bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of APCs Through CD40 or Toll-Like Receptor 9 Overcomes Tolerance and Precipitates Autoimmune Disease
Authors: Ichikawa Hiroshi T. , Williams Lucas P. , Segal Benjamin M.
Published: April 2014

Some autoreactive T cells normally escape thymic selection and persist in the periphery. This is true of myelin-reactive CD4+ T cells, the effectors of experimental autoimmune encephalomyelitis (EAE) in laboratory animals and the presumed mediators of multiple sclerosis in humans. Nonetheless, most individuals do not succumb to autoimmune disease. There is growing evidence that while peripheral APCs stimulate immune responses against foreign Ags in the setting of tissue destruction and “danger,” they actually maintain tolerance against self Ags under steady state conditions. We hypothesized that tolerance against candidate autoantigens could be reversed by activation of APCs via CD40 or Toll-like receptor 9 signaling. Adult SJL mice injected i.p. with a peptide fragment of proteolipid protein (a candidate autoantigen in multiple sclerosis) emulsified in IFA fail to mount lymphoproliferative or cytokine responses and are protected from EAE upon subsequent challenge with the Ag combined with adjuvants. Here we report that tolerized proteolipid protein-specific lymph node cells regain the ability to divide, differentiate along a Th1 lineage, and transfer EAE when reactivated in the presence of agonistic Abs against CD40 or CpG oligonucleotides. The effects of both anti-CD40 and CpG oligonucleotides are dependent upon induction of IL-12. Our findings suggest two mechanisms to explain the well-documented association between infectious illnesses and flare-ups of multiple sclerosis. Microbial pathogens could 1) release molecules that bind Toll-like receptors, and/or 2) stimulate microbe-specific T cells to express CD40 ligand, thereby licensing APCs that bear both microbial and autoantigens to break tolerance.
Generation of CD3+CD8low Thymocytes in the HIV Type 1-Infected Thymus
Authors: Keir Mary E. , Rosenberg Michael G. , Sandberg Johan K. , Jordan Kimberly A. , Wiznia Andrew , Nixon Douglas F. , Stoddart Cheryl A. , McCune Joseph M.
Published: April 2014

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4+ T cells and in the generation of dysfunctional CD8+ T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-α secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3−/intCD4+CD8+ thymocytes correlates with the generation of mature single-positive CD4−CD8+ thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4−CD8+ thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8low phenotype on circulating CD3+CD8+ T cells. Furthermore, CD8low peripheral T cells from these HIV-1+ pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-α-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3+CD8low T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8+ T cells in HIV-1-infected patients.
Cutting Edge: Identification of the Targets of Clonal Deletion in an Unmanipulated Thymus
Authors: Cho Hyung J. , Edmondson Samuel G. , Miller Arden D. , Sellars MacLean , Alexander Shawn T. , Somersan Selin , Punt Jennifer A.
Published: April 2014

Autoreactive thymocytes can be eliminated by clonal deletion during their development in the thymus. The precise developmental stage(s) at which clonal deletion occurs in a normal thymus has been difficult to assess, in large part because of the absence of a specific marker for TCR-mediated apoptosis. In this report, we reveal that Nur77 expression can be used as a specific marker of clonal deletion in an unmanipulated thymus and directly identify TCRintCD4+CD8+ and semimature CD4+CD8− thymocytes as the principal targets of deletion. These data indicate that clonal deletion normally occurs at a relatively late stage of development, as cells mature from CD4+CD8+ thymocytes to single-positive T cells.
Innate Immunity Mediated by the Cytokine IL-1 Homologue 4 (IL-1H4/IL-1F7) Induces IL-12-Dependent Adaptive and Profound Antitumor Immunity
Authors: Gao Wentao , Kumar Sanjay , Lotze Michael T. , Hanning Charles , Robbins Paul D. , Gambotto Andrea
Published: April 2014

Recently, several novel members of the IL-1 family have been identified. The possible therapeutic utility and the underlying biologic role of these new members remain unclear. In the present study we analyzed the anti-tumor activity of human IL-1 homologue 4(IL-1H4; renamed IL-F7) by adenovirus-mediated gene transfer (AdIL-1H4) directly into murine tumors. In vitro expression analysis showed that IL-1H4 was a secretory protein. Treatment of an established MCA205 mouse fibrosarcoma by single intratumoral injection of AdIL-1H4 resulted in significant growth suppression. Furthermore, complete inhibition of tumor growth was observed following multiple injections of AdIL-1H4. The anti-tumor activity of IL-1H4 was abrogated in nude and SCID mice and in IL-12-, IFN-γ-, or Fas ligand-deficient mice. In contrast, IL-1H4 was able to confer substantial anti-tumor effects in NKT-deficient mice. These results suggest that IL-1H4 could play an important role in the link between innate and adaptive immunity and may be useful for tumor immunotherapy.
Detection of Low-Avidity CD4+ T Cells Using Recombinant Artificial APC: Following the Antiovalbumin Immune Response
Authors: Mallet-Designe Valérie I. , Stratmann Thomas , Homann Dirk , Carbone Francis , Oldstone Michael B. A. , Teyton Luc
Published: April 2014

