bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine Deprivation of Naive CD8+ T Cells Promotes Minimal Cell Cycling but Maximal Cytokine Synthesis and Autonomous Proliferation Subsequently: A Mechanism of Self-Regulation
Authors: Sad Subash , Krishnan Lakshmi
Published: December 2022

Naive CD8+ T cells differentiate into effectors secreting various cytokines that aid their function. IL-2, but not IL-15, promoted this differentiation of naive CD8+ T cells into effectors. However, the amount of IL-2 present during differentiation had a dichotomous effect on their subsequent function. High concentrations of IL-2 enhanced proliferation and cell cycling initially, but the effectors subsequently failed to produce cytokines and proliferate autonomously, although CD28 expression was maintained. In contrast, suboptimal amounts of IL-2 during priming promoted apoptosis, little proliferation and cell cycling, yet the CD8+ effectors generated produced high levels of cytokines and proliferated autonomously. Interestingly, the effects of IL-2 on naive CD8+ T cells were totally opposite those on naive CD4+ T cells. Although IL-2 impaired cytokine synthesis by CD8+ T cells, the expression of LFA1 and CD44 as well as Fas-dependent cytotoxicity were enhanced. However, loss of cytokine synthesis was not due to increased cytotoxicity, as inhibition occurred even in the absence of perforin/FasL. Interestingly, CD8+ effectors secreting reduced amounts of cytokines exhibited enhanced IL-2Rα, but reduced IL-2Rβ, expression. Furthermore, sorted CD8+ IL-2Rαhigh cells secreted less cytokines than IL-2Rαlow cells. These results suggest that the presence of excessive IL-2 during the activation of naive CD8+ T cells, while promoting cell cycling initially, may compromise long-term immunity.
Norepinephrine-Mediated Inhibition of Antitumor Cytotoxic T Lymphocyte Generation Involves a β-Adrenergic Receptor Mechanism and Decreased TNF-α Gene Expression
Authors: Kalinichenko Vladimir V. , Mokyr Margalit B. , Graf Lloyd H. , Cohen Rhonna L. , Chambers Donald A.
Published: December 2022

We have previously shown that norepinephrine (NE) inhibits the in vitro generation of anti-MOPC-315 CTL activity by spleen cells from BALB/c mice rejecting a large MOPC-315 tumor as a consequence of low-dose melphalan (l-phenylalanine mustard (l-PAM)) treatment (l-PAM TuB spleen cells). Since TNF-α plays a key role in the generation of antitumor CTL activity in this system, we determined whether NE mediates this inhibition through inhibition of TNF-α production. Here, we show that NE inhibits the production of TNF-α protein and mRNA by l-PAM TuB spleen cells stimulated in vitro with mitomycin C-treated tumor cells. Flow cytometric analysis of intracellular expression of TNF-α revealed substantial NE-mediated decreases in the percentages of TNF-α+ cells among CD4+ and CD8+ T cells and F4/80+ activated macrophages. NE inhibition of CTL generation was largely overcome by addition of TNF-α to the stimulation cultures. When the β-adrenergic antagonist propranolol was added to the stimulation cultures of l-PAM TuB spleen cells at a concentration that prevented NE-induced cAMP elevation, the NE-mediated decrease in TNF-α mRNA and NE-mediated inhibition of CTL generation were reversed. Collectively, these results suggest that NE inhibits antitumor CTL generation, at least in part, by inhibiting TNF-α synthesis through a mechanism(s) involving β-adrenergic receptor signaling.
CD45 Is Essential for FcεRI Signaling by ZAP70, But Not Syk, in Syk-Negative Mast Cells
Authors: Zhang Juan , Siraganian Reuben P.
Published: December 2022

The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore FcεRI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FcεRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated FcεRIγ-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling.
Phenotypical and Functional Characterization of the CD8+ T Cell Repertoire ofHLA-A2.1Transgenic,H-2Kb°Db°Double Knockout Mice
Authors: Ureta-Vidal Abel , Firat Hüseyin , Pérarnau Béatrice , Lemonnier François A.
Published: December 2022

