bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen Secreted from Noncytosolic Listeria monocytogenes Is Processed by the Classical MHC Class I Processing Pathway
Authors: Zwickey Heather L. , Potter Terry A.
Published: January 2023

Intracellular bacteria can reside in a vacuolar compartment, or they can escape the vacuole and become free living in the cytoplasm. The presentation of Ag by class I MHC molecules has been defined primarily for Ag present in the cytoplasm. It was therefore thought that Ags from bacteria that remain in a vacuole would not be presented by MHC class I molecules. Although some studies have provided data to support this idea, it is not necessarily true for all intracellular bacteria. For example, we have previously demonstrated that an epitope from the p60 protein secreted by LLO− Listeria monocytogenes, which does not reside in the cytoplasm, can be presented by MHC class I molecules to a T cell clone specific for the epitope, p60217–225. We have further examined the route by which Ag secreted by LLO− L. monocytogenes is presented by MHC class I molecules. Using pharmacological inhibitors, we demonstrate that MHC class I presentation of the p60 epitope derived from by LLO− L. monocytogenes requires phagolysosome fusion and processing by the proteasome. Lysosomal cathepsins, however, are not required for processing of the p60 epitope. Similarly, processing of the AttM epitope, secreted by LLO− L. monocytogenes and presented by H2-M3, also requires phagolysosome fusion and cleavage by the proteasome. Thus, p60 and AttM secreted by LLO−  L. monocytogenes are processed via the classical class I pathway for presentation by MHC class I molecules.
Opposing Roles of CD28:B7 and CTLA-4:B7 Pathways in Regulating In Vivo Alloresponses in Murine Recipients of MHC Disparate T Cells
Authors: Blazar Bruce R. , Taylor Patricia A. , Panoskaltsis-Mortari Angela , Sharpe Arlene H. , Vallera Daniel A.
Published: January 2023

Blockade with B7 antagonists interferes with CD28:B7 and CTLA-4:B7 interactions, which may have opposing effects. We have examined the roles of CD28:B7 and CTLA-4:B7 on in vivo alloresponses. A critical role of B7:CD28 was demonstrated by markedly compromised expansion of CD28-deficient T cells and diminished graft-versus-host disease lethality of limited numbers of purified CD4+ or CD8+ T cells. When high numbers of T cells were infused, the requirement for CD28:B7 interaction was lessened. In lethally irradiated recipients, anti-CTLA-4 mAb enhanced in vivo donor T cell expansion, but did not affect, on a per cell basis, anti-host proliferative or CTL responses of donor T cells. Graft-versus-host lethality was accelerated by anti-CTLA-4 mAb infusion given early post-bone marrow transplantation (BMT), mostly in a CD28-dependent fashion. Donor T cells obtained from anti-CTLA-4 mAb-treated recipients were skewed toward a Th2 phenotype. Enhanced T cell expansion in mAb-treated recipients was strikingly advantageous in the graft-versus-leukemia effects of delayed donor lymphocyte infusion. In two different systems, anti-CTLA-4 mAb enhanced the rejection of allogeneic T cell-depleted marrow infused into sublethally irradiated recipients. We conclude that blockade of the selective CD28-B7 interactions early post-BMT, which preserve CTLA-4:B7 interactions, would be preferable to blocking both pathways. For later post-BMT, the selective blockade of CTLA-4:B7 interactions provides a potent and previously unidentified means for augmenting the GVL effect of delayed donor lymphocyte infusion.
Genetic Dissection of Sle Pathogenesis:Sle3on Murine Chromosome 7 Impacts T Cell Activation, Differentiation, and Cell Death
Authors: Mohan Chandra , Yu Ying , Morel Laurence , Yang Ping , Wakeland Edward K.
Published: January 2023

Polyclonal, generalized T cell defects, as well as Ag-specific Th clones, are likely to contribute to pathology in murine lupus, but the genetic bases for these mechanisms remain unknown. Mapping studies indicate that loci on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) confer disease susceptibility in the NZM2410 lupus strain. B6.NZMc7 mice are C57BL/6 (B6) mice congenic for the NZM2410-derived chromosome 7 susceptibility interval, bearing Sle3. Compared with B6 controls, B6.NZMc7 mice exhibit elevated CD4:CD8 ratios (2.0 vs 1.34 in 1- to 3-mo-old spleens); an age-dependent accumulation of activated CD4+ T cells (33.4% vs 21.9% in 9- to 12-mo-old spleens); a more diffuse splenic architecture; and a stronger immune response to T-dependent, but not T-independent, Ags. In vitro, Sle3-bearing T cells show stronger proliferation, increased expansion of CD4+ T cells, and reduced apoptosis (with or without anti-Fas) following stimulation with anti-CD3. With age, the B cells in this strain acquire an activated phenotype. Thus, the NZM2410 allele of Sle3 appears to impact generalized T cell activation, and this may be causally related to the low grade, polyclonal serum autoantibodies seen in this strain. Epistatic interactions with other loci may be required to transform this relatively benign phenotype into overt autoimmunity, as seen in the NZM2410 strain.
Induction of HLA Class I-Restricted CD8+ CTLs Specific for the Major Outer Membrane Protein of Chlamydia trachomatis in Human Genital Tract Infections
Authors: Kim Seon-Kyeong , Angevine Mark , Demick Karen , Ortiz Linette , Rudersdorf Richard , Watkins David , DeMars Robert
Published: January 2023

HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein (MOMP) of Chlamydia trachomatis are present in the peripheral blood of humans who acquired genital tract infections with the organism. Three HLA-A2-restricted epitopes and two HLA-B51-restricted epitopes were identified in serovar E-MOMP. One of the five epitopes spans a variable segment of MOMP and is likely a serovar E-specific epitope. The other four epitopes are localized in constant segments and are C. trachomatis species specific. CTL populations specific for one or more of the four constant segment epitopes were isolated from all 10 infected subjects tested, regardless of infecting serovars, but from only one of seven uninfected subjects tested. The CTLs failed to recognize corresponding peptides derived from Chlamydia pneumoniae MOMP, further suggesting that they indeed resulted from genital tract infections with C. trachomatis. Significantly, ME180 human cervical epithelial cells productively infected with C. trachomatis were killed by the MOMP peptide-specific CTLs. Further investigations of the ability of such CTLs to lyse normal infected epithelial cells and their presence at inflamed sites in the genital tract will help understand the protective or pathological role of CTLs in chlamydial infections. The MOMP CTL epitopes may be explored as potential components of a subunit vaccine against sexually transmitted diseases caused by C. trachomatis. Moreover, the knowledge provided here will facilitate studies of HLA class I pathways of chlamydial Ag processing and presentation in physiologically relevant human APCs.
Intracellular Expression and Release of FcεRIα by Human Eosinophils
Authors: Seminario Maria-Cristina , Saini Sarbjit S. , MacGlashan Donald W. , Bochner Bruce S.
Published: January 2023

Although FcεR have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, FcεRI, and FcεRII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance FcεR on other cells) failed to induce any detectable surface FcεR. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for FcεRIα showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR γ-chain, but no FcεRIβ. Surface biotinylation followed by immunoprecipitation again failed to detect surface FcεRIα, although surface FcRγ was easily detected. Since we were able to detect intracellular FcεRIα, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of FcεRIα into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of FcεRIα that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of FcεRIα remains to be determined.
Mucosally Induced Systemic T Cell Unresponsiveness to Ovalbumin Requires CD40 Ligand-CD40 Interactions
Authors: Kweon Mi-Na , Fujihashi Kohtaro , Wakatsuki Yoshio , Koga Toshiya , Yamamoto Masafumi , McGhee Jerry R. , Kiyono Hiroshi
Published: January 2023

CD40 ligand (CD40L) gene-disrupted (CD40L−/−) mice were employed to examine the role of costimulatory signals via CD40L-CD40 interactions in mucosally induced tolerance. CD40L−/− and control (CD40L+/+) mice of the same C57BL/6 × 129/J background were immunized orally with 25 mg of OVA before systemic challenge with OVA in CFA. While CD40L+/+ mice showed reductions in Ag-specific T cell responses including delayed-type hypersensitivity (DTH) and proliferative responses, CD40L−/− mice underwent normal T cell responses. Further, cytokine analysis of splenic CD4+ T cells showed that both Th1-type (e.g., IFN-γ and IL-2) and Th2-type (e.g., IL-4, IL-5, IL-6, and IL-10) responses were maintained in CD40L−/− mice orally immunized with OVA, whereas these cytokine responses in CD40L+/+ mice were significantly reduced. In addition, splenic CD4+ T cells from CD40L−/− mice orally immunized with OVA provided B cell help in Ag-specific Ab-forming cells when the cells were cultured with naive B cells in the presence of Ag and CD40L-transfected cell lines. In contrast, an identical culture condition containing splenic CD4+ T cells from orally tolerized CD40L+/+ mice did not exhibit helper activity. Taken together, these findings indicate that CD40L and CD40 interactions are essential for the induction of systemic T cell unresponsiveness to orally administered Ag.
Two Human Neonatal IgM Antibodies Encoded by Different Variable-Region Genes Bind the Same Linear Peptide: Evidence for a Stereotyped Repertoire of Epitope Recognition
Authors: Messmer Bradley T. , Sullivan James J. , Chiorazzi Nicholas , Rodman Toby C. , Thaler David S.
Published: January 2023

Two monoclonal IgM Abs have been produced from lymphocytes isolated from two human umbilical cord bloods. These mAbs recognize a conformational epitope present in a CNBr digestion fraction of lactoferrin. Linear epitopes recognized by each mAb were selected from several phage display peptide libraries. In each case, phages displaying a peptide with a motif defined by [WF],G,[EQS],N were recovered. Phages displaying that motif bound equally well to either mAb but did not bind to control IgM. A peptide bearing this motif competed with the phage-displayed peptides for binding to either mAb. The same peptide also competes with a component of the CNBr digestion fraction of lactoferrin for Ab binding in ELISA. The Abs use different families of VH, JH, and VK gene cassettes but use the same JK cassette. All segments are virtually identical to their germline gene counterparts. This work provides further evidence that certain innate specificities are stereotyped among individuals.
Role for the Rac1 Exchange Factor Vav in the Signaling Pathways Leading to NK Cell Cytotoxicity
Authors: Galandrini Ricciarda , Palmieri Gabriella , Piccoli Mario , Frati Luigi , Santoni Angela
Published: January 2023