Subtle differences oppose CD4+ to CD8+ T cell physiologies that lead to different arrays of effector functions. Interestingly, this dichotomy has also unexpected practical consequences such as the inefficacy of many MHC class II tetramers in detecting specific CD4+ T cells. As a mean to study the CD4+ anti-OVA response in H-2d and H-2b genetic backgrounds, we developed I-Ad- and I-Ab-OVA recombinant MHC monomers and tetramers. We were able to show that in this particular system, despite normal biological activity, MHC class II tetramers failed to stain specific T cells. This failure was shown to be associated with a lack of cooperation between binding sites within the tetramer as measured by surface plasmon resonance. This limited cooperativeness translated into a low “functional avidity” and very transient binding of the tetramers to T cells. To overcome this biophysical barrier, recombinant artificial APC that display MHC molecules in a lipid bilayer were developed. The plasticity and size of the MHC-bearing fluorescent liposomes allowed binding to Ag-specific T cells and the detection of low numbers of anti-OVA T cells following immunization. The same liposomes were able, at 37°C, to induce the full reorganization of the T cell signaling molecules and the formation of an immunological synapse. Artificial APC will allow T cell detection and the dissection of the molecular events of T cell activation and will help us understand the fundamental differences between CD4+ and CD8+ T cells.
A Novel Polymorphic CAAT/Enhancer-Binding Protein β Element in theFasLGene Promoter Alters Fas Ligand Expression: A Candidate Background Gene in African American Systemic Lupus Erythematosus Patients
Authors: Wu Jianming , Metz Christine , Xu Xiulong , Abe Riichiro , Gibson Andrew W. , Edberg Jeffrey C. , Cooke Jennifer , Xie Fenglong , Cooper Glinda S. , Kimberly Robert P.
Published: April 2014

A single-nucleotide polymorphism (SNP), identified at nucleotide position −844 in the 5′ promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein β (C/EBPβ). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPβ and demonstrated that the two alleles of this element have different affinities for C/EBPβ. In luciferase reporter assays, the −844C genotype had twice the basal activity of the −844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in −844C than in −844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the −844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The −844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.
Cutting Edge: Bacterial Lipoprotein Induces Endotoxin-Independent Tolerance to Septic Shock
Authors: Wang Jiang Huai , Doyle Majella , Manning Brian J. , Blankson Siobhan , Wu Qiong Di , Power Colm , Cahill Ronan , Redmond H. Paul
Published: April 2014

Tolerance to bacterial cell wall components is an adaptive host response. Endotoxin/LPS tolerance is characterized by a survival advantage against subsequent lethal LPS challenge. However, it is uncertain whether LPS tolerance can afford protection against other septic challenges. In this study, we show that tolerance induced by bacterial lipoprotein (BLP) protects mice against not only BLP-induced lethality, but also LPS-, live bacteria-, and polymicrobial sepsis-induced lethality. In contrast, LPS tolerance offers no survival benefit against the latter two challenges. Furthermore, induction of BLP tolerance results in overexpression of complement receptor type 3 and FcγIII/IIR on neutrophils (polymorphonuclear neutrophils) and peritoneal macrophages, with increased bacterial recognition and bactericidal activity, whereas LPS-tolerized mice exhibit an impaired ability to ingest and to kill bacteria. These results indicate that BLP tolerance is a novel adaptive host response associated with a unique protective effect during septic shock.
Induction of Telomerase Activity and Maintenance of Telomere Length in Virus-Specific Effector and Memory CD8+ T Cells
Authors: Hathcock Karen S. , Kaech Susan M. , Ahmed Rafi , Hodes Richard J.
Published: April 2014

