bluuebunny/crossref_metadata_2025 huggingface dataset:
Independent regulation of cutaneous lymphocyte-associated antigen expression and cytokine synthesis phenotype during human CD4+ memory T cell differentiation.
Authors: Teraki Y , Picker L J
Published: January 2023

Although considerable attention has been paid to the development of cytokine synthesis heterogeneity during memory T cell differentiation, little information is available on how this function is coregulated with homing receptor expression. The development of skin-homing, CD4+ memory T cells in the human provides an excellent model for such investigation, since 1) the skin supports both Th1- and Th2-predominant responses in different settings, and 2) the skin-homing capability of human memory T cells correlates with and appears to depend on expression of the skin-selective homing receptor cutaneous lymphocyte-associated Ag (CLA). In this study, we used multiparameter FACS analysis to examine expression of CLA vs IFN-gamma, IL-4, and IL-2 synthesis capabilities among fresh peripheral blood CD4+ memory T cells, and Th1 vs Th2 memory T cells generated in vitro from purified CD4+ naive precursors by cyclic activation in polarizing culture conditions. Among normal peripheral blood T cells, CLA expression was essentially identical among the IFN-gamma- vs IL-4-producing CD4+ memory subsets, clearly indicating the existence of in vivo mechanisms capable of producing both Th1 vs Th2 skin-homing T cells. In vitro differentiation of naive CD4+ T cells confirmed the independent regulation of CLA and all three cytokines examined, regulation that allowed differential production of IFN-gamma-, IL-4-, and IL-2-producing, CLA+ memory subsets. These studies also 1) demonstrated differences in regulatory factor activity depending on the differentiation status of the responding cell, and 2) revealed CLA expression to be much more rapidly reversible on established memory cells than cytokine synthesis capabilities.
Cytokine induction of the ability of human monocyte CD44 to bind hyaluronan is mediated primarily by TNF-alpha and is inhibited by IL-4 and IL-13.
Authors: Levesque M C , Haynes B F
Published: January 2023

Ligation of CD44 by hyaluronan (HA) is a key proinflammatory event that regulates lymphocyte and monocyte adhesion and cytokine production. While most immune cells express CD44, few immune cells constitutively bind HA. We have previously shown that monocyte CD44 acquires the ability to bind HA after in vitro culture of PBMC in human serum and, therefore, we have investigated a series of human cytokines and bacterial LPS for their ability to induce and/or inhibit monocyte CD44 to bind HA. We found that IL-1alpha, IL-1beta, IL-3, granulocyte macrophage (GM)-CSF, and TNF-alpha, as well as bacterial LPS, all directly induced peripheral blood monocytes to bind HA. In contrast, IL-2 and IL-15 up-regulated monocyte CD44 HA-binding in PBMC suspensions, but not in purified monocyte suspensions. An anti-TNF-alpha-neutralizing Ab inhibited IL-1alpha-, IL-1beta-, IL-3-, and GM-CSF-mediated monocyte HA binding. In addition, treatment of IL-2- and IL-15-stimulated PBMC cultures with an anti-TNF-alpha Ab prevented IL-2- and IL-15-induced monocyte HA binding, thus identifying TNF-alpha as a lymphocyte-derived factor that acted on monocytes to induce HA binding. In contrast, IL-4 and IL-13 were potent inhibitors of monocyte CD44-HA binding induced by either human serum or by IL-1alpha, IL-1beta, IL-3, GM-CSF, or TNF-alpha. IL-10 had dual effects on monocyte CD44-HA binding. Alone, IL-10 induced HA binding to PBMC monocyte CD44, while in contrast, IL-10 inhibited IL-1-induced monocyte CD44 binding to HA. Taken together, these studies identify a network of T cell and monocyte-derived cytokines that regulate HA binding to peripheral blood monocyte CD44, primarily through TNF-alpha.
Extracellular ATP and UTP control the generation of reactive oxygen intermediates in human macrophages through the opening of a charybdotoxin-sensitive Ca2+-dependent K+ channel.
Authors: Schmid-Antomarchi H , Schmid-Alliana A , Romey G , Ventura M A , Breittmayer V , Millet M A , Husson H , Moghrabi B , Lazdunski M , Rossi B
Published: January 2023

Human monocyte-derived macrophages possess a NADPH oxidase that catalyzes superoxide formation upon phagocytosis. Extracellular ATP per se does not activate NADPH oxidase but potentiates superoxide generation triggered by opsonized zymosan. UTP can substitute for ATP with the same efficiency, suggesting that ATP mediates its effects specifically through P2U receptors. Extracellular UTP stimulates a rapid increase in cytoplasmic Ca2+ concentration in monocytic cells, which results from a release of intracellular Ca2+ stores. Moreover, UTP-induced calcium increase is sufficient to activate a charybdotoxin-sensitive Ca2+-dependent outward K+ channel (K(Ca)). The activity of this channel develops between 0.1 and 1.0 microM free cytoplasmic Ca2+ concentration; it is half-blocked by 10 nM charybdotoxin but insensitive to iberiotoxin. Under asymmetrical K+ conditions, this K(Ca) channel does not depend on membrane potential and is characterized by a linear single-current voltage relationship in the voltage range of -100 to +50 mV, giving a unitary conductance of 10 pico-Siemens. Interestingly, ATP/UTP-induced oxygen radicals release was inhibited by charybdotoxin in the same range of concentration as the UTP-induced K(Ca) channel. Furthermore, we show that ATP or UTP fail to enhance oxygen radicals production before K(Ca) channel is expressed (3 days). The electrogenic nature of the NADPH oxidase, i.e., its level of activation, being dependent on the plasmic membrane potential, might provide the causal link between the reactive oxygen intermediates generation and the opening of the K(Ca) channel.
Identification and characterization of a novel beta chemokine containing six conserved cysteines.
Authors: Hedrick J A , Zlotnik A
Published: January 2023