Here we investigate the activation of and a possible role for the hematopoietic Rac1 exchange factor, Vav, in the signaling mechanisms leading to NK cell-mediated cytotoxicity. Our data show that direct contact of NK cells with a panel of sensitive tumor targets leads to a rapid and transient tyrosine phosphorylation of Vav and to its association with tyrosine-phosphorylated Syk. Vav tyrosine phosphorylation is also observed following the activation of NK cells through the low-affinity Fc receptor for IgG (FcγRIII). In addition, we demonstrate that both direct and Ab-mediated NK cell binding to target cells result in the activation of nucleotide exchange on endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide treatment leads to an impairment of NK cytotoxicity, with FcγRIII-mediated killing being more sensitive to the abrogation of Vav expression. These results provide new insight into the signaling pathways leading to cytotoxic effector function and define a role for Vav in the activation of NK cell-mediated killing.
The Jun Kinase Cascade Is Responsible for Activating the CD28 Response Element of the IL-2 Promoter: Proof of Cross-Talk with the IκB Kinase Cascade
Authors: Kempiak Stephan J. , Hiura Timothy S. , Nel Andre E.
Published: January 2023

Costimulation of TCR/CD3 and CD28 receptors leads to activation of the Jun kinase (JNK) cascade, which plays a key role in T cell activation, including activation of the IL-2 promoter. We demonstrate that the JNK cascade plays a central role in the activation of the CD28 response element (CD28RE) in the IL-2 promoter. This response element is linked to an activating protein-1 (AP-1) site, which functions synergistically with the CD28RE. The role of the JNK cascade in the activation of this composite element is twofold: 1) activation of the AP-1 site through transcriptional activation of c-Jun, and 2) activation of the CD28RE through selective cross-talk with IκB kinase-β (IKKβ). Dominant-negative versions of JNK kinase, c-Jun, and IKKβ interfered in CD3- plus CD28-induced CD28RE/AP-1 luciferase activity in Jurkat cells. In contrast, the dominant-active JNK kinase kinase, MEKK1, induced CD28RE/AP-1 luciferase activity, in parallel with induction of c-Jun and c-Rel binding to this combined promoter site. Dominant-active MEKK1 also induced transfected IKKβ, but not IKKα, activity. In contrast to the JNK cascade, the extracellular signal-regulated kinase (ERK) cascade did not exert an affect on the CD28RE/AP-1 site, but did contribute to activation of the distal NF-AT/AP-1 site.
The T Cell-Specific CXC Chemokines IP-10, Mig, and I-TAC Are Expressed by Activated Human Bronchial Epithelial Cells
Authors: Sauty Alain , Dziejman Michelle , Taha Rame A. , Iarossi Albert S. , Neote Kuldeep , Garcia-Zepeda Eduardo A. , Hamid Qutayba , Luster Andrew D.
Published: January 2023

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-γ (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-γ, but not TNF-α or IL-1β, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-α and IL-1β synergized with IFN-γ induction in all three cell types. High levels of IP-10 protein (>800 ng/ml) were detected in supernatants of IFN-γ/TNF-α-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-γ is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-γ is highly expressed) play an important role in the recruitment of activated T cells.
Common Intra-Articular T Cell Expansions in Patients with Reactive Arthritis: Identical β-Chain Junctional Sequences and Cytotoxicity Toward HLA-B27
Authors: Dulphy Nicolas , Peyrat Marie-Alix , Tieng Vannary , Douay Corinne , Rabian Claire , Tamouza Ryad , Laoussadi Saddek , Berenbaum Francis , Chabot Agnes , Bonneville Marc , Charron Dominique , Toubert Antoine
Published: January 2023

Spondyloarthropathies constitute a group of autoimmune diseases of special interest because of their tight association with the MHC class I molecule HLA-B27 and the bacterial triggering of some clinical forms called reactive arthritis (ReA). One current hypothesis is the presentation by HLA-B27 of a so-called arthritogenic peptide to T cells. To better focus on the relevant T cell populations within the joint, we performed an extensive β-chain T cell repertoire analysis of synovial fluid compared with PBL in seven patients, four of whom were characterized as having ReA triggered by Yersinia enterocolitica, Chlamydia trachomatis, or Shigella sonnei. Analysis of the size diversity of the β-chain complementarity-determining region 3 (CDR3) allowed us to evaluate the degree of T cell clonality in the samples. Oligoclonal T cell expansions were frequently observed in the joint. In one patient, CDR3 amino acid sequences of major expansions using two different BV genes were identical. One dominant T cell expansion and several CDR3 amino acid sequences were identical in two different patients. Furthermore, one sequence was identical with a sequence reported independently in a Salmonella-induced ReA patient. Together, these data indicate a surprisingly high degree of conservation in the T cell responses in recent-onset ReA triggered by different micro-organisms. A CD8+ synovial line expressing shared clonotypes was established and reacted toward several B*2705 lymphoblastoid cell lines, therefore supporting a molecular mimicry phenomenon at the T cell level in the disease mechanism.
Three Different MHC Class I Molecules Bind the Same CTL Epitope of the Influenza Virus in a Primate Species with Limited MHC Class I Diversity
Authors: Evans David T. , Knapp Leslie A. , Jing Peicheng , Piekarczyk Marian S. , Hinshaw Virginia S. , Watkins David I.
Published: January 2023