Acute viral infections induce extensive proliferation and differentiation of virus-specific CD8+ T cells. One mechanism reported to regulate the proliferative capacity of activated lymphocytes is mediated by the effect of telomerase in maintaining the length of telomeres in proliferating cells. We examined the regulation of telomerase activity and telomere length in naive CD8+ T cells and in virus-specific CD8+ T cells isolated from mice infected with lymphocytic choriomeningitis virus. These studies reveal that, compared with naive CD8+ T cells, which express little or no telomerase activity, Ag-specific effector and long-lived memory CD8+ T cells express high levels of telomerase activity. Despite the extensive clonal expansion that occurs during acute lymphocytic choriomeningitis virus infection, telomere length is maintained in both effector and memory CD8+ T cells. These results suggest that induction of telomerase activity in Ag-specific effector and memory CD8+ T cells is important for the extensive clonal expansion of both primary and secondary effector cells and for the maintenance and longevity of the memory CD8+ T cell population.
Human γδ-T Lymphocytes Express and Synthesize Connective Tissue Growth Factor: Effect of IL-15 and TGF-β1 and Comparison with αβ-T Lymphocytes
Authors: Workalemahu Grefachew , Foerster Martin , Kroegel Claus , Braun Ruedi K.
Published: April 2014

T lymphocytes bearing the γδ-TCR accumulate during wound healing and inflammation. However, the role of γδ-T lymphocytes in fibrogenic tissue reactions is not well understood. Therefore, we addressed the question of whether human γδ-T cells express and synthesize connective tissue growth factor (CTGF), a factor known to regulate fibrogenesis and wound healing. In addition, the lymphoblastic leukemia T cell line (Loucy) that possesses characteristics typical of γδ-T cells was used as a model to evaluate the regulation of CTGF gene expression. Blood γδ-T cells isolated from healthy donors were grown in the presence of IL-15/TGF-β1 for 48 h and assessed for the expression and synthesis of CTGF. Nonstimulated human blood γδ-T cells and Loucy γδ-T cells expressed low levels of CTGF mRNA. Costimulation of the cells with IL-15 and TGF-β1 resulted in a substantially increased level of CTGF mRNA expression within 4–8 h, and it remained elevated for at least 48 h. In contrast, no CTGF mRNA was detected when nonstimulated and stimulated human CD4+ αβ-T cells were analyzed. In addition, Western blot analysis of human γδ-T cell lysates prepared 4 days following stimulation with IL-15 and TGF-β1 revealed a 38-kDa CTGF protein in cell lysates of human γδ-T cells. Detection was confirmed using Colo 849 fibroblasts, which can constitutively express high levels of CTGF. In conclusion, we herein present novel evidence that in contrast to CD4+ αβ-T cells human γδ-T cells are capable of expressing CTGF mRNA and synthesizing its corresponding protein, which supports the concept that γδ-T cells may contribute to wound healing or tissue fibrotic processes.
Critical Paracrine Interactions Between TNF-α and IL-10 Regulate Lipopolysaccharide-Stimulated Human Choriodecidual Cytokine and Prostaglandin E2 Production
Authors: Sato Timothy A. , Keelan Jeffrey A. , Mitchell Murray D.
Published: April 2014

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 μg/ml) induced a rapid increase in TNF-α production, peaking at 4 h. In contrast, IL-10, IL-1β, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-α was a primary regulator of IL-1β, IL-10, and PGE2 production, while IL-1β stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-α and PGE2 production. IL-10 treatment markedly decreased TNF-α and IL-1β production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-α may be a potential target for treatment intervention; IL-10 could be one such therapeutic.
Differential Expression of Platelet-Activating Factor Acetylhydrolase in Macrophages and Monocyte-Derived Dendritic Cells
Authors: Al-Darmaki Salma , Schenkein Harvey A. , Tew John G. , Barbour Suzanne E.
Published: April 2014

Although macrophages (Mφ) and monocyte-derived dendritic cells (MDDC) come from a common precursor, they are distinct cell types. This report compares the two cell types with respect to the metabolism of platelet-activating factor (PAF), a biologically active lipid mediator. These experiments were prompted by our studies of localized juvenile periodontitis, a disease associated with high IgG2 production and a propensity of monocytes to differentiate into MDDC. As the IgG2 Ab response is dependent on PAF, and MDDC selectively induce IgG2 production, we predicted that PAF levels would be higher in MDDC than in Mφ. To test this hypothesis, human MDDC were prepared by treating adherent monocytes with IL-4 and GM-CSF, and Mφ were produced by culture in M-CSF. Both Mφ and MDDC synthesized PAF; however, MDDC accumulated significantly more of this lipid. We considered the possibility that PAF accumulation in MDDC might result from reduced turnover due to lower levels of PAF acetylhydrolase (PAFAH), the enzyme that catabolizes PAF. Although PAFAH increased when monocytes differentiated into either cell type, MDDC contained significantly less PAFAH than did Mφ and secreted almost no PAFAH activity. The reduced levels of PAFAH in MDDC could be attributed to lower levels of expression of the enzyme in MDDC and allowed these cells to produce PGE2 in response to exogenous PAF. In contrast, Mφ did not respond in this manner. Together, these data indicate that PAF metabolism may impinge on regulation of the immune response by regulating the accessory activity of MDDC.
Models for Antigen Receptor Gene Rearrangement. III. Heavy and Light Chain Allelic Exclusion
Authors: Kalmanovich Gil , Mehr Ramit
Published: April 2014