The chemokines are emerging as a diverse and expanding family of cytokines. In this study, we report the identification of a novel beta (CC) chemokine that exhibits an unusual pattern of six conserved cysteines and was thus designated 6Ckine. The primary amino acid sequence of 6Ckine is highly conserved in humans, mice, and pigs and contains the expected four cysteines characteristic of the beta chemokine family plus two additional carboxyl-terminal cysteines. Expression of human 6Ckine appears to be restricted to lymphoid organs, particularly lymph node, spleen, and appendix, while murine 6Ckine has a broader tissue distribution with the highest mRNA levels found in spleen and lung. Recombinant murine 6Ckine was chemotactic in vitro for thymocytes and activated T cells, but not for B cells, macrophages, or neutrophils. Together these data demonstrate that 6Ckine is a novel member of the chemokine superfamily.
Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3-like superfamily: implications for vaccine development.
Authors: Threlkeld S C , Wentworth P A , Kalams S A , Wilkes B M , Ruhl D J , Keogh E , Sidney J , Southwood S , Walker B D , Sette A
Published: January 2023

Recent data demonstrate that HLA class I alleles can be grouped into superfamilies based on similarities of their peptide-binding motifs. In this study, we have tested the immunogenicity and antigenicity of peptides capable of degenerate binding to multiple HLA class I molecules of the A3-like superfamily. The assay systems utilized included both primary in vitro cultures of lymphocytes from healthy donors, as well as in vitro restimulation of lymphocytes from HIV-infected individuals. Several of the peptides capable of binding more than one HLA A3-like class I molecule were also found to be immunogenic in the context of this same group of A3-like molecules (degenerate CTL recognition). Furthermore, some of the CTL lines thus generated demonstrated promiscuous recognition of the cognate epitope in the context of MHC molecules from more than one member of the superfamily. The fine Ag specificity of this phenomenon was further analyzed using two promiscuous CTL clones derived from A3 and A11 individuals, respectively, and specific for an epitope in the HIV-1 reverse transcriptase. By the use of single-amino acid-substitution analogues, it was demonstrated that the fine specificity of the TCR is largely maintained between MHC-matched and MHC-mismatched presentation of peptide within the A3-like superfamily. These results indicate that the similar peptide-binding specificities among different members of the A3-like superfamily can be reflected in a remarkable similarity in the peptide-MHC complex structures engaged by the TCR and responsible for T cell activation.
Augmentation of mature CD4+ T cell responses to isolated antigenic class II proteins by fibronectin and intercellular adhesion molecule-1.
Authors: Brunmark A , O'Rourke A M
Published: January 2023

Mature CD4+ T cells can undergo stable adhesion to isolated antigenic MHC complexes, and upon TCR engagement exhibit up-regulated adhesion to the integrin ligands fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1). Here, we have examined T cell responses to purified antigenic class II complexes, alone or coimmobilized in the presence of FN or ICAM-1. T cell adhesion to immobilized peptide-MHC complexes alone stimulated suboptimal, but marked levels of IFN-gamma and IL-2 secretion, and this was accompanied by cell proliferation. T cell adhesion to both FN and ICAM-1 strongly augmented cytokine release and T cell proliferation. Activation of Vbeta3+ and Vbeta8+ T cell lines by isolated staphylococcal enterotoxin-MHC complexes was also examined, and surprisingly, a Vbeta8+ T cell line displayed significant cell adhesion or later response to staphylococcal enterotoxin B-MHC complexes only when Ag was coimmobilized with ICAM-1 or FN. The results demonstrate that adhesion of CD4+ T cells to ICAM-1 or FN activated by natural TCR ligands can strongly augment T cell signaling and downstream responses. Moreover, for some Ags, T cell interaction with accessory ligands may be critical in attaining a threshold level of receptor occupancy for cell activation.
Cytotoxic CD8+ T cells recognize EBV antigen but poorly kill autologous EBV-infected B lymphoblasts: immunodominance is elicited by a peptide epitope that is presented at low levels in vitro.
Authors: Shi Y , Smith K D , Kurilla M G , Lutz C T
Published: January 2023