Homozygous HLA-A2.1 transgenic H-2Kb°Db° double knockout (KO) mice were created. Their potential to develop HLA-A2.1-restricted cytolytic responses was compared with that of their classical transgenic counterparts, which still express H-2Kb, Db molecules. On cell surfaces, both strains express similar amounts of chimeric (α1α2 domains of human, α3 cytoplasmic domains of mouse) HLA-A2.1 molecules in noncovalent association with mouse β2-microglobulin. Compared with mice that are totally deprived of histocompatibility class Ia molecules (H-2Kb°Db° double KO), the expression of HLA-A2.1 in transgenic/double KO mice resulted in sizeable increase in the periphery of CD8+ T cells with a normally diversified TCR repertoire. A biased education in favor of HLA-A2.1, ascribable to the absence of H-2 class Ia molecules, was evidenced in these transgenic/double KO mice by their improved capacity to mount HLA-restricted cytolytic responses, regardless of whether they were virally infected or injected with synthetic epitopic peptide. HLA class I transgenic, H-2 class Ia KO mice should represent useful animal models for the preclinical evaluation of vaccine formulations aiming at the induction of HLA class I-restricted CTL responses.
A Role for the Transmembrane Domain in the Trimerization of the MHC Class II-Associated Invariant Chain
Authors: Ashman Jonathan B. , Miller Jim
Published: December 2022

MHC class II and invariant chain (Ii) associate early in biosynthesis to form a nonameric complex. Ii first assembles into a trimer and then associates with three class II αβ heterodimers. Although the membrane-proximal region of the Ii luminal domain is structurally disordered, the C-terminal segment of the luminal domain is largely α-helical and contains a major interaction site for the Ii trimer. In this study, we show that the Ii transmembrane domain plays an important role in the formation of Ii trimers. The Ii transmembrane domain contains an unusual patch of hydrophilic residues near the luminal interface. Substitution of these polar residues with nonpolar amino acids resulted in a decrease in the efficiency of Ii trimerization and subsequent class II association. Moreover, N-terminal fragments of Ii were found to trimerize independently of the luminal α-helical domain. Progressive C-terminal truncations mapped a homotypic association site to the first 80 aa of Ii. Together, these results implicate the Ii transmembrane domain as a site of trimer interaction that can play an important role in the initiation of trimer formation.
Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages
Authors: Marecki Sylvia , Atchison Michael L. , Fenton Matthew J.
Published: December 2022

IFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form specific complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1-independent H-2Ld MHC class I promoter. In NIH-3T3 fibroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1β promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites. Synergistic activation of the IL-1β promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages.
Role of IL-10 in a Neonatal Mouse Listeriosis Model
Authors: Genovese Francesco , Mancuso Giuseppe , Cuzzola Maria , Biondo Carmelo , Beninati Concetta , Delfino Demetrio , Teti Giuseppe
Published: December 2022

This study was undertaken to test the hypothesis that altered IL-10 production plays a role in the increased susceptibility of neonates to listeriosis. Plasma IL-10 levels were measured in neonatal and adult mice at various times after infection with Listeria monocytogenes. Relative to adults, neonatal mice had markedly increased IL-10 levels early in the course of infection with Listeria using a 90% lethal dose. Higher neonatal IL-10 responses were also observed after injecting adults and pups with equal doses of killed organisms. Splenic macrophages from neonates produced higher IL-10 levels than those of adults after in vitro stimulation with killed bacteria, confirming in vivo observations. Moreover, IL-10 blockade had differential effects in neonates and adults infected with live Listeria. In adult mice, anti-IL-10 Abs decreased bacterial burden early in the course of infection, but were no longer effective at 6 days or later after challenge. In the pups, however, the same treatment had beneficial effects both early and late during infection and resulted in increased survival. Collectively, our data suggest that an overproduction of IL-10 by macrophages may at least partially explain the increased susceptibility of neonates to listeriosis, and provide further evidence that cytokine production is different in adults and neonates.
Human Dendritic Cells Very Efficiently Present a Heterologous Antigen Expressed on the Surface of Recombinant Gram-Positive Bacteria to CD4+ T Lymphocytes
Authors: Corinti Silvia , Medaglini Donata , Cavani Andrea , Rescigno Maria , Pozzi Gianni , Ricciardi-Castagnoli Paola , Girolomoni Giampiero
Published: December 2022

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-γ release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-α, IL-1β, IL-12, TGF-β, and IL-10) and of the chemokines IL-8, RANTES, IFN-γ-inducible protein-10, and monokine induced by IFN-γ. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.
Migration of Antigen-Presenting B Cells from Peripheral to Mucosal Lymphoid Tissues May Induce Intestinal Antigen-Specific IgA Following Parenteral Immunization
Authors: Coffin Susan E. , Clark Stephanie L. , Bos Nico A. , Brubaker Jeffery O. , Offit Paul A.
Published: December 2022

Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.
IL-4-Producing γδ T Cells That Express a Very Restricted TCR Repertoire Are Preferentially Localized in Liver and Spleen
Authors: Gerber David J. , Azuara Véronique , Levraud Jean-Pierre , Huang Shu Ying , Lembezat Marie-Pierre , Pereira Pablo
Published: December 2022

IL-4-producing γδ thymocytes in normal mice belong to a distinct subset of γδ T cells characterized by low expression of Thy-1. This γδ thymocyte subset shares a number of phenotypic and functional properties with the NK T cell population. Thy-1dull γδ thymocytes in DBA/2 mice express a restricted repertoire of TCRs that are composed of the Vγ1 gene product mainly associated with the Vδ6.4 chain and exhibit limited junctional sequence diversity. Using mice transgenic for a rearranged Vγ1Jγ4Cγ4 chain and a novel mAb (9D3) specific for the Vδ6.3 and Vδ6.4 murine TCRδ chains, we have analyzed the peripheral localization and functional properties of γδ T cells displaying a similarly restricted TCR repertoire. In transgenic mice, IL-4 production by peripheral γδ T cells was confined to the γδ+9D3+ subset, which contains cells with a TCR repertoire similar to that found in Thy-1dull γδ thymocytes. In normal DBA/2 mice such cells represent close to half of the γδ T cells present in the liver and around 20% of the splenic γδ T cells.
The Role of the Common Cytokine Receptor γ-Chain in Regulating IL-2-Dependent, Activation-Induced CD8+ T Cell Death
Authors: Dai Zhenhua , Arakelov Alexandr , Wagener Maylene , Konieczny Bogumila T. , Lakkis Fadi G.
Published: December 2022

IL-2-dependent, activation-induced T cell death (AICD) plays an important role in peripheral tolerance. Using CD8+ TCR-transgenic lymphocytes (2C), we investigated the mechanisms by which IL-2 prepares CD8+ T cells for AICD. We found that both Fas and TNFR death pathways mediate the AICD of 2C cells. Neutralizing IL-2, IL-2Rα, or IL-2Rβ inhibited AICD. In contrast, blocking the common cytokine receptor γ-chain (γc) prevented Bcl-2 induction and augmented AICD. IL-2 up-regulated Fas ligand (FasL) and down-regulated γc expression on activated 2C cells in vitro and in vivo. Adult IL-2 gene-knockout mice displayed exaggerated γc expression on their CD8+, but not on their CD4+, T cells. IL-4, IL-7, and IL-15, which do not promote AICD, did not influence FasL or γc expression. These data provide evidence that IL-2 prepares CD8+ T lymphocytes for AICD by at least two mechanisms: 1) by up-regulating a pro-apoptotic molecule, FasL, and 2) by down-regulating a survival molecule, γc.
SLP-76 Binding to p56lck: A Role for SLP-76 in CD4-Induced Desensitization of the TCR/CD3 Signaling Complex
Authors: Sanzenbacher Ralf , Kabelitz Dieter , Janssen Ottmar
Published: December 2022

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185–194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.
B Cell Antigen Receptor-Mediated Activation of Cyclin-Dependent Retinoblastoma Protein Kinases and Inhibition by Co-Cross-Linking with Fcγ Receptors
Authors: Tanguay Debra , Pavlovic Sandra , Piatelli Michael J. , Bartek Jiri , Chiles Thomas C.
Published: December 2022

