. Comparative analysis demonstrated failure to induce various aspects of the ILC gene program in the absence of TOX, implicating this nuclear factor as a key early determinant of ILC lineage specification.
The p38-interacting protein negatively regulates monocyte/macrophage differentiation (HEM5P.239)
Authors: Li Yingqiu , yu xiao , Xu Anlong
Published: December 2022

Monocyte/macrophage differentiation is a central event in the immune response. Here, we investigated the role of p38IP (p38-interacting protein) in monocyte/macrophage differentiation. During monocyte/macrophage differentiation, we found that p38IP is downregulated. Forward knockdown of p38IP by RNA interference induced G1/S arrest, promoting monocytes differentiation into macrophages and the maturation of macrophages as well. Further analysis revealed that p38IP negatively regulates monocyte/macrophage differentiation. Moreover, we found that the downregulation of p38IP is caused by the upregulated miRNA during differentiation. Together, this study reveals the critical role and mechanism for p38IP in regulation of monocyte/macrophage differentiation.
BCAP is a negative regulator of myeloid cell development (HEM5P.225)
Authors: Duggan Jeffrey , Gessay Griffin , Krishnamurty Akshay , Hamerman Jessica
Published: December 2022

B cell adaptor for PI3-kinase (BCAP) is a signaling adaptor protein expressed in hematopoietic cells, including myeloid cells. Here we examined the role of BCAP in myeloid cell homeostasis and development. We found an increased number of monocytes in the bone marrow of BCAP-/- mice versus WT mice, while splenic myeloid populations were similar in number in WT and BCAP-/- mice. Also, mixed chimeras generated with a 1:1 ratio of WT and BCAP-/- bone marrow exhibited a skewing towards BCAP-/- monocytes and neutrophils in the bone marrow, blood and spleen, showing a competitive advantage of BCAP-/- myeloid cells. Thus we hypothesized that BCAP regulates myeloid cell development. WT and BCAP-/- bone marrow had similar numbers of myeloid progenitors, including LSK (Lin-Sca1+cKit+) and CMP (Common Myeloid Progenitor) cells. However, in mixed bone marrow chimeras the progenitor populations were skewed toward BCAP-/- cells, showing that BCAP-/- progenitors out-compete their WT counterparts. In an in vitro myeloid colony forming unit assay, sorted BCAP-/- progenitors produced more cells than WT progenitors, supporting a cell-intrinsic role of BCAP in regulating myeloid differentiation. Also, during infection with Listeria monocytogenes, we observed increased splenic bacterial clearance and numbers of monocytes and neutrophils by day 2 post-infection in BCAP-/- mice compared to WT mice. Together, these data show that BCAP is a negative regulator of myeloid cell development.
Quantitative Studies in Anaphylaxis
Authors: Coulson E. J. , Stevens Henry
Published: December 2022

Alum-precipitated ovalbumin had a sensitizing capacity in guinea-pigs of from 4 to nearly 400 times greater than that of ovalbumin in saline solution, depending upon the route of administration of the sensitizing dose.

For ovalbumin in saline solution the sensitizing capacity of the antigen by intraäbdominal administration was about 1½ times greater than by intravenous injection and nearly 2½ times greater that by subcutaneous administration. Only the latter difference was statistically significant.

For alum-precipitated ovalbumin the sensitizing capacity by subcutaneous administration was 2½ times greater than by intraäbdominal administration and 60 times greater than by intravenous injection. Both of these differences were statistically significant.
A More Accurate Method for the Statistical Evaluation of the Effects of Vaccination
Authors: Carlinfanti E.
Published: December 2022

A statistical method of determining the prophylactic efficacy of vaccines is described which can be applied also to prophylactic inoculation in man. This is done by calculating and comparing the doses of living bacteria lethal for inoculated and uninoculated individuals.
Immunologic Studies of Japanese Encephalitis Virus in Japan
Authors: Buescher Edward L. , Scherer William F. , Grossberg Sidney E. , Chanock Robert M. , Van Philpot Buren
Published: December 2022

The antibody response patterns of 99 patients with proved or presumed JE have been studied. Neutralizing and HI antibodies were found to appear early in the course of disease. Although maximal amounts of neutralizing antibody were generally attained by the first week after onset of symptoms, HI antibody tended to increase through the third post onset week. CF antibody, on the other hand, was not commonly detected in the first two weeks of disease, and was not uniformly demonstrable in all patients. When present, significant increases in CF antibody were commonly demonstrated after the third week of disease. The significance of the dissociation in time of appearance of these antibody types, with particular reference to the practical problems of laboratory diagnosis of JE, is discussed. For routine diagnosis both CF and HI tests should be performed. The former is of greatest value when the initial serum is obtained relatively late after onset of disease. When this initial specimen is obtained within the first week of disease, the HI test provides the most rapid diagnosis. The neutralization test is of little diagnostic value because significant increases in titer are rarely demonstrable, and the test is time-consuming and expensive. The residual neutralizing antibody 5 years after overt infection with JE was found to be the same titer as that observed at the time of infection. CF antibody, although not detectable after 5 years in the majority of individuals, still was present in approximately 25% of persons studied. HI antibody for the most part remained detectable, though the range of titers was lower than that seen immediately after infection.
Effect of Active and Inactive Vaccinia Virus Preparations on Human Amnion Cell Cultures
Authors: Bernkopf H. , Nishmi M. , Rosin A.
Published: December 2022

Cultures of trypsinized human amnion cells react in a morphologically different manner to the inoculation of active and inactive irradiated preparations of vaccinia virus. The most characteristic changes which accompany virus multiplication are rounding of the cells and degenerative changes in both cytoplasm and nucleus. In pronounced contrast inactive virus preparations lead to an extreme lengthening and thinning of the cytoplasm of the cell whereas the nucleus remains intact. In the latter ease the previously homogenous cell sheet is finally transformed into a netlike structure. As reported previously for mouse macrophage cultures, the inactive virus particles themselves seem to be responsible for these changes which are believed to be the result of a toxic viral effect.
The Solubility and Other Physicochemical Properties of Human γ-Globulin Labeled with Fluorescein Isothiocyanate
Authors: Frommhagen Laurence H.
Published: December 2022

The solubility of human γ-globulin, unlike that of albumin and α-β-globulins, is modified by the addition of fluorescein isothiocyanate (FITC). The solubility, as well as electrophoretic behavior, of fluorescein-labeled γ-globulin demonstrates a considerable heterogeneity of labeling at the higher conjugation ratios and a more homogeneous labeling at low conjugation ratios. The value of optimum labeling of immune globulin preparations in order to assure specific staining was again underscored.