The extent of allelic exclusion in Ig genes is very high, although not absolute. Thus far, it has not been clearly established whether rapid selection of the developing B cell as soon as it has achieved the first productively rearranged, functional heavy chain is the only mechanism responsible for allelic exclusion. Our computational models of Ag receptor gene rearrangement in B lymphocytes are hereby extended to calculate the expected fractions of heavy chain allelically included newly generated B cells as a function of the probability of heavy chain pairing with the surrogate light chain, and the probability that the cell would test this pairing immediately after the first rearrangement. The expected fractions for most values of these probabilities significantly exceed the levels of allelic inclusion in peripheral B cells, implying that in most cases productive rearrangement and subsequent cell surface expression of one allele of the heavy chain gene probably leads to prevention of rearrangement completion on the other allele, and that additional mechanisms, such as peripheral selection disfavoring cells with two productively rearranged heavy chain genes, may also play a role. Furthermore, we revisit light chain allelic exclusion by utilizing the first (to our knowledge) computational model which addresses and enumerates B cells maturing with two productively rearranged κ light chain genes. We show that, assuming that there are no selection mechanisms responsible for abolishing cells expressing two light chains, the repertoire of newly generated B lymphocytes exiting the bone marrow must contain a significant fraction of such κ double-productive B cells.
Self-Specific MHC Class II-Restricted CD4−CD8− T Cells That Escape Deletion and Lack Regulatory Activity
Authors: Viret Christophe , Janeway Charles A.
Published: April 2014

In the presence of the I-Eα protein, transgenic (Tg) mice expressing the 1H3.1 αβ TCR that is specific for the Eα52–68:I-Ab complex display drastic intrathymic deletion. Although peripheral T cells from these mice remained unresponsive to the Eα52–68:I-Ab complex, they contained a subpopulation able to specifically react to this complex in the presence of exogenous IL-2, indicating that some 1H3.1 αβ TCR Tg T cells have escaped clonal deletion and efficiently populated the periphery. IL-2-dependent, Eα52–68:I-Ab complex-responsive T cells were CD4−CD8− and expressed the 1H3.1 αβ TCR. Such T cells could develop intrathymically, did not show sign of regulatory/suppressor activity, displayed a typical naive phenotype, and seemed to persist in vivo over time. CD4−CD8− TCR Tg T cells were also detected when the surface density of the deleting ligand was increased on MHC class II+ cells. In addition, the development of CD4−CD8− 1H3.1 αβ TCR Tg T cells could be supported by I-Ab molecules. These observations indicate that CD4 surface expression neither specifies, nor is required for, the thymic export of mature thymocytes expressing a MHC class II-restricted αβ TCR. The data also show that, although the avidity of the interaction involved in intrathymic deletion is significantly lower than that involved in mature T cell activation, its range can be large enough to be influenced by the presence or absence of coreceptors. Finally, the margin created by the absence of CD4 coreceptor was substantial because it could accommodate various amounts of the deleting ligand on thymic stromal cells.
Cutting Edge: Dissociation Between Autoimmune Response and Clinical Disease After Vaccination with Dendritic Cells
Authors: Bondanza Attilio , Zimmermann Valérie S. , Dell’Antonio Giacomo , Dal Cin Elena , Capobianco Annalisa , Sabbadini Maria Grazia , Manfredi Angelo A. , Rovere-Querini Patrizia
Published: April 2014

Autoimmunity represents a caveat to the use of dendritic cells (DCs) as adjuvant for human vaccines. We derived DCs from normal BALB/c mice or from mice prone to autoimmunity (NZB × NZW) F1. We allowed DCs to phagocytose apoptotic thymocytes and vaccinated syngeneic animals. All mice developed anti-nuclear and anti-dsDNA Abs. Autoantibodies in normal mice were transient, without clinical or histological features of autoimmunity or tissue involvement. In contrast, autoimmunity was maintained in susceptible mice, which underwent renal failure and precociously died. The data suggest that DC vaccination consistently triggers autoimmune responses. However, clinical autoimmunity develops in susceptible subjects only.
A Division-Linked Mechanism for the Rapid Generation of Ig-Secreting Cells from Human Memory B Cells
Authors: Tangye Stuart G. , Avery Danielle T. , Hodgkin Philip D.
Published: April 2014