Cross-linking the B cell Ag receptor (BCR) to surface Fc receptors for IgG (FcγR) inhibits G1-to-S progression; the mechanism by which this occurs is not completely known. We investigated the regulation of three key cell cycle regulatory components by BCR-FcγR co-cross-linking: G1-cyclins, cyclin-dependent kinases (Cdks), and the retinoblastoma gene product (Rb). Rb functions to suppress G1-to-S progression in mammalian cells. Rb undergoes cell-cycle-dependent phosphorylation, leading to its inactivation and thereby promoting S phase entry. We demonstrate in this paper for the first time that BCR-induced Rb phosphorylation is abrogated by co-cross-linking with FcγR. The activation of Cdk4/6- and Cdk2-dependent Rb protein kinases is concomitantly blocked. FcγR-mediated inhibition of Cdk2 activity results in part from an apparent failure to express Cdk2 protein. By contrast, inhibition of Cdk4/6 activities is not due to suppression of Cdk4/6 or cyclins D2/D3 expression or inhibition of Cdk-activating kinase activity. Cdk4- and Cdk6-immune complexes recovered from B cells following BCR-FcγR co-cross-linking are devoid of coprecipitated D-type cyclins, indicating that inhibition of their Rb protein kinase activities is due in part to the absence of bound D-type cyclin. Thus, BCR-derived activation signals that up-regulate D-type cyclin and Cdk4/6 protein expression remain intact; however, FcγR-mediated signals block cyclin D-Cdk4/6 assembly or stabilization. These results suggest that assembly or stabilization of D-type cyclin holoenzyme complexes 1) is an important step in the activation of Cdk4/6 by BCR signals, and 2) suffice in providing a mechanism to account for inhibition of BCR-stimulated Rb protein phosphorylation by FcγR.
Roles of Integrins and CD44 on the Adhesion and Migration of Fetal Liver Cells to the Fetal Thymus
Authors: Kawakami Naoto , Nishizawa Fumiko , Sakane Naoki , Iwao Mutsumi , Tsujikawa Kazutake , Ikawa Masahito , Okabe Masaru , Yamamoto Hiroshi
Published: December 2022

Adhesion and migration of mouse fetal liver (FL) cells to the thymus were investigated using cells from green fluorescent protein transgenic (GFP+) mice. FL cells from GFP+ embryos at 12 gestational days (E12) of mice were incubated with 2′-deoxyguanosine-treated fetal thymus lobe (from E14) by thymic repopulation (hanging drop) culture methods. GFP+ cells were observed in the thymus lobe at the end of the repopulation culture period. A large part of the infiltrated cells expressed CD44 until day 2 of culture on a permeable membrane, then lost the expression. CD25 expression was observed from day 1 to day 4. Around day 8, GFP+ cells became both CD4+ and CD8+. The results support the early observation of the sequential expression of CD44, CD25, and CD4/8 during the early stages of thymocyte development. When anti-CD44 mAb was added at the beginning of the repopulation culture period, GFP+ FL cells adhered to the surface of the thymus lobe but did not migrate into the thymus. Pretreatment of the thymus with hyaluronidase or hyaluronate produced results similar to the results of anti-CD44 treatment. On the other hand, the addition of anti-integrin α4 mAb inhibited adhesion to the thymus, and almost no GFP+ cells were seen on the surface of the thymus lobe. The data suggest that integrin α4 and CD44 play different roles, i.e., integrin α4 is required for the adhesion of FL cells to the thymus lobe and CD44 is required for the migration of the cells into the thymus.
A Novel Role for HEB Downstream or Parallel to the Pre-TCR Signaling Pathway During αβ Thymopoiesis
Authors: Barndt Robert , Dai Mei-Fang , Zhuang Yuan
Published: December 2022

TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4−CD8−TCR− double negative (DN) stage to CD4+CD8+ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-α, TCR-β, and CD4. Targeted disruption of HEB results in a 5- to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4low/−CD8+CD5lowHSA+TCRlow/− immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the αβ T cell lineage. Introduction of an αβ TCR transgene into the HEBko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEBko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.
Cytosolic Phospholipase A2 Activation Is Essential for β1 and β2 Integrin-Dependent Adhesion of Human Eosinophils
Authors: Zhu Xiangdong , Muñoz Nilda M. , Kim Kwang Pyo , Sano Hiroyuki , Cho Wonhwa , Leff Alan R.
Published: December 2022