One of the most remarkable features of the MHC class I loci of most outbred mammalian populations is their exceptional diversity, yet the functional importance of this diversity remains to be fully understood. The cotton-top tamarin (Saguinus oedipus) is unusual in having MHC class I loci that exhibit both limited polymorphism and sequence variation. To investigate the functional implications of limited MHC class I diversity in this outbred primate species, we infected five tamarins with influenza virus and defined the CTL epitopes recognized by each individual. In addition to an immunodominant epitope of the viral nucleoprotein (NP) that was recognized by all individuals, two tamarins also made a response to the same epitope of the matrix (M1) protein. Surprisingly, these two tamarins used different MHC class I molecules, Saoe-G*02 and -G*04, to present the M1 epitope. In addition, CTLs from one of the tamarins recognized target cells that expressed neither Saoe-G*02 nor -G*04, but, rather, a third MHC class I molecule, Saoe-G*12. Sequence analysis revealed that Saoe-G*12 differs from both Saoe-G*02 and -G*04 by only two nucleotides and was probably generated by recombination between these two alleles. These results demonstrate that at least three of the tamarin’s MHC class I molecules can present the same epitope to virus-specific CTLs. Thus, four of the tamarin’s 12 MHC class I molecules bound only two influenza virus CTL epitopes. Therefore, the functional diversity of cotton-top tamarin’s MHC class I loci may be even more limited than their genetic diversity suggests.
Triggering of Effector Functions on a CD8+ T Cell Clone Upon the Aggregation of an Activatory CD94/kp39 Heterodimer
Authors: Bellón Teresa , Heredia Agustín B. de , Llano Manuel , Minguela Alfredo , Rodriguez Antonio , López-Botet Miguel , Aparicio Pedro
Published: January 2023

Some T lymphocytes express the CD94 Ag, which is known to form heterodimers with members of the NKG2 family. We have studied the expression pattern and function of CD94 heterodimers in different αβ or γδ T cell clones. Most of the CD94+NKG2A− T cells have a low to intermediate expression of CD94 Ag. The cross-linking of the CD94/NKG2 heterodimer in one of these CD8 αβ CD94+NKG2A− T cell clones (K14B06) was able to: 1) increase the intracellular concentration of Ca2+, 2) induce the up-regulation of CD25 Ag expression and the secretion of IFN-γ, and 3) trigger redirected cytotoxicity in a TCR-independent manner. This activatory property was not shared by any other costimulatory molecule expressed by the K14B06 T cell clone, including CD8, CD28, CD45, CD69, or CD2 Ags. The immunoprecipitation of CD94 heterodimer showed a 39-kDa band with a similar m.w. to the activatory heterodimer found on some NK clones. A novel form of the NKG2 family (NKG2H) was identified in K14B06. NKG2H protein represents an alternative spliced form of the NKG2E gene, displaying a charged residue in the transmembrane portion and a cytoplasmic tail that lacks immunoreceptor tyrosine-based inhibitory motifs. The expression of NKG2H in the cell membrane through its association to CD94 and DAP-12 molecules supports that it could form part of the activatory CD94/Kp39 heterodimer present on K14B06 cells.
Engagement of Natural Cytotoxicity Programs Regulates AP-1 Expression in the NKL Human NK Cell Line
Authors: Bernard Karine , Cambiaggi Anna , Guia Sophie , Bertucci François , Granjeaud Samuel , Tagett Rebecca , N’Guyen Catherine , Jordan Bertrand R. , Vivier Eric
Published: January 2023

NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.
Tolerization of Mice toSchistosoma mansoniEgg Antigens Causes Elevated Type 1 and Diminished Type 2 Cytokine Responses and Increased Mortality in Acute Infection
Authors: Fallon Padraic G. , Dunne David W.
Published: January 2023