Memory B cells, when re-exposed to Ag and T cell help, differentiate into Ig-secreting cells (ISC) at the same time as maintaining a residual pool of non-Ig-secreting cells with memory capabilities. To investigate the mechanism underlying this dual process, we followed the fate of human B cells activated in vitro with the T cell-derived signals CD40 ligand (CD40L), IL-2, and IL-10 using CFSE to monitor cell division. A substantial number of ISCs detected by ELISPOT, intracellular Ig staining, and Ig secretion could be generated from memory but not naive B cells. The proportion of ISCs increased with successive cell divisions and was markedly enhanced by IL-10 at each division. Within ISCs, two distinct populations were detected after withdrawal of CD40L. The first had acquired the plasma cell marker CD38 and continued to proliferate despite the absence of CD40L. In contrast, the second population remained CD38−, ceased dividing, and underwent rapid apoptosis. The former most likely represent the immediate precursors of long-lived plasma cells, which preferentially home to the bone marrow in vivo, whereas the latter contain short-lived ISCs responsible for the initial Ab response to stimulation with Ag and T cell help. Taken together, the results point to a division-based mechanism responsible not only for regulating differentiation of short- and long-lived ISCs from memory B cells, but for preserving the memory B cell pool for reactivation upon subsequent Ag exposure.
Activated STAT4 and a Functional Role for IL-12 in Human Peyer’s Patches
Authors: Monteleone Giovanni , Holloway Judith , Salvati Virginia M. , Pender Sylvia L.-F. , Fairclough Peter D. , Croft Nicholas , MacDonald Thomas T.
Published: April 2014

T cells in the Peyer’s patches (PP) of the human ileum are exposed to a myriad of dietary and bacterial Ags from the gut lumen. Recall proliferative responses to common dietary Ags are readily demonstrable by PP T cells from healthy individuals, and the cytokine response is dominated by IFN-γ. Consistent with Th1 skewing, PP cells spontaneously secrete IL-12p70, and IL-12p40 protein can be visualized underneath the PP dome epithelium. In this study, we have analyzed IL-12 signaling in PP and investigated whether IL-12 plays a functional role. CD3+ T lymphocytes isolated from PP and adjacent ileal mucosa spontaneously secrete IFN-γ with negligible IL-4 or IL-5. RNA transcripts for IL-12Rβ2, the signaling component of the IL-12R, are present in purified CD4+ and CD8+ T PP lymphocytes. Active STAT4, a transcription factor essential for IL-12-mediated Th1 differentiation, is readily detectable in biopsies from PP and ileal mucosa and STAT4-DNA binding activity is demonstrable by EMSA. Nuclear proteins from CD3+ T PP lymphocytes contain STAT4 and T-bet, a transcription factor selectively expressed in Th1 cells. Stimulation of freshly isolated PP cells with staphylococcal enterotoxin B dramatically enhanced the production of IFN-γ, an effect which was largely inhibited by neutralizing anti-IL-12 Ab. These data show that IL-12 in human PP is likely to be responsible for the Th1-dominated cytokine response of the human mucosal immune system.
Roles of Vasoactive Intestinal Peptide (VIP) in the Expression of Different Immune Phenotypes by Wild-Type Mice and T Cell-Targeted Type II VIP Receptor Transgenic Mice
Authors: Voice Julia K. , Grinninger Carola , Kong Yvonne , Bangale Yogesh , Paul Sudhir , Goetzl Edward J.
Published: April 2014

Vasoactive intestinal peptide (VIP) and its two G protein-coupled receptors, VPAC1 and VPAC2, are quantitatively prominent and functionally critical in the immune system. Transgenic (T) mice constitutively expressing VPAC2 selectively in CD4 T cells, at levels higher than those found after maximal induction in CD4 T cells of wild-type (N) mice, have elevated blood concentrations of IgE, IgG1, and eosinophils; enhanced immediate-type hypersensitivity; and reduced delayed-type hypersensitivity. In contrast, VPAC2-null (K) mice manifest decreased immediate-type hypersensitivity and enhanced delayed-type hypersensitivity. The phenotypes are attributable to opposite skewing of the Th2/Th1 cytokine ratio, but no studies were conducted on the roles of T cell-derived VIP and altered expansion of the Th subsets. Dependence of the Th phenotype of T mice, but not of N or K mice, on T cell-derived VIP now is proven by showing that eliminating VIP from TCR-stimulated T cell cultures with VIPase IgG normalizes the elevated number of IL-4-secreting CD4 T cells, decreases the secretion of IL-4 and IL-10, and increases the secretion of IFN-γ. Flexible responsiveness of CD4 T cells from N and K mice, but not T mice, to exogenous VIP in vitro and in vivo is shown by increased numbers of IL-4-secreting CD4 T cells, greater secretion of IL-4 and IL-10, and lesser secretion of IFN-γ after TCR stimulation with VIP. The level of VIP recognized by CD4 T cells thus is a major determinant of the relative contributions of Th subsets to the immune effector phenotype.
Induction of the Early Growth Response Gene 1 Promoter by TCR Agonists and Partial Agonists: Ligand Potency Is Related to Sustained Phosphorylation of Extracellular Signal-Related Kinase Substrates
Authors: Xi Hongkang , Kersh Gilbert J.
Published: April 2014