CD8+ T cells play a crucial role in surveillance against EBV-associated malignancies. EBV-specific T cells traditionally have been identified by their ability to kill autologous EBV-transformed B lymphoblastoid cell lines (LCL). Here we report CD8+ cloned and bulk T cells that specifically recognize EBV nuclear Ag EBNA-3C, but do not efficiently kill autologous or HLA-matched LCL. The low cytolysis of these T cells was due to the extremely low density of the antigenic epitope (LDFVRFMGV, EBNA-3C amino acids 285-293) on autologous LCL. The T cells efficiently killed target cells in the presence of < 1 pM synthetic EBNA-3C peptide and, therefore, recognize peptide/HLA complexes with high avidity. Donor T cells with this phenotype were stimulated by autologous LCL and dominated the in vitro EBV-specific response. This indicates that low abundance viral peptides can induce a dominant T cell response.
The Chemical Nature of the Constituent in Fowl Serum Responsible for Non-Specific Precipitations
Authors: Schilling Geo. S.
Published: January 2023

In a previous communication (1), observations were reported on the effect of employing antigens of greater density than those commonly used in the agglutination test for determining infection in fowls. It was found that antigens containing larger quantities of bacterial bodies were more stable as regards the element in fowl sera, which induces non-specific precipitations. Further, it was suggested that the constituent responsible for such precipitation, commonly spoken of as the cloudy reaction, was lipoidal in constitution. This paper reports further studies on the physio-chemical properties of this factor.

Fatty and proteid materials have been variously charged with causing this interference in the agglutination reaction. A review of the literature of the chemical constituents of fowl sera discloses that Warner and Edmond (2) found that there is a higher percentage of fat in the whole blood of pullets and hens in heavy lay than in females not in lay and in male birds.
Cutting Edge: Involvement of SHP-2 in Multiple Aspects of IL-2 Signaling: Evidence for a Positive Regulatory Role
Authors: Gadina Massimo , Stancato Louis M. , Bacon Chris M. , Larner Andrew C. , O’Shea John J.
Published: January 2023

Binding of IL-2 to its receptor activates several biochemical pathways, but precisely how these pathways are linked is incompletely understood. Here, we report that SHP-2, an SH2-domain containing tyrosine phosphatase, associates with different molecules of the IL-2 signaling cascade. Upon IL-2 stimulation, SHP-2 was coimmunoprecipitated with Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. In contrast, SHP-2 was constitutively associated with JAK1 and JAK3. Finally, SHP-2 expression amplified STAT-dependent transcriptional activation whereas a dominant negative allele inhibited transactivation and the IL-2-induced activation of MAPK (mitogen-activated protein kinase). These results demonstrate the involvement of SHP-2 in multiple pathways of the IL-2 signaling cascade and provide evidence for its positive regulatory role.
Cutting Edge: CD40 Ligation Prevents Neonatal Induction of Transplantation Tolerance
Authors: Flamand Véronique , Donckier Vincent , Demoor François-Xavier , Le Moine Alain , Matthys Patrick , Vanderhaeghen Marie-Line , Tagawa Yoh-ichi , Iwakura Yoichiro , Billiau Alfons , Abramowicz Daniel , Goldman Michel
Published: January 2023

To investigate the consequences of CD40 engagement on the neonatal induction of transplantation tolerance, BALB/c mice were injected at birth with (A/J × BALB/c) F1 spleen cells together with activating anti-CD40 mAb and grafted 4 wk later with A/J skin. Whereas A/J allografts were accepted in mice neonatally injected with F1 cells and control Ab, they were acutely rejected in mice injected with F1 cells and anti-CD40 mAb. Neonatal administration of anti-CD40 mAb resulted in enhanced anti-A/J CTL activity, increased IFN-γ, and decreased IL-4 production by donor-specific T cells in vitro. Experiments using anti-cytokine mAb and IFN-γ-deficient mice demonstrated that CD40 ligation prevents neonatal allotolerance through an IFN-γ- and IL-12-dependent pathway. Finally, we found that newborn T cells express less CD40L than adult T cells upon TCR engagement. Taken together these data indicate that insufficiency of CD40/CD40L interactions contribute to neonatal transplantation tolerance.
Transplacental Priming of the Human Immune System to Environmental Allergens: Universal Skewing of Initial T Cell Responses Toward the Th2 Cytokine Profile
Authors: Prescott Susan L. , Macaubas Claudia , Holt Barbara J. , Smallacombe Troy B. , Loh Richard , Sly Peter D. , Holt Patrick G.
Published: January 2023

The expression of Th2-skewed immunity against soluble protein Ags present in the normal environment is recognized as the primary cause of allergic inflammation in atopics. In contrast, nonallergic normal individuals display low level Th1-skewed immunity against the same Ags (“allergens”), which is perceived as conferring protection against Th2-dependent allergic sensitization. The type of T cell memory that develops against these Ags is currently believed to be the result of complex interactions between environmental and genetic susceptibility factors, which occur postnatally when the naive immune system directly confronts the outside environment. The results of the present study challenge this general concept. We demonstrate here for the first time that Th2-skewed responses to common environmental allergens, comprising IL-4, IL-5, IL-6, IL-9, and IL-13, are present in virtually all newborn infants and are dominated by high level production of IL-10. Moreover, these responses are demonstrable within 24 h of culture initiation, arguing against a significant contribution from covert in vitro T cell priming and/or differentiation. These findings imply that the key etiologic factor in atopic disease may not be the initial acquisition of allergen-specific Th2-skewed immunity per se, but instead may be the efficiency of immune deviation mechanisms, which in normal (nonatopic) individuals redirect these fetal immune responses toward the Th1 cytokine phenotype.
Flt3 Ligand Plus IL-7 Supports the Expansion of Murine Thymic B Cell Progenitors That Can Mature Intrathymically
Authors: McKenna Hilary J. , Morrissey Philip J.
Published: January 2023