The granuloma that surrounds the Schistosoma mansoni egg is the cause of pathology in murine schistosomiasis, and its formation is driven by egg Ag-stimulated type 1 and type 2 cytokines. To determine the role of egg-driven immune responses during schistosome infection we rendered CBA/Ca mice unresponsive to schistosome eggs by combined cyclophosphamide treatment and thymectomy. In the early acute stages of schistosome infection, egg-tolerized mice suffered high mortalities. Granuloma size and deposition of collagen in the liver were significantly reduced in egg-tolerized mice. Similarly, limited granuloma responses were detected in the intestines of these mice, and this was associated with a >90% reduction in egg excretion. Histologically, egg-tolerized mice had exacerbated hepatocyte damage, with extensive microvesicular steatosis. Elevated plasma transaminase levels confirmed the damage to hepatocytes. Infected egg-tolerized mice had impaired proliferation responses to egg Ag but intact responses to worm Ag. Tolerized mice had diminished Ab responses to egg Ag and had a type 1 cytokine isotype pattern to worm Ag, with elevated IgG2a and diminished IgG1 and IgE. Egg-tolerized mice failed to down-regulate type 1 cytokines that are normally elicited during early schistosome infection. Hepatic granuloma cells from egg-tolerized mice were also type 1 cytokine dominated, with elevated frequencies of Tc1/Th1 and reduced Tc2/Th2 cells. This study demonstrates that mice tolerized to schistosome eggs have elevated type 1 cytokine responses with diminished type 2 responses and reduced anti-egg Ab during schistosome infection, and these effects are detrimental to the host.
Cutting Edge: Effects of an Allergy-Associated Mutation in the Human IL-4Rα (Q576R) on Human IL-4-Induced Signal Transduction
Authors: Wang Helen Y. , Shelburne Chris P. , Zamorano Jose , Kelly Ann E. , Ryan John J. , Keegan Achsah D.
Published: January 2023

A mutation in the human (hu) IL-4Rα, Q576R, has been linked with allergy in humans. Increased sensitivity of patients cells with this mutation to IL-4 suggest that a Q576R change enhances IL-4 signaling. To directly test this hypothesis, we analyzed the ability of huIL-4Rα cDNA bearing the Q576R and Y575F mutations to signal tyrosine phosphorylation, DNA-binding activity, proliferation, protection from apoptosis, and CD23 induction in response to huIL-4 in murine cells. Responses generated by the Q576R and Y575F mutants were similar to those of the wild-type receptor, using various concentrations of huIL-4 and times of stimulation. These results indicate that neither the Q576R nor the Y575F mutations have a significant direct effect on IL-4 signal transduction, and that hypersensitive induction of CD23 in cells derived from human allergy patients may be due to different and/or additional alterations in the IL-4 signaling pathway.
Phosphatidylinositol 3′-Kinase, But Not S6-Kinase, Is Required for Insulin-Like Growth Factor-I and IL-4 To Maintain Expression of Bcl-2 and Promote Survival of Myeloid Progenitors
Authors: Minshall Christian , Arkins Sean , Dantzer Robert , Freund Gregory G. , Kelley Keith W.
Published: January 2023

Phosphatidylinositol 3′-kinase (PI 3-kinase) catalyzes the formation of 3′ phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway.
Differential Expression and Cross-Regulatory Function of RANTES During Mycobacterial (Type 1) and Schistosomal (Type 2) Antigen-Elicited Granulomatous Inflammation
Authors: Chensue Stephen W. , Warmington Kelly S. , Allenspach Eric J. , Lu Bao , Gerard Craig , Kunkel Steven L. , Lukacs Nicholas W.
Published: January 2023

The role of RANTES in Th1 and Th2 cell-mediated immune responses has been enigmatic. To approach this question, we analyzed RANTES expression and function in murine models of types 1 and 2 cell-mediated pulmonary granulomas elicited with Mycobacterium bovis or Schistosoma mansoni egg Ag-coated beads, respectively. Compared with type 2, type 1 lesions had up to 4-fold greater RANTES protein and mRNA production. Type 1 draining lymph nodes also produced up to 7-fold higher levels of RANTES. Anti-RANTES Ab treatments had opposite effects, decreasing type 1 lesion area by 25% and augmenting type 2 lesions by 50%. The latter was associated with increased IL-4, IL-5, IL-10, and IL-13 production by lymph nodes. Infusion of rRANTES (1 mg/kg/day) did not affect type 1 lesions, but reduced type 2 lesion area by 27% and eosinophils by 40%. Lymph node cultures from RANTES-treated mice had augmented type 1 and impaired type 2 responses. In vitro, RANTES caused selective, dose-related inhibition of IL-4 that was largely dependent on CCR1 receptors. In conclusion, RANTES plays different roles in types 1 and 2 granuloma formation, promoting the former and mediating cross-regulatory inhibition of the latter. Moreover, RANTES may have therapeutic potential in the treatment of established type 2 hypersensitivity.
Depletion of IL-10- and TGF-β-Producing Regulatory γδ T Cells by Administering a Daunomycin-Conjugated Specific Monoclonal Antibody in Early Tumor Lesions Augments the Activity of CTLs and NK Cells
Authors: Seo Naohiro , Tokura Yoshiki , Takigawa Masahiro , Egawa Kohji
Published: January 2023