We examined the role of cytosolic phospholipase A2 (cPLA2) during human eosinophil adherence to ICAM-1- or VCAM-1-coated plates. IL-5-stimulated eosinophils adhered to ICAM-1 through the β2 integrin CD11b/CD18, while nonstimulated eosinophils did not. By contrast, nonstimulated eosinophils adhered to VCAM-1 through the β1-integrin VLA-4/CD29. Both IL-5-induced adhesion to ICAM-1 and spontaneous adhesion to VCAM-1 corresponded temporally to cPLA2 phosphorylation, which accompanied enhanced catalytic activity of cPLA2. The structurally unrelated cPLA2 inhibitors, arachidonyl trifluoromethylketone and surfactin, significantly inhibited eosinophil adhesion to ICAM-1 and VCAM-1 in a concentration-dependent manner. Inhibition of secretory PLA2, 5-lipoxygenase, or cyclooxygenase did not affect eosinophil adhesion. Addition of arachidonic acid to eosinophils after cPLA2 inhibition with arachidonyl trifluoromethylketone or surfactin did not reverse the blockade of adhesion to ICAM-1 or VCAM-1. However, CV-6209, a receptor-specific antagonist of platelet-activating factor, inhibited all integrin-mediated adhesion. The activated conformation of CD11b as identified by the mAb, CBRM1/5, as well as quantitative surface CD11b expression were up-regulated after IL-5 stimulation. However, cPLA2 inhibition neither prevented CBRM1/5 expression nor blocked surface Mac-1 up-regulation caused by IL-5. Our data suggest that cPLA2 activation and its catalytic product platelet-activating factor play an essential role in regulating β1 and β2 integrin-dependent adhesion of eosinophils. This blockade occurs even in the presence of up-regulated eosinophil surface integrin.
Adenosine Inhibits Macrophage Colony-Stimulating Factor-Dependent Proliferation of Macrophages Through the Induction of p27kip-1Expression
Authors: Xaus Jordi , Valledor Annabel F. , Cardó Marina , Marquès Laura , Beleta Jorge , Palacios José M. , Celada Antonio
Published: December 2022

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 μM. Only specific agonists that can activate A2B adenosine receptors such as 5′-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine), or A3 (N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 μM) and forskolin (IC50 7 μM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.
Genetic Evidence for Lyn as a Negative Regulator of IL-4 Signaling
Authors: Janas Michelle L. , Hodgkin Philip , Hibbs Margaret , Tarlinton David
Published: December 2022

IL-4 has multiple effects on B lymphocytes, many of which are concentration dependent. This is particularly so for Ig isotype switching, where different thresholds of IL-4 stimulation are needed to induce switching from IgM to either IgG1 or IgE. In this report we describe a critical role for the tyrosine kinase Lyn in setting IL-4 signaling thresholds in mouse B lymphocytes. Upon CD40 ligand stimulation of lyn−/− B cells, 10-fold less IL-4 was required to induce switching from IgM to IgG1 and IgE and an increased proportion of B cells isotype switched at each IL-4 concentration. These in vitro results correlate with the in vivo findings that in lyn−/− mice, IgG1 Ab-forming cells develop prematurely in ontogeny and that adult lyn−/− mice have an abnormally high proportion of IgG1-expressing B cells in their spleens. Adult lyn−/− mice also have significantly higher levels of IgE in their serum. These results identify Lyn as a molecule involved in modulating the IL-4 signal in B cells and provide insights into its regulation and how a B cell signaling imbalance may contribute to atopy.
NK Cell Triggering by the Human Costimulatory Molecules CD80 and CD86
Authors: Wilson Julia L. , Charo Jehad , Martín-Fontecha Alfonso , Dellabona Paolo , Casorati Giulia , Chambers Benedict J. , Kiessling Rolf , Bejarano Maria-Teresa , Ljunggren Hans-Gustaf
Published: December 2022

NK cell-mediated effector functions are regulated by a delicate balance between positive and negative signals. Receptors transmitting negative signals upon engagement with target cell MHC class I molecules have been characterized in detail in recent years. In contrast, less information is available about receptor-ligand interactions involved in the transmission of positive or “triggering” signals to NK cells. Recently, it has been described that murine NK cells are triggered by the costimulatory molecules CD80, CD86, and CD40. Using NK cell lines derived from PBMC as effectors, we demonstrate that the human CD80 and CD86 gene products can function as triggering molecules for NK cell-mediated cytotoxicity. Expression of human CD80 or CD86 molecules in murine B16.F1 melanoma cells rendered these significantly more susceptible to lysis by human NK cell lines. Blocking of the transfected gene products with specific mAb reduced lysis levels to that of nontransfected control cell lines. Triggering of human NK cells by CD80 and CD86 appeared to be independent of CD28 and CTLA-4, at least as determined by the reagents used in the present study, because the expression of these molecules could not be detected on the NK cell lines by either flow cytometry or in redirected lysis assays. Thus, human NK cells may use receptors other than CD28 and CTLA-4 in their interactions with CD80 and CD86 molecules. Alternatively, interactions may involve variants of CD28 (and possibly CTLA-4) that are not recognized by certain anti-CD28 mAb.
CD2 and CD3 Associate Independently with CD5 and Differentially Regulate Signaling Through CD5 in Jurkat T Cells
Authors: Carmo Alexandre M. , Castro Mónica A. A. , Arosa Fernando A.
Published: December 2022