Flt3 ligand (flt3L) has potent effects on hemopoietic progenitors, dendritic cells, and B lymphopoiesis. We have investigated the effects of flt3L on intrathymic precursors. The addition of flt3L + IL-7 to lobe submersion cultures of murine fetal thymic lobes resulted in the expansion of an immature population of Thy-1low, CD44high, HSAhigh cells. This population contained cells with precursor activity, as determined by their capacity to repopulate deoxyguanosine-treated fetal thymic lobes. Upon reentry to the thymic lobe, flt3L + IL-7-cultured Thy-1low, CD44high, HSAhigh cells underwent expansion and differentiation into B cells. Two weeks after fetal thymic organ culture following thymic lobe reconstitution, intrathymic cells were Thy-1−, B220+, and a subset was sIgM+. The intrathymic B cells shared features of adult thymic B cells, including CD5 expression and proliferative responses to IL-4 + IL-5 + CD40 ligand, but not to LPS or soluble anti-IgM. Ig production was noted upon stimulation with IL-4 + IL-5 + LPS and IL-4 + IL-5 + CD40 ligand. In conclusion, we have demonstrated that flt3L + IL-7 supports the expansion of a subset of progenitors present in the fetal thymus. The cultured progenitors can repopulate a fetal thymic lobe and develop into mature functional B cells, demonstrating that the fetal thymus is able to support B cell as well as T cell development.
Consequences of Intrathymic TCR Engagement by Partial Agonist on Selection Events and Peripheral T Cell Activation Program
Authors: Auphan Nathalie , Simon Anna Katharina , Asnagli Hélène , Phillips Roderick J. , Rincon Mercedes , Ghosh Sankar , Flavell Richard A. , Schmitt-Verhulst Anne-Marie
Published: January 2023

Functions elicited from mature T cells depend on the nature of the Ag. Thus, an agonist induces a larger set of cytokine responses than a partial agonist. Additionally, Ags present in the thymus influence both the selection of TCRs generated by gene rearrangement and the potential functional program of developing thymocytes. This can be approached by analysing the development of T cells in mice expressing the same transgenic TCR (tgTCR) under different conditions of intrathymic selection. H-2Kbm8 was found to act as a partial agonist for CD8+ T cells expressing a tgTCR specific for the H-2Kb alloantigen. Intrathymic exposure to full or to partial agonist affected the development of thymocytes at different stages, consistent with the respective CD8-independent and -dependent characteristic of the tgTCR/Ag interaction. The presence of the partial agonist led to the accumulation of a major population of thymocytes (tgTCRhighCD4−CD8low) originating from TCR engagement at the immature single-positive CD8low stage as evidenced by: 1) results from reaggregated thymic organ culture in the presence of H-2k/bm8 thymic stromal cells; 2) the absence of CD4+ thymocytes, the development of which depends on rearrangements of endogenous TCRα genes; and 3) the identification of the CD8low thymocytes as cycling cells. Peripheral CD8low T cells selected in an H-2k/bm8 thymus expressed a partial functional program in response to H-2Kb, akin to the response of CD8high T cells to a partial agonist. The analysis of the molecular bases for partial reactivity revealed a correlation with inefficient AP-1, but efficient NF-κB transactivation.
Activated Rat Macrophages Produce a Galectin-1-Like Protein That Induces Apoptosis of T Cells: Biochemical and Functional Characterization
Authors: Rabinovich Gabriel A. , Iglesias María M. , Modesti Nidia M. , Castagna Leonardo F. , Wolfenstein-Todel Carlota , Riera Clelia M. , Sotomayor Claudia E.
Published: January 2023

Galectins, a family of closely related β-galactoside-binding proteins, show specific immunomodulatory properties. We have recently identified the presence of a galectin-like protein in rat peritoneal macrophages by means of a cross-reactivity with a polyclonal Ab raised against a galectin purified from adult chicken liver. Galectin expression was up-regulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells. In an attempt to further explore its functional significance, rat macrophage galectin was purified from activated macrophages by a single-step affinity chromatography on a lactosyl-Sepharose matrix. The eluted fraction was resolved as a single protein band of ∼15,000 Da by SDS-PAGE that immunoreacted strongly with the anti-chicken galectin serum. Gel filtration studies revealed that the protein behaved like a dimer under native conditions, and saccharides bearing a β-d-galactoside configuration were able to inhibit the hemagglutinating activity displayed by the purified galectin. In agreement with its isoelectric point of ∼4.8, the amino acid analysis showed a definitive acidic pattern. Internal amino acid sequencing of selected peptides obtained by proteolytic cleavage revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin-1 subfamily. Finally, biochemical and ultrastructural evidence, obtained by genomic DNA fragmentation and transmission electron microscopy, are also provided to show its potential implications in the apoptotic program of T cells. This effect was quantified by using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling assay and was found to be associated to the specific carbohydrate-binding properties of galectin.
Purification and Characterization of Lymphocyte Chymase I, a Granzyme Implicated in Perforin-Mediated Lysis
Authors: Woodard Susan L. , Fraser Stephanie A. , Winkler Ulrike , Jackson Delwin S. , Kam Chih-Min , Powers James C. , Hudig Dorothy
Published: January 2023