It has been demonstrated that γδ T cells accumulating in early tumor lesions and those purified from spleen cells of tumor-bearing mice attenuate the activity of CTLs and NK cells. We, therefore, investigated whether depletion of γδ T cells from early lesions of tumors results in restoration of CTL and NK cell activities and subsequent regression of tumors. A daunomycin-conjugated anti-γδTCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently deplete γδ T cells. An in vitro study revealed that Dau-UC7 specifically lysed γδTCR+ cells and effectively inhibited splenic γδ T cells from tumor-bearing mice to produce cytotoxic cell-suppressive factors. Furthermore, intralesional injections of Dau-UC7 at an early stage of tumor development led to augmentation of tumor-specific CTL as well as NK cell activities and to the resultant regression or growth inhibition of the tumors. On analysis of cytokine profile, γδ T cells transcribed mRNAs for IL-10 and TGF-β, but not IL-4 or IFN-γ, suggesting the T regulatory 1-like phenotype. Finally, a blocking study with mAbs showed that the inhibitory action of γδ T cells on CTLs and NK cells was at least partly mediated by IL-10 and TGF-β. These results clearly demonstrated the novel mechanism by which T regulatory 1-like γδ T cells suppress anti-tumor CTL and NK activities by their regulatory cytokines in early tumor formation.
The CD3ε Subunit of the TCR Contains Endocytosis Signals
Authors: Borroto Aldo , Lama Juan , Niedergang Florence , Dautry-Varsat Alice , Alarcón Balbino , Alcover Andrés
Published: January 2023

Ligand binding to TCR induces its internalization and cell surface down-modulation. These phenomena contribute to the extinction of activation signals. Due to the multicomponent nature of the TCR-CD3 complex, its internalization may be mediated by one or several of its subunits. Although it has been reported that CD3γ and CD3δ contain endocytosis motifs involved in the internalization of the TCR-CD3 complex, other subunits could also be involved in this process. For instance, CD3ε and CDζ display amino acid sequences reminiscent of internalization motifs. To investigate whether CD3ε bears endocytosis signals, we have analyzed the internalization capacity of a panel of deletion and point mutants of CD3ε that were expressed on the cell surface independently of other TCR-CD3 subunits. Here we report that CD3ε displays endocytosis determinants. These data indicate that CD3ε could contribute to the internalization and cell surface down-regulation of TCR-CD3 complexes. Moreover, the existence of endocytosis signals in this polypeptide could serve to retrieve unassembled CD3ε subunits or partial CD3 complexes from the plasma membrane, thus restricting the expression on the cell surface to fully functional TCR-CD3 complexes.
A Single-Chain IL-12 IgG3 Antibody Fusion Protein Retains Antibody Specificity and IL-12 Bioactivity and Demonstrates Antitumor Activity
Authors: Peng Lisan S. , Penichet Manuel L. , Morrison Sherie L.
Published: January 2023

IL-12 is a heterodimeric cytokine with many actions on innate and cellular immunity that may have antitumor and antimetastatic effects. However, systemic administration of IL-12 can be toxic. Tumor-specific Abs provide a means to selectively target a metastatic/residual nodule and deliver therapeutic quantities of an immunostimulatory molecule like IL-12 with lower systemic levels and ideally, toxicity. We report the construction and characterization of an Ab fusion protein in which single-chain murine IL-12 is fused to an anti-Her2/neu Ab at the amino terminus (mscIL-12.her2.IgG3). The use of single-chain IL-12 in the fusion protein simplifies vector construction, ensures equimolar concentrations of the two IL-12 subunits, and may confer greater stability to the fusion protein. SDS-PAGE analysis shows this 320-kDa protein is secreted and correctly assembled. FACS analysis demonstrates that this fusion protein binds to cells transfected with the Her2/neu Ag, thus retaining Ab specificity; this fusion protein also binds to a cell line and to PHA-activated PBMC that express the IL-12R, thus demonstrating cytokine receptor specificity. T cell proliferation assays and NK cytotoxicity assays demonstrate that this fusion protein exhibits IL-12 bioactivity comparable to recombinant murine IL-12. In vivo studies demonstrate that this fusion protein has antitumor activity. These results are significant and suggest that this IL-12 Ab fusion protein can effectively combine the therapeutic potential of IL-12 with the tumor-targeting ability of the Ab and may provide a viable alternative to systemic administration of IL-12.
Constitutive Nuclear Translocation of NF-κB in B Cells in the Absence of IκB Degradation
Authors: Doerre Stefan , Corley Ronald B.
Published: January 2023

Members of the NF-κB/Rel family of transcription factors are involved in many aspects of B lymphocyte development and function. NF-κB is constitutively active in these cells, in contrast with most other cell types. In the inactive form, NF-κB/Rel proteins are sequestered in the cytoplasm by members of the IκB family of NF-κB inhibitors. When activated, NF-κB is translocated to the nucleus, a process that involves the phosphorylation and proteasomal degradation of IκB proteins. Thus, NF-κB activation is accompanied by the rapid turnover of IκB proteins. We show that while this “classical” mode of NF-κB activation is a uniform feature of IgM+ B cell lines, all IgG+ B cells analyzed contain nuclear NF-κB yet have stable IκBα, IκBβ, and IκBε. Furthermore, Iκβε levels are at least 10 times lower in IgG+ B cells than in IgM+ B cells, an additional indication that the regulation of constitutive NF-κB activity in these two types of B cells is fundamentally different. These data imply the existence of a novel mechanism of NF-κB activation in IgG+ B cells that operates independently of IκB degradation. They further suggest that different isoforms of the B cell receptor may have distinct roles in regulating NF-κB activity.
Estrogen Protects Against Cellular Infiltration by Reducing the Expressions of E-Selectin and IL-6 in Endotoxin-Induced Uveitis
Authors: Miyamoto Noriko , Mandai Michiko , Suzuma Izumi , Suzuma Kiyoshi , Kobayashi Kaori , Honda Yoshihito
Published: January 2023