In T lymphocytes, the CD2 and CD5 glycoproteins are believed to be involved in the regulation of signals elicited by the TCR/CD3 complex. Here we show that CD2 and CD3 independently associate with CD5 in human PBMC and Jurkat cells. CD5 coprecipitates with CD2 in CD3-deficient cells and, conversely, coprecipitates with CD3 in cells devoid of CD2. In unstimulated CD2+ CD3+ Jurkat cells, CD5 associates equivalently with CD2 and CD3 and is as efficiently phosphorylated in CD2 as in CD3 immune complexes. However, upon activation the involvement of CD5 is the opposite in the CD2 and CD3 pathways. CD5 becomes rapidly tyrosine phosphorylated after CD3 stimulation, but is dephosphorylated upon CD2 cross-linking. These opposing effects correlate with the decrease in the activity of the SH2 domain-containing protein phosphatase 1 (SHP-1) following CD3 activation vs an enhanced activity of the phosphatase after CD2 triggering. The failure of CD5 to become phosphorylated on tyrosine residues in the CD2 pathway has no parallel with the lack of use of ζ-chains in CD2 signaling; contrasting with comparable levels of association of CD2 or CD3 with CD5, ζ associates with CD2 only residually and is nevertheless slightly phosphorylated after CD2 stimulation. The modulation of CD5 phosphorylation may thus represent a level of regulation controlled by CD2 in signal transduction mechanisms in human T lymphocytes.
Contrasting the In Situ Behavior of a Memory B Cell Clone During Primary and Secondary Immune Responses
Authors: Vora Kalpit A. , Tumas-Brundage Kathleen , Manser Tim
Published: December 2022

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the participation of a predominant anti-arsonate clonotype in the primary and secondary responses in the spleens of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region somatic hypermutation is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity-enhancing mutations.
An Extensive Region of an MHC Class I α2 Domain Loop Influences Interaction with the Assembly Complex
Authors: Yu Yik Y. L. , Turnquist H¯eth R. , Myers Nancy B. , Balendiran Ganesaratnam K. , Hansen Ted H. , Solheim Joyce C.
Published: December 2022

Presentation of antigenic peptides to CTLs at the cell surface first requires assembly of MHC class I with peptide and β2-microglobulin in the endoplasmic reticulum. This process involves an assembly complex of several proteins, including TAP, tapasin, and calreticulin, all of which associate specifically with the β2-microglobulin-assembled, open form of the class I heavy chain. To better comprehend at a molecular level the regulation of class I assembly, we have assessed the influence of multiple individual amino acid substitutions in the MHC class I α2 domain on interaction with TAP, tapasin, and calreticulin. In this report, we present evidence indicating that many residues surrounding position 134 in H-2Ld influence interaction with assembly complex components. Most mutations decreased association, but one (LdK131D) strongly increased it. The Ld mutants, with the exception of LdK131D, exhibited characteristics suggesting suboptimal intracellular peptide loading, similar to the phenotype of Ld expressed in a tapasin-deficient cell line. Notably, K131D was less peptide inducible than wild-type Ld, which is consistent with its unusually strong association with the endoplasmic reticulum assembly complex.
Protective Immunity Using Recombinant Human IL-12 and Alum as Adjuvants in a Primate Model of Cutaneous Leishmaniasis
Authors: Kenney Richard T. , Sacks David L. , Sypek Joseph P. , Vilela Luciano , Gam Albert A. , Evans-Davis Kamela
Published: December 2022