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37°C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.
Synergistic Effect of Type II Phospholipase A2 and Platelet-Activating Factor on Mac-1 Surface Expression and Exocytosis of Gelatinase Granules in Human Neutrophils: Evidence for the 5-Lipoxygenase-Dependent Mechanism
Authors: Takasaki Jun , Kawauchi Yasushi , Masuho Yasuhiko
Published: January 2023

Stimulation of human neutrophils with inflammatory mediators such as TNF-α or platelet-activating factor (PAF) induces translocation of adhesion molecule Mac-1 (CD11b/CD18) from secretory vesicles to the plasma membrane. Type II phospholipase A2 (PLA2-II) also induces translocation of Mac-1 from secretory vesicles. However, there are more Mac-1 molecules in gelatinase granules and specific granules than in secretory vesicles. Therefore, different combinations of PLA2-II and other mediators were examined for their ability to induce gelatinase granules and specific granules to induce Mac-1 surface expression. The combination of PLA2-II and PAF synergistically increased Mac-1 surface expression, and the effect was greater than the combinations of PLA2-II with TNF-α, IL-8, or FMLP. Additionally, the combination of PLA2-II and PAF induced exocytosis of both secretory vesicles and gelatinase granules, which did not occur with either PLA2-II alone or PAF alone. The induction was accompanied by marked production of leukotriene B4. AA861, an inhibitor of 5-lipoxygenase, did not inhibit exocytosis of secretory vesicles but did inhibit exocytosis of gelatinase granules and decrease Mac-1 surface expression. It was also found that Ca2+ influx is essential for 5-lipoxygenase activation, because Ni2+, which blocks the influx of extracellular Ca2+, inhibited the production of leukotriene B4. These results suggest that stimulation by the combination of PLA2-II and PAF, unlike stimulation by each mediator alone, causes exocytosis of gelatinase granules via the 5-lipoxygenase pathway, resulting in a synergistic increase in neutrophil Mac-1 surface expression during inflammatory processes.
Expression of Receptors for C5a Anaphylatoxin (CD88) on Human Bronchial Epithelial Cells: Enhancement of C5a-Mediated Release of IL-8 upon Exposure to Cigarette Smoke
Authors: Floreani Anthony A. , Heires Art J. , Welniak Lisbeth A. , Miller-Lindholm Amanda , Clark-Pierce Laurel , Rennard Stephen I. , Morgan Edward L. , Sanderson Sam D.
Published: January 2023

Results are presented that demonstrate a heightened responsiveness of human bronchial epithelial cells (HBECs) toward the complement-derived anaphylatoxin C5a when these cells are exposed to cigarette smoke. This C5a response is possible because we show at both the protein and mRNA levels that HBECs constitutively express receptors for C5a (C5aR, CD88). Control (untreated) HBECs responded to C5a (50 nM) by releasing the proinflammatory cytokine IL-8 at low but significant levels. However, exposure of HBECs to 5% cigarette smoke extract (CSE) for at least 15 min resulted in an increase in the ability of an anti-human C5aR Ab to bind to the cell surface. CSE-treated HBECs responded in a dose-dependent fashion to human recombinant C5a and to a conformationally biased decapeptide agonist of C5a (YSFKPMPLaR) by releasing IL-8. The levels of IL-8 released in response to C5a were significantly greater in CSE-treated HBECs than in control HBECs. Moreover, this C5a-mediated release of IL-8 from CSE-treated HBECs was significantly reduced in the presence of the anti-human C5aR Ab. These results indicate that HBECs constitutively express C5aRs and that exposure to environmental irritants such as cigarette smoke modulates the expression and responsiveness of these C5aRs toward the C5a-mediated release of IL-8.
Escherichia coli Bound to the Primate Erythrocyte Complement Receptor via Bispecific Monoclonal Antibodies Are Transferred to and Phagocytosed by Human Monocytes in an In Vitro Model
Authors: Kuhn Susan E. , Nardin Alessandra , Klebba Philip E. , Taylor Ronald P.
Published: January 2023

We have prepared cross-linked, bispecific mAb complexes (heteropolymers) that facilitate rapid and quantitative binding of a prototype pathogen, Escherichia coli, to the complement receptor (CR1) on primate erythrocytes. Incubation of the erythrocyte-heteropolymer-E. coli complexes with freshly isolated human mononuclear cells leads to rapid removal of the E. coli from the erythrocytes, and phagocytosis and killing of the bacteria. The erythrocytes are not lysed or phagocytosed during this transfer reaction, but both heteropolymer and CR1 are removed from the erythrocytes along with the E. coli. These findings parallel observations made in previous in vivo experiments in which heteropolymers were used to facilitate clearance of innocuous prototype pathogens in a monkey model. It should now be possible to extend the heteropolymer paradigm to a live pathogen in a primate model.
Intrinsic Differences in L-Selectin Expression Levels Affect T and B Lymphocyte Subset-Specific Recirculation Pathways
Authors: Tang Mimi L. K. , Steeber Douglas A. , Zhang Xiu-Qin , Tedder Thomas F.
Published: January 2023