Anterior uveitis associated with Behcet’s disease and ankylosing spondylitis preferentially occurs in adult men, which may suggest the effects of sex hormones on acute anterior uveitis. Recently, estrogen receptors in the vascular endothelium have been reported to be involved in several pathological conditions. In the present study, we examined the gender differences in susceptibility to endotoxin-induced uveitis (EIU) and the effects of estrogen on anterior inflammation. EIU was induced in adult male, female, and ovariectomized female Lewis rats (200 g) by hind footpad injection of 200 μg of LPS. In EIU, cellular infiltration was more marked in male than in female rats, and ovariectomy increased cellular infiltration. Treatment with 10 μg of 17β-estradiol significantly reduced the cell number in male and ovariectomized female rats with EIU. Estrogen receptor immunoreactivity was found in the nucleus of vascular endothelium and in some stromal cells of the iris-ciliary body. Semiquantitative PCR revealed that E-selectin and IL-6 gene expressions were increased in rats following LPS injection, and an overdose of tamoxifen, an estrogen receptor antagonist, reversed the effect of 17β-estradiol on E-selectin, but not its effect on IL-6. These observations suggested that the down-modulation of these inflammatory genes by estrogen may contribute to the reduction in cellular infiltration in acute anterior uveitis.
Mouse Monocyte-Derived Chemokine Is Involved in Airway Hyperreactivity and Lung Inflammation
Authors: Gonzalo Jose-Angel , Pan Yang , Lloyd Clare M. , Jia Gui-Quan , Yu Gary , Dussault Barry , Powers Christine A. , Proudfoot Amanda E. I. , Coyle Anthony J. , Gearing David , Gutierrez-Ramos Jose-Carlos
Published: January 2023

The cloning, expression, and function of the murine (m) homologue of human (h) monocyte-derived chemokine (MDC) is reported here. Like hMDC, mMDC is able to elicit the chemotactic migration in vitro of activated lymphocytes and monocytes. Among activated lymphocytes, Th2 cells were induced to migrate most efficiently. mMDC mRNA and protein expression is modulated during the course of an allergic reaction in the lung. Neutralization of mMDC with specific Abs in a model of lung inflammation resulted in prevention of airway hyperreactivity and significant reduction of eosinophils in the lung interstitium but not in the airway lumen. These data suggest that mMDC is essential in the transit/retention of leukocytes in the lung tissue rather than in their extravasation from the blood vessel or during their transepithelial migration into the airways. These results also highlight the relevance of factors, such as mMDC, that regulate the migration and accumulation of leukocytes within the tissue during the development of the key physiological endpoint of asthma, airway hyperreactivity.
Altered Memory T Cell Differentiation in Patients with Early Rheumatoid Arthritis
Authors: Skapenko Alla , Wendler Jörg , Lipsky Peter E. , Kalden Joachim R. , Schulze-Koops Hendrik
Published: January 2023

The chronic immune response in rheumatoid arthritis (RA) might be driven by activated Th1 cells without sufficient Th2 cell differentiation to down-modulate inflammation. To test whether disordered memory T cell differentiation contributes to the typical Th1-dominated chronic inflammation in RA we investigated differentiation of resting CD4+ memory T cells in patients with early (6 wk to 12 mo) untreated RA and in age- and sex-matched healthy controls in vitro. No difference in cytokine secretion profiles of freshly isolated memory T cells was detected between patients and controls. A cell culture system was then employed that permitted the differentiation of Th effectors from resting memory T cells by short term priming. Marked differences were found in response to priming. Th2 cells could be induced in all healthy controls by priming with anti-CD28 in the absence of TCR ligation. By contrast, priming under those conditions resulted in Th2 differentiation in only 9 of 24 RA patients. Exogenous IL-4 could overcome the apparent Th2 differentiation defect in seven patients but was without effect in the remaining eight patients. In all patients a marked decrease in IL-2-producing cells and a significant increase in well-differentiated Th1 cells that produced IFN-γ but not IL-2 were evident after priming with anti-CD3 and anti-CD28. The data suggest that CD4+ memory T cells from patients with early untreated RA manifest an intrinsic abnormality in their ability to differentiate into specific cytokine-producing effector cells that might contribute to the characteristic Th1-dominated chronic (auto)immune inflammation in RA.
Recognition of a Shared Human Prostate Cancer-Associated Antigen by Nonclassical MHC-Restricted CD8+ T Cells
Authors: Housseau Franck , Bright Robert K. , Simonis Toni , Nishimura Michael I. , Topalian Suzanne L.
Published: January 2023