Protection from cutaneous leishmaniasis, a chronic ulcerating skin lesion affecting millions, has been achieved historically using live virulent preparations of the parasite. Killed or recombinant Ags that could be safer as vaccines generally require an adjuvant for induction of a strong Th1 response in murine models. Murine rIL-12 as an adjuvant with soluble Leishmania Ag has been shown to protect susceptible mice. We used 48 rhesus macaques to assess the safety, immunogenicity, and efficacy of a vaccine combining heat-killed Leishmania amazonensis with human rIL-12 (rhIL-12) and alum (aluminum hydroxide gel) as adjuvants. The single s.c. vaccination was found to be safe and immunogenic, although a small transient s.c. nodule developed at the site. Groups receiving rhIL-12 had an augmented in vitro Ag-specific IFN-γ response after vaccination, as well as increased production of IgG. No increase in IL-4 or IL-10 was found in cell culture supernatants from either control or experimental groups. Delayed hypersensitivity reactions were not predictive of protection. Intradermal forehead challenge infection with 107 metacyclic L. amazonensis promastigotes at 4 wk demonstrated protective immunity in all 12 monkeys receiving 2 μg rhIL-12 with alum and Ag. Partial efficacy was seen with lower doses of rhIL-12 and in groups lacking either adjuvant. Thus, a single dose vaccine with killed Ag using rhIL-12 and alum as adjuvants was safe and fully effective in this primate model of cutaneous leishmaniasis. This study extends the murine data to primates, and provides a basis for further human trials.
IL-15 Prolongs the Duration of CD8+ T Cell-Mediated Immunity in Mice Infected with a Vaccine Strain ofToxoplasma gondii
Authors: Khan Imtiaz A. , Casciotti Lori
Published: December 2022

Immunization of mice with a vaccine (ts-4) strain of Toxoplasma gondii is known to induce complete protection against subsequent lethal infection. Ts-4-mediated protection has been reported to be primarily dependent on IFN-γ-producing CD8+ T cells. However, duration of CD8+ T cell-mediated immunity in the ts-4-vaccinated animals is not known. In the present study, the kinetics of the CD8+ T cell response in mice immunized with the ts-4 strain of T. gondii was evaluated. Optimal CD8+ T cell immunity persisted at least 6 mo after vaccination, and mice at this time point continued to overcome lethal challenge with a more virulent strain. However, at 9 mo postimmunization, CD8+ T cell immunity was severely diminished and the mice succumbed to Toxoplasma challenge. Pretreatment of animals, vaccinated 9 mo earlier, with rIL-15 prevented the mortality induced by Toxoplasma challenge. The protective effect of IL-15 treatment was due to a rise in the frequency of Ag-specific CD8+ T cells. CD8+ T cells from IL-15-administered animals showed increased proliferation and IFN-γ production in response to antigenic restimulation. These findings suggest that rIL-15 can reverse the decline in the long-term CD8+ T cell immune response in mice immunized with vaccine strain of T. gondii.
Protein Kinase C (PKC) Isoforms Translocate to Triton-Insoluble Fractions in Stimulated Human Neutrophils: Correlation of Conventional PKC with Activation of NADPH Oxidase
Authors: Nixon Jennifer B. , McPhail Linda C.
Published: December 2022

The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-δ (29%) and small amounts of PKC-α (0.6%), but not PKC-βII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-α, PKC-βII, and PKC-δ increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-δ maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-α and PKC-βII plateaued at 300 nM PMA. Translocation of PKC-δ was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-α and PKC-βII was biphasic, plateauing at 2–3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-α and PKC-βII, but not with translocation of PKC-δ. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-α, PKC-βII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-α and/or PKC-βII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-δ may modulate other PMN responses that involve cytoskeletal components.
CC Chemokine Receptor 5 Cell-Surface Expression in Relation to CC Chemokine Receptor 5 Genotype and the Clinical Course of HIV-1 Infection
Authors: de Roda Husman Ana-Maria , Blaak Hetty , Brouwer Margreet , Schuitemaker Hanneke
Published: December 2022