Responses to partial agonist TCR signals include positive selection of thymocytes, survival of naive T cells, and homeostatic proliferation. As part of an effort to understand the molecular basis of these processes, we have determined how agonist and partial agonist ligands act differently to induce a change in gene expression. We have found that the early growth response gene 1 (Egr1) promoter is activated by agonist and partial agonist ligands, but the partial agonist induces 10-fold lower promoter activity. Both agonist and partial agonist ligands require all six serum response elements in the Egr1 promoter to reach maximum induction. Although slightly fewer cells respond to the partial agonist, all of the responding cells have reduced activity compared with the cells responding to agonist. The factors binding to the serum response elements of the Egr1 promoter form a ternary complex (TC) consisting of serum response factor and either Elk-1 or serum response factor accessory protein-1a. Formation of a stable TC and inducible promoter activity are both dependent on extracellular signal-related kinase activation. Examination of TC formation over time reveals that this complex is induced well by partial agonist ligands, but it is not sustained, whereas agonist stimulation induces longer lived TCs. Therefore, the data suggest that both agonist and partial agonist ligands can induce formation of multiple TC on the Egr1 promoter, but the ability of the agonist ligand to maintain these complexes for an extended time results in the increased potency of the agonist.
Minimal Requirement of Tyrosine Residues of Linker for Activation of T Cells in TCR Signaling and Thymocyte Development
Authors: Zhu Minghua , Janssen Erin , Zhang Weiguo
Published: April 2014

Linker for activation of T cells (LAT) is a membrane-associated adaptor protein that is phosphorylated on multiple tyrosines upon TCR cross-linking. Previous studies show that LAT is essential for TCR-mediated signaling and thymocyte development. In this study, we expressed a series of LAT Tyr to Phe mutants in LAT-deficient J.CaM2.5 cells and examined their tyrosine phosphorylation; association with Grb2, Gads, and phospholipase C (PLC)-γ1; and function in T cell activation. Our results showed that the five membrane-distal tyrosines were phosphorylated upon T cell activation. Grb2, Gads, and PLC-γ1 associated with LAT preferentially via different sets of tyrosine residues; however, they failed to interact with LAT mutants containing only one tyrosine. We also determined the minimal requirement of LAT tyrosine residues in T cell activation and thymocyte development. Our results showed that a minimum of three tyrosines is required for LAT to function in T cell activation and thymocyte development. LAT mutants that were capable of binding Grb2 and PLC-γ1 could reconstitute T cell activation in LAT-deficient cells and thymocyte development in LAT-deficient mice.
Regulation of Cell Type-Specific Mouse FcεRI β-Chain Gene Expression by GATA-1 Via Four GATA Motifs in the Promoter
Authors: Maeda Keiko , Nishiyama Chiharu , Tokura Tomoko , Akizawa Yushiro , Nishiyama Makoto , Ogawa Hideoki , Okumura Ko , Ra Chisei
Published: April 2014

The FcR β-chain, a subunit of two related multisubunit receptor complexes, the FcεRI and FcγRIII, amplifies the mast cell response and is necessary for the cell surface expression of FcεRI in mouse. The transient reporter assay indicated that −69/+4 region is required for cell type-specific transcriptional regulation of mouse β-chain gene. EMSA using Abs against transcription factors or competitive oligonucleotides demonstrated that −58/−40 region (containing overlapping three GATA-1 sites, −53/−48, −46/−51, and −42/−47) and −31/−26 region (containing one GATA-1 site) are recognized by GATA-1. The promoter activity of β-chain was decreased by nucleotide replacements of the GATA-1 sites in mouse mast cell line PT18. Furthermore, exogenously produced GATA-1 up-regulated the promoter activity in CV-1 cells, which are negative in the β-chain production and the up-regulation was apparently suppressed by GATA-1 site mutations. These results indicate that cell type-specific transcription of mouse β-chain gene is regulated by GATA-1.
Serial Analysis of Gene Expression in Circulating γδ T Cell Subsets Defines Distinct Immunoregulatory Phenotypes and Unexpected Gene Expression Profiles
Authors: Meissner Nicole , Radke Jay , Hedges Jodi F. , White Michael , Behnke Michael , Bertolino Shannon , Abrahamsen Mitchell , Jutila Mark A.
Published: April 2014