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-γ-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR αβ+ T cell clone were isolated that secreted IFN-γ and TNF-α in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR αβ, CD3, Vβ12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but β2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR αβ+ T cells via a nonclassical MHC I-like molecule yet to be defined.
Effects of recombinant vectors expressing Chlamydia pneumoniae’s major outer membrane protein (MOMP) on humoral and T cell-mediated immunity. (129.1)
Authors: Elkadri Rehab Ahmed shafik , Grobler Anne-Katrien , Bermudez-Fajardo Alexandra , Puolakkainen Mirja , Stewart Graham , Oviedo-Orta Ernesto
Published: January 2023

Chlamydia pneumoniae is an obligatory intracellular pathogen. C. pneumoniae's major outer membrane protein (MOMP) is a 40 kDa porin and represents. The antigenic and immunomodulatory properties of this protein remain controversial.our aim is to assess the effect of recombinant vectors expressing MOMP on the induction of humoral and T cell-mediated immunity. : We used Mycobacterium vaccae and pcDNA3.1 expressing MOMP. Plasmids containing untruncated MOMP and truncated MOMP were created and used to express MOMP in M. vaccae.. In this study, we found that the signal sequence of MOMP effect on the viablity of M. vaccae. rMOMP expressed either by M. vaccae or by DNA plasmid is capable of significantly induce regulatory Treg of both CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+phenotype. DNA immunisation induced a significant up-regulation of IL-4 and IL-10 in mice indicating a shift towards a predominant Th2 immune response. Additionally, DNA immunisation was also capable of significantly suppres the secretion of IFN-gamma. We also found that M. vaccae expressing MOMP significantly increases IL-10 secretion but without having a significant effect on IFN-γ and IL-4 secretion. We conclude that recombinant vectors expressing MOMP have the ability to induce predominantly T cell-mediated immunosuppressive responses which shed light on the effect of this protein on host immune responses that may explain Chlamydia's long term survival and also be responsible for the pathogen's immune evasion mechanisms.
Development of a non-living vaccine against Burkholderia mallei (129.2)
Authors: Deeraksa Arpaporn , Qazi Omar , Whitlock Gregory C , Judy Barbara M , Torres Alfredo G
Published: January 2023

Burkholderia mallei is a gram-negative highly pathogenic bacterium, responsible for glanders. B. mallei is considered potential bioterrorism agents, classified as such in list B by the Centers for Disease Control and Prevention. Currently there is no vaccine available against B. mallei. In this study, we aimed to identify the protective proteins of B. mallei. The candidate proteins were selected based on the previous data of proteomics and linear expression library (LEE) whole-genome screens coupled with known structures or functions of the proteins in other bacteria. The selected proteins were BMA-4, BMA-8, BMA-9, BMA-10, BMA-11 and BMA-12. The genes encoding each protein were cloned into pCDNA3.1 and pET28a. The His-tag proteins were then purified. BALB/c mice were vaccinated with DNA encoding each protein and then boosted 3 weeks later with the recombinant proteins. Two weeks after boosting, mice were infected with B. mallei ATCC23344. The result showed that, 19 days after infection, there was 100% survival in the BMA2821 and BMAA0768 groups, however only 78% survival in the control group. The study needs to be repeated and the result will be discussed.
Anti-viral CD8+ T cell Effector Activity Is Regulated by Hematopoietic Cells in the Influenza-Infected Lung Parenchyma (130.16)
Authors: Hufford Matthew Thomas , Sun Jie , Kim Taeg , Braciale Thomas J
Published: January 2023

CD8+ T cell effector activity is instrumental in the resolution of influenza infection, as well as additional respiratory viral infections, by their ability to eliminate infected cells and to release potent cytokines. CD8+ T cells localized to the lung airways are believed to be responsible for this anti-viral activity because CD8+ T cells located in the lung parenchyma have limited access to the respiratory epithelium, the principal site of influenza replication. To test this hypothesis, we utilized the detection of CD8+ T cell IFN? production ex vivo and in vivo as a convenient means to identify influenza-specific CD8+ T cells and to define the location of antigen presentation within the influenza-infected lungs. Surprisingly, influenza-specific CD8+ T cells collected from the airways produced limited amounts of IFN? in vivo. In contrast, significant numbers of influenza-specific CD8+ T cells in the lung parenchyma were actively producing IFN?. Because IFN? production by CD8+ T cells is principally TCR dependent, data indicated antigen engagement occurs in the lung parenchyma, not the airways. Because the lung parenchyma is enriched in hematopoietic (CD45+) cells compared to the airways, it was hypothesized that these cells were the primary antigen presenting cells in the lungs. To test this hypothesis, H-2Kb-/-Db-/- mice were irradiated and reconstituted with H-2Kb+/+Db+/+ bone marrow. Despite the absence of MHC class I on respiratory epithelial cells, CD8+ T cells in the lung parenchyma produced significant amounts of IFN? in vivo. These observations challenge conventional knowledge on the mechanism by which CD8+ T cells clear virally-infected cells in the respiratory epithelium.