CCR5 cell-surface expression was studied in relation to CCR5 genotype and clinical course of HIV-1 infection. HIV-1 infected CCR5+/+ individuals had higher percentages of CCR5-expressing CD4+ T cells as compared with HIV-1-infected CCR532/+ individuals. For both genotypic groups, the percentages of CCR5-expressing cells were higher than for the uninfected counterparts (CCR5+/+, HIV+ 28% and HIV− 15% (p < 0.0001); CCR532/+, HIV+ 21% and HIV− 10% (p = 0.001), respectively). In HIV-1-infected individuals, high percentages of CCR5-expressing cells were associated with low CD4+ T cell numbers (p = 0.001), high viral RNA load in serum (p = 0.046), and low T cell function (p = 0.054). As compared with nonprogressors with similar CD4+ T cell numbers, individuals who did progress to AIDS had a higher percentage of CCR5-expressing CD4+ T cells (32% vs 21% (p = 0.002). Longitudinal analysis of CCR5+/+ individuals revealed slight, although not statistically significant, increases in CCR5-expressing CD4+ T cells and CD4+ T cell subsets characterized by the expression of CD45 isoforms, during the course of HIV-1 infection. Preseroconversion, the percentage of CCR5-expressing CD4+ T cells was higher in individuals who subsequently developed AIDS (28%) than in those who did not show disease progression within a similar time frame (20%; p = 0.059). Our data indicate that CCR5 expression increases with progression of disease, possibly as a consequence of continuous immune activation associated with HIV-1 infection. In turn, CCR5 expression may influence the clinical course of infection.
Isolation, Characterization and Sequence Analysis of Five IgG Monoclonal Anti-β2-Glycoprotein-1 and Anti-Prothrombin Antigen-Binding Fragments Generated by Phage Display
Authors: Chukwuocha Reginald U. , Hsiao Elie T. , Shaw Peter , Witztum Joseph L. , Chen Pojen P.
Published: December 2022

We have isolated five monoclonal IgG anti-β2-glycoprotein-1 (anti-β2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three β2GP-1-enriched mAbs (B14, B22, B27) reacted with β2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with β2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 × 10−6 M for β2GP-1 vs 3.2 × 10−6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between β2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to β2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to β2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-β2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients.
C1q-Containing Immune Complexes Purified from Sera of Juvenile Rheumatoid Arthritis Patients Mediate IL-8 Production by Human Synoviocytes: Role of C1q Receptors
Authors: Khalkhali-Ellis Zhila , Bulla Gary A. , Schlesinger Larry S. , Kirschmann Dawn A. , Moore Terry L. , Hendrix Mary J. C.
Published: December 2022

Immune complexes that vary in size and composition are present in the sera and synovial fluid of juvenile rheumatoid arthritis (JRA) patients. They are believed to be potent inducers of the ongoing inflammatory process in JRA. However, the precise composition and role of these complexes in the pathophysiology of JRA remain unclear. We hypothesized that circulating ICs have the potential to interact with resident joint synovial fibroblasts (synoviocytes) and induce the expression of inflammatory cytokines. To test this hypothesis, cultures of synoviocytes from healthy individuals were treated with ICs isolated from the sera of JRA patients. Studies reported in this work demonstrate that IgM affinity-purified ICs from the sera of JRA patients contain IgM, C1q, IgG, and C3 to a variable extent. These ICs induce IL-8 mRNA and protein production in normal synoviocytes. Our data indicate that C1q in these ICs mediates, in part, IL-8 induction in synoviocytes. This is based on our findings of C1q-binding proteins for collagen stalks (cC1qR) and globular heads (gC1q-binding protein) of C1q in synoviocytes. In addition, collagen stalk and to some extent globular head fragments of C1q inhibit IC-mediated IL-8 induction in synoviocytes. Together, these findings provide evidence for a novel mechanism of IL-8 production by synoviocytes, which could play a key role in inflammation by recruiting leukocytes to synovial tissue and fluid—and subsequently contributing to joint disease.
Cutting Edge: Role of C-C Chemokine Receptor 5 in Organ-Specific and Innate Immunity toCryptococcus neoformans
Authors: Huffnagle Gary B. , McNeil Lisa K. , McDonald Roderick A. , Murphy Juneann W. , Toews Galen B. , Maeda Nobuyo , Kuziel William A.
Published: December 2022

After intratracheal inoculation of the AIDS-associated pathogen Cryptococcus neoformans, 12-wk survival was >90% for CCR5+/+ mice but <25% for CCR5−/− mice. There were no defects in lung leukocyte recruitment (wk 5), pulmonary clearance, or delayed-type hypersensitivity in CCR5−/− mice. However, CCR5−/− mice had defects in leukocyte recruitment into the brain and, strikingly, in elimination of cryptococcal polysaccharide from the brain. In nonimmune CCR5−/− mice, there was a significant defect in macrophage recruitment after challenge with shed cryptococcal products (C. neoformans filtrate Ag) but not other nonspecific stimuli. Thus, CCR5 plays specific roles in innate immunity and organ-specific leukocyte trafficking during host defense against C. neoformans.