Lymphocyte migration into lymphoid organs is regulated by tissue-specific adhesion molecules such as L-selectin and the α4β7 integrin. Whether L-selectin also regulates lymphocyte subset-specific migration into specific lymphoid tissues was examined in this study by comparing the migration of CD4+ T cells, CD8+ T cells, and B cells from L-selectin-deficient and wild-type mice. T cells were the predominant lymphocyte subset entering PLN, MLN, Peyer’s patches, and spleen during short term (1-h) migration assays. However, both B cell and CD4+ and CD8+ T cell entries into PLN, MLN, and Peyer’s patches were dramatically impaired (73–98%) by loss of L-selectin. Lymphocyte expression of α4β7 integrin did not compensate for the loss of L-selectin, since both B and T cells predominantly migrated into the spleen in the absence of L-selectin. The more efficient migration of T cells into peripheral lymphoid tissues relative to that of B cells was partly explained by the finding that T cells expressed L-selectin at 50 to 100% higher levels than B cells. In addition, a 50% reduction in L-selectin expression by lymphocytes from hemizygous L-selectin+/− mice resulted in a 50 to 70% decrease in short term lymphocyte migration into peripheral lymphoid tissues relative to that of wild-type lymphocytes. Thus, the differential migration of T and B lymphocyte subsets to lymphoid tissues is regulated in part by subset-specific differences in L-selectin expression levels.
IFN-γ Is Produced by Polymorphonuclear Neutrophils in Human Uterine Endometrium and by Cultured Peripheral Blood Polymorphonuclear Neutrophils
Authors: Yeaman Grant R. , Collins Jane E. , Currie Janet K. , Guyre Paul M. , Wira Charles R. , Fanger Michael W.
Published: January 2023

Cytokines present in the human uterus play an important role both in modulating immune responses to infectious challenge and in the establishment and maintenance of pregnancy. In particular, successful implantation and pregnancy is thought to require the establishment of a Th2 environment, while Th1 cytokines are associated with pregnancy loss and infertility. On the other hand, a Th1 response appears to be required for the resolution of acute infection. Using novel confocal microscopic analysis of fresh sections of human tissue, we have investigated the production of IFN-γ, a Th1 cytokine, in human endometria. Extracellular IFN-γ, mostly associated with matrix components, was located immediately beneath the luminal epithelium and along the glandular epithelium proximal to the lumen. As evidenced by intracellular staining, IFN-γ is produced by both stromal cells and intraepithelial lymphocytes through all stages of the menstrual cycle. Surprisingly, the stromal cell containing intracellular IFN-γ was identified as a polymorphonuclear neutrophil on the basis of its reactivity with a panel of mAbs and its nuclear morphology. We further found that polymorphonuclear neutrophils isolated from normal donors produce IFN-γ in response to stimulation with LPS, IL-12, and TNF-α. Taken together, these findings suggest that polymorphonuclear neutrophils are capable of producing IFN-γ both in vitro and in vivo, indicating that their role in shaping immune responses may be more extensive than previously thought. Furthermore, these studies strongly suggest that polymorphonuclear neutrophils play an important role in determining immune responsiveness within the female reproductive tract.
Two Types of Anti-TL (Thymus Leukemia) CTL Clones with Distinct Target Specificities: Differences in Cytotoxic Mechanisms and Accessory Molecule Requirements
Authors: Tsujimura Kunio , Takahashi Toshitada , Iwase Shigeru , Matsudaira Yasue , Kaneko Yoko , Yagita Hideo , Obata Yuichi
Published: January 2023

TCRαβ CTL clones recognizing mouse thymus leukemia (TL) Ags were established and categorized into two groups: those killing any TL+ target cells (type I) and those killing only TL+ Con A blasts (type II). Cold target inhibition assays showed that the antigenic determinant(s) recognized by type II clones are expressed not only on TL+ Con A blasts but also on other TL+ target cells. The relation of the target specificity to the killing machinery and the accessory molecules involved in cytotoxicity were therefore analyzed using four representative clones selected from each type. Of the target cells tested, Fas was only expressed on Con A blasts, indicating that Fas ligand (FasL)-dependent cytotoxicity is limited to such cells. All four type II and one of four type I clones expressed FasL on the surface, while both types contained perforin in the cytoplasm. Blocking studies using neutralizing anti-FasL mAbs and concanamycin A (CMA), a selective inhibitor of the perforin pathway, suggested that type I clones kill target cells by way of perforin, while type II clones kill TL+ Con A blasts through FasL together with perforin. For their cytotoxicity, type I CTLs require a signal through CD8, while type II require LFA-1/ICAM-1 interactions. Type II clones also need a costimulatory signal through an unknown molecule for perforin-dependent cytotoxicity. These results taken together suggest that the difference in the target specificity of anti-TL CTL clones is due to variation in the killing machineries and the dependence on accessory molecules.
A Pathogenic Role of IL-12 in Blood-Stage Murine Malaria Lethal Strain Plasmodium berghei NK65 Infection
Authors: Yoshimoto Takayuki , Takahama Yasuhiro , Wang Chrong-Reen , Yoneto Toshihiko , Waki Seiji , Nariuchi Hideo
Published: January 2023