Gene expression profiles were compared in circulating bovine GD3.5+ (CD8−) and GD3.5− (predominantly CD8+) γδ T cells using serial analysis of gene expression (SAGE). Approximately 20,000 SAGE tags were generated from each library. A comparison of the two libraries demonstrated 297 and 173 tags representing genes with 5-fold differential expression in GD3.5+ and GD3.5− γδ T cells, respectively. Consistent with their localization into sites of inflammation, GD3.5+ γδ T cells appeared transcriptionally and translationally more active than GD3.5− γδ cells. GD3.5− γδ T cells demonstrated higher expression of the cell proliferation inhibitor BAP 37, which was associated with their less activated gene expression phenotype. The immune regulatory and apoptosis-inducing molecule, galectin-1, was identified as a highly abundant molecule and was higher in GD3.5+γδ T cells. Surface molecules attributed to myeloid cells, such as CD14, CD68, and scavenger receptor-1, were identified in both populations. Furthermore, expression of B lymphocyte-induced maturation protein, a master regulator of B cell and myeloid cell differentiation, was identified by SAGE analysis and was confirmed at the RNA level to be selectively expressed in γδ T cells vs αβ T cells. These results provide new insights into the inherent differences between circulating γδ T cell subsets.
The Role of OX40 Ligand Interactions in the Development of the Th2 Response to the Gastrointestinal Nematode Parasite Heligmosomoides polygyrus
Authors: Ekkens Melinda J. , Liu Zhugong , Liu Qian , Whitmire Jeannette , Xiao Shiyun , Foster Anthony , Pesce John , VanNoy Jansie , Sharpe Arlene H. , Urban Joseph F. , Gause William C.
Published: April 2014

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L−/− mice. The host-protective memory response was compromised in OX40L−/− mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L+/+ mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L−/− mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L+/+ and OX40L−/− mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.
Human CD4+ T Cells Present Within the Microenvironment of Human Lung Tumors Are Mobilized by the Local and Sustained Release of IL-12 to Kill Tumors In Situ by Indirect Effects of IFN-γ
Authors: Hess Stephen D. , Egilmez Nejat K. , Bailey Nicola , Anderson Timothy M. , Mathiowitz Edith , Bernstein Steven H. , Bankert Richard B.
Published: April 2014

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-γ. Serum levels of human IFN-γ in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-γ abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-γ and IFN-γ-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-γ production, and the associated changes in gene expression are all dependent upon CD4+ T cells.
Transgenic Expression of Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12 Enhances Myeloid Progenitor Cell Survival/Antiapoptosis In Vitro in Response to Growth Factor Withdrawal and Enhances Myelopoiesis In Vivo
Authors: Broxmeyer Hal E. , Cooper Scott , Kohli Lisa , Hangoc Giao , Lee Younghee , Mantel Charlie , Clapp D. Wade , Kim Chang H.
Published: April 2014

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and Gαi proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.
Chimeras of Labile Toxin One and Cholera Toxin Retain Mucosal Adjuvanticity and Direct Th Cell Subsets Via Their B Subunit
Authors: Boyaka Prosper N. , Ohmura Mari , Fujihashi Kohtaro , Koga Toshiya , Yamamoto Masafumi , Kweon Mi-Na , Takeda Yoshifumi , Jackson Raymond J. , Kiyono Hiroshi , Yuki Yoshikazu , McGhee Jerry R.
Published: April 2014

Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB5-type enterotoxins. Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes. Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4+) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-γ and IL-4-independent Th2-type responses. To address the relative importance of the A or B subunits in CD4+ Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed. Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT. Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity. The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4+ T cells secreting IFN-γ and IL-4-independent Th2-type responses, with plasma IgG2a anti-OVA Abs. Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4+ Th2 cells secreting high levels of IL-4, but not IFN-γ. The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-γ production by activated T cells. Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.
Ablation of CD8 and CD4 T Cell Responses by High Viral Loads
Authors: Fuller Michael J. , Zajac Allan J.
Published: April 2014