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-γ, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-α, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-γ. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-γ in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-γ production rather than the protection.
Cutting Edge: JAK3 Activation and Rescue of T Cells from HIV gp120-Induced Unresponsiveness
Authors: Selliah Nithianandan , Finkel Terri H.
Published: January 2023

In early HIV disease, immunodeficiency is characterized by the inability of CD4+ T cells to produce a critical cytokine, IL-2, and to express the receptor for IL-2 (IL-2R) in response to antigenic or mitogenic stimulation. The shared common γ-chain (γc) of IL-2R and its associated Janus kinase, JAK3, are indispensable for normal T cell function. Here, we show that the inhibition of IL-2R expression and proliferation induced by ligation of CD4 by HIV envelope glycoprotein, gp120, is correlated with inhibition of expression and activation of JAK3. Stimulation through the γc-related cytokine receptors restores JAK3 expression and activation and rescues CD4-mediated T cell unresponsiveness. Collectively, these data argue that inhibition of JAK3 expression and activation may, in part, explain the T cell dysfunction seen in early HIV disease. In addition, rescue from gp120-mediated T cell unresponsiveness by activation of JAK3 suggests a novel therapeutic approach for enhancing immune function in HIV disease.
Specific Immune Induction Following DNA-Based Immunization Through In Vivo Transfection and Activation of Macrophages/Antigen-Presenting Cells
Authors: Chattergoon Michael A. , Robinson Tara M. , Boyer Jean D. , Weiner David B.
Published: January 2023

The initiation of an adaptive immune response requires Ag presentation in combination with the appropriate activation signals. Classically, Ag presentation and immune activation occur in the lymph node and spleen, where a favorable organ architecture and rich cellular help can enhance the process. Recently, several investigators have reported the use of DNA expression cassettes to elicit cellular and humoral immunity against diverse pathogens. Although the immune mechanisms involved are still poorly understood, plasmid inoculation represents a model system for studying immune function in response to invading pathogens. In this report, we demonstrate the presence of activated macrophages or dendritic cells in the blood lymphocyte pool and peripheral tissues of animals inoculated with DNA expression cassettes. These cells are directly transfected in vivo, present Ag, and display the surface proteins CD80 and CD86. Our studies indicate that these cells function as APC and can activate naive T lymphocytes. They may represent an important first step APC in genetic immunization and natural infection.
Differential Involvement of a Fas-CPP32-Like Protease Pathway in Apoptosis of TCR/CD9-Costimulated, Naive T Cells and TCR-Restimulated, Activated T Cells
Authors: Park Cheung-Seog , Yashiro Yumi , Tai Xu-Guang , Toyo-oka Kazuhito , Hamaoka Toshiyuki , Yagita Hideo , Okumura Ko , Neben Steven , Fujiwara Hiromi
Published: January 2023

Our previous study showed that CD9 costimulation of TCR-triggered naive T cells elicits activation ([3H]TdR incorporation) that is similar to CD28 costimulation; however, unlike CD28 costimulation, CD9 costimulation results in apoptosis of these previously activated T cells. Here, we investigated whether the apoptosis occurring after TCR/CD9 stimulation is associated with a death pathway involving Fas stimulation and Fas-mediated caspase activation as observed in activation-induced cell death (AICD). In contrast to AICD, the apoptosis resulting from TCR/CD9 stimulation in C57BL/6 T cells was independent of Fas, because this form of apoptosis was not prevented by anti-Fas ligand mAb and was also induced in MRL/lpr T cells. AICD was observed at 12 h after the restimulation of activated T cells with anti-CD3 and reached a peak level at 24 h after this restimulation. CPP32-like protease activity was detected during AICD. Although TCR/CD9 stimulation-associated apoptosis was observed at 24 h after the stimulation of naive T cells and reached a peak level at 36 h after this stimulation, CPP32-like protease activity in these T cells was only marginal at all time points. Nevertheless, both forms of apoptosis were prevented similarly by two different peptide-based caspase inhibitors. These results indicate that the apoptosis that follows the T cell activation which is induced as a result of CD9 costimulation does not involve a Fas-CPP32-like protease pathway, but suggest that different caspase members are likely to be critical in this form of apoptosis.
Intracellular Formation and Cell Surface Expression of a Complex of an Intact Lysosomal Protein and MHC Class II Molecules
Authors: Arunachalam Balasubramanian , Pan Mary , Cresswell Peter
Published: January 2023