To evaluate the impact of sustained viral loads on anti-viral T cell responses we compared responses that cleared acute lymphocytic choriomeningitis virus infection with those that were elicited but could not resolve chronic infection. During acute infection, as replicating virus was cleared, CD8 T cell responses were down-regulated, and a pool of resting memory cells developed. In chronically infected hosts, the failure to control the infection was associated with pronounced and prolonged activation of virus-specific CD8 T cells. Nevertheless, there was a progressive diminution of their effector activities as their capacity to produce first IL-2, then TNF-α, and finally IFN-γ was lost. Chronic lymphocytic choriomeningitis virus infection was also associated with differential contraction of certain CD8 T cell responses, resulting in altered immunodominance. However, this altered immunodominance was not due to selective expansion of T cells expressing particular TCR Vβ segments during chronic infection. High viral loads were not only associated with the ablation of CD8 T cell responses, but also with impaired production of IL-2 by virus-specific CD4 T cells. Taken together, our data show that sustained exposure to high viral loads results in the progressive functional inactivation of virus-specific T cell responses, which may further promote virus persistence.
Identification of Heat Shock Protein 60 as the Ligand on Histoplasma capsulatum That Mediates Binding to CD18 Receptors on Human Macrophages
Authors: Long Kristin H. , Gomez Francisco J. , Morris Randall E. , Newman Simon L.
Published: April 2014

Histoplasma capsulatum (Hc), is a facultative intracellular fungus that binds to CD11/CD18 receptors on macrophages (Mφ). To identify the ligand(s) on Hc yeasts that is recognized by Mφ, purified human complement receptor type 3 (CR3, CD11b/CD18) was used to probe a Far Western blot of a detergent extract of Hc cell wall and cell membrane. CR3 recognized a single 60-kDa protein, which was identified as heat shock protein 60 (hsp60). Biotinylation of viable yeasts, followed by precipitation with streptavidin-coated beads, and Western blotting with anti-hsp60 demonstrated that hsp60 was on the surface of Hc yeasts. Electron and confocal microscopy revealed that hsp60 resided on the yeast cell wall in discrete clusters. Recombinant hsp60 (rhsp60) inhibited attachment of Hc yeasts to Mφ. Recombinant hsp60 and Abs to CD11b and CD18 inhibited binding of yeasts to Chinese hamster ovary cells transfected with CR3 (CHO3). Polystyrene beads coated with rhsp60 bound to Mφ, and attachment was inhibited by Abs to CD11 and CD18. Freeze/thaw extract (F/TE), a preparation of Hc yeast surface proteins that contained hsp60, inhibited the attachment of Hc yeasts to Mφ. Depletion of hsp60 from F/TE removed the capacity of F/TE to block binding of Hc to Mφ. Interestingly, rhsp60 did not inhibit binding of Hc yeasts to dendritic cells (DC), which recognize Hc via very late Ag 5. Moreover, F/TE inhibited attachment of Hc to DC even when depleted of hsp60. Thus, Hc hsp60 appears to be a major ligand that mediates attachment of Hc to Mφ CD11/CD18, whereas DC recognize Hc via a different ligand(s).
HIV Mucosal Vaccine: Nasal Immunization with gp160-Encapsulated Hemagglutinating Virus of Japan-Liposome Induces Antigen-Specific CTLs and Neutralizing Antibody Responses
Authors: Sakaue Gaku , Hiroi Takachika , Nakagawa Yoko , Someya Kenji , Iwatani Kohich , Sawa Yoshiki , Takahashi Hidemi , Honda Mitsuo , Kunisawa Jun , Kiyono Hiroshi
Published: April 2014

Nasal immunization of normal mice with HIVgp160-encapsulated hemagglutinating virus of Japan (HVJ)-liposome induced high titers of gp160-specific neutralizing IgG in serum and IgA in nasal wash, saliva, fecal extract, and vaginal wash, along with both Th1- and Th2-type responses. HIVgp160-specific IgG- and IgA-producing cells were also detected in mononuclear cells isolated from spleen, nasal cavity, salivary gland, intestinal lamina propria, and vaginal tissue of nasally immunized mice. In addition, CD8+ CTLs were induced in mice nasally immunized with gp160-HVJ-liposome. These findings suggest that two layers of effective HIV-specific humoral and cellular immunity, in mucosal and systemic sites, were induced by this nasal vaccine. In immunodeficient mice, nasal immunization with gp160-HVJ-liposome induced Ag-specific immune responses for the systemic and mucosal compartments of both Th1 (IFN-γ−/−) and Th2 (IL-4−/−). In vitro Ag-specific serum IgG Ab and vaginal wash samples possessing IgA and IgG Abs that had been induced by nasal immunization with gp160-HVJ-liposome were able to neutralize a clinically isolated strain of HIV-MN strain isolated from Japanese hemophiliac patients. Taken together, these results suggest that, for the prevention and control of AIDS, nasally administered gp160-HVJ-liposome is a powerful immunization tool that induces necessary Ag-specific immune responses at different stages of HIV infection.