The generation of invariant chain-free MHC class II molecules and their association with endocytically generated peptides are thought to occur in specialized lysosome-like compartments called MIICs (MHC class II compartments). A number of in vitro studies have shown that large denatured proteins can bind to class II molecules, and that class II association can protect the bound segment of protein from proteolytic degradation. In this work, we present what we believe is the first example of an intact endogenous protein (IP30) binding in an allele-dependent fashion to class II molecules in vivo. IP30 is an IFN-γ-inducible 35-kDa glycoprotein that localizes in MIICs. In this study, we show that intact IP30 binds to certain HLA-DR alleles via an N-terminal prosequence. The association takes place in the endocytic pathway following removal of invariant chain from class II molecules and before their cell surface expression. We also show that DR-IP30 complexes are SDS stable. The potential precursor-product relationship between DR-IP30 complexes and the DR-peptide complex is discussed.
T Cell Renewal Rates, Telomerase, and Telomere Length Shortening
Authors: De Boer Rob J. , Noest André J.
Published: January 2023

Measurements on the average telomere lengths of normal human naive and memory T cells suggested that 1) naive and memory human T cells have similar division rates, and 2) that the difference between naive and memory cells reflects the degree of clonal expansion during normal immune reactions. Here we develop mathematic models describing how the population average of telomere length depends on the cell division rates of naive and memory T cells during clonal expansion and normal renewal. The results show that 1) telomeres shorten with twice the cell division rate, 2) that the conventional approach of estimating telomere length shortening per mean population doubling gives rise to estimates that are 39% larger than the “true” loss per cell division, 3) that naive and memory T cells are expected to shorten their telomeres at rates set by the division rate of the naive T cells only, i.e., irrespective of the division rate of memory T cells, 4) that the measured difference in the average telomere length between naive and memory T cells may largely reflect the difference in renewal rates between these subpopulations rather than the clonal expansion, and 5) that full telomerase compensation during clonal expansion is consistent with all data on the shortening of telomere length in, and between, naive and memory T cells. Thus we reconcile the apparent contradictions between the demonstrated difference in division rates between human naive and memory T cells and their similar rates of telomere shortening, and the demonstrated telomere shortening in the presence of telomerase activity.
Dipeptidyl Peptidase I and Granzyme A Are Coordinately Expressed During CD8+ T Cell Development and Differentiation
Authors: Mabee Christopher L. , McGuire Michael J. , Thiele Dwain L.
Published: January 2023

Dipeptidyl peptidase I (DPPI) is a granule protease that plays a requisite role in processing the proenzyme form of the CTL granule serine proteases (granzymes). This study assesses DPPI mRNA and enzyme expression during T lymphocyte ontogeny and CTL differentiation. The most immature CD3−CD4−CD8− thymocytes were found to express >40-fold higher levels of DPPI mRNA, although levels of DPPI enzymatic activity in CD3−CD4−CD8− thymocytes were only modestly higher than those seen for CD4+CD8+ or CD4+CD8− thymocytes. More mature CD8+CD4− thymocytes and CD8+ splenocytes expressed significantly higher levels of DPPI mRNA and enzymatic activity than CD4+CD8+ or CD4+CD8− thymocytes. Granzyme A mRNA expression was observed in DPPI expressing CD3−CD4−CD8− and CD8+CD4− thymocytes and was also observed in CD8+CD4− splenocytes; however, expression was not observed in CD4+CD8+ or CD4+CD8− thymocytes. Both DPPI mRNA and granzyme A mRNA expression in CD8+ T cells decreased to very low or undetectable levels during the first 48 h after allostimulation in MLCs. However, peak levels of both DPPI and granzyme A expression were observed later in the course of CD8+ T cell responses to alloantigen, with DPPI mRNA expression peaking on either day 3 or day 4 and granzyme A expression peaking at the end of a 5-day MLR. These data indicate that DPPI is expressed at all stages of T cell ontogeny and differentiation in which granzyme A mRNA is detected; consequently, DPPI appears to be available for the processing and activation of granzyme A during both CD8+ T cell development and differentiation.
Molecular Mechanisms of the Induction of IL-12 and Its Inhibition by IL-10
Authors: Aste-Amezaga Miguel , Ma Xiaojing , Sartori Alexandrina , Trinchieri Giorgio
Published: January 2023

Exogenously added IL-10 rapidly inhibited Staphylococcus aureus- or LPS-induced cytokine mRNA expression in human PBMCs and monocytes, with a maximal effect observed when IL-10 was added from 20 h before until 1 h after the addition of the inducers. Nuclear run-on assays revealed that the inhibition of IL-12 p40, IL-12 p35, and TNF-α was at the gene transcriptional level and that the addition of IL-10 to S. aureus- or LPS-treated PBMCs did not affect mRNA stability. The inhibitory activity of IL-10 was abrogated by cycloheximide (CHX), suggesting the involvement of a newly synthesized protein(s). The addition of CHX at 2 h before S. aureus or LPS also inhibited the accumulation of IL-12 p40 mRNA, but did not inhibit IL-12 p35 and TNF-α mRNA. This finding suggests that p40 transcription is regulated through a de novo synthesized protein factor(s), whereas the addition of CHX at 2 h after S. aureus activation caused superinduction of the IL-12 p40, IL-12 p35, and TNF-α genes. These results indicate that in human monocytes, the mechanism(s) of IL-10 suppression of both IL-12 p40 and IL-12 p35 genes is primarily seen at the transcriptional level, and that the induction of the IL-12 p40 and p35 genes have different requirements for de novo protein synthesis.