bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral CD4+ Lymphocytes Derived from Fetal versus Adult Thymic Precursors Differ Phenotypically and Functionally
Authors: Adkins Becky
Published: April 2014

There is growing evidence that the differentiation processes in the fetal and adult thymus are not identical. However, there is little information on whether these developmental differences influence the properties of mature cells that exit the thymus and seed peripheral lymphoid organs. We have addressed this issue by comparing the development of Ag-specific Th1/Th2 function by fetal vs adult thymic derived CD4+ cells in the same adoptive adult hosts. Host mice were irradiated and transplanted with 14- to 15-day fetal thymic lobes from Thy-1 congenic mice. Ag (keyhole limpet hemocyanin)-specific Th1/Th2 responses of fetal-derived (donor) or adult-derived (host) CD4+ cells were analyzed by ELISA following primary or secondary immunization. Fetal-derived cells produced up to 10-fold more of both Th1 (IFN-γ) and Th2 (IL-4) cytokines than did adult-derived cells. Comparisons of the IL-4:IFN-γ ratios showed that the responses of fetal-derived cells were Th2-skewed in an Ag dose-dependent manner. At low doses of Ag, the fetal-derived ratio was ∼5 times higher than the adult-derived ratio. As the Ag dose was increased, the differences between the ratios of the fetal- and adult-derived responses were minimized. These relative responses were established initially during the primary effector phase but were maintained for weeks, into the memory phase of the immune response. Importantly, fetal-derived CD4+ cells showed these properties whether the fetal thymic precursors matured within the fetal or adult thymic microenvironment. These results demonstrate that cells arising from fetal thymic precursors are functionally different both qualitatively and quantitatively from adult-derived cells.
CD8 T Cell Clonal Expansion and Development of Effector Function Require Prolonged Exposure to Antigen, Costimulation, and Signal 3 Cytokine
Authors: Curtsinger Julie M. , Johnson Christopher M. , Mescher Matthew F.
Published: April 2014

Full activation of naive CD8 T cells requires Ag, costimulation, and a third signal that can be provided by IL-12. Brief exposure (6 h) to Ag and B7-1 is sufficient to stimulate multiple rounds of cell division, but clonal expansion and development of effector function are minimal even when signal 3 is present. Full activation instead requires concerted signaling by Ag, B7-1, and IL-12 for greater than 40 h. Thus, the gene expression program required for cell division can be initiated by brief interaction with Ag and costimulation, but maintaining the expression of the genes needed for survival and effector function requires prolonged signaling by a signal 3 cytokine in concert with Ag and costimulation.
Individual Analysis of Mice Vaccinated against a Weakly Immunogenic Self Tumor-Specific Antigen Reveals a Correlation between CD8 T Cell Response and Antitumor Efficacy
Authors: Rosato Antonio , Zoso Alessia , Milan Gabriella , Macino Beatrice , Dalla Santa Silvia , Tosello Valeria , Di Carlo Emma , Musiani Piero , Whalen Robert G. , Zanovello Paola
Published: April 2014

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.
Granulocyte-Macrophage Colony-Stimulating Factor-Based Melanoma Cell Vaccines Immunize Syngeneic and Allogeneic Recipients via Host Dendritic Cells
Authors: Schneeberger Achim , Lührs Petra , Kutil Raphaela , Steinlein Peter , Schild Hansjörg , Schmidt Walter , Stingl Georg
Published: April 2014

Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the β-galactosidase (βgal) gene into M3-GM cells. Their administration activated βgal-specific, Ld-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-βgal injection sites revealed the presence of cells presenting the respective Ld-binding βgal peptide epitope. Based on their capacity to activate βgal-specific CTL, they were identified as being CD11c+ dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.
T Cell Activation-Induced Mitochondrial Hyperpolarization Is Mediated by Ca2+- and Redox-Dependent Production of Nitric Oxide
Authors: Nagy Gyorgy , Koncz Agnes , Perl Andras
Published: April 2014

Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Δψm) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Δψm or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca2+ levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca2+ release, ROI levels, and NO production were diminished by inositol 1,4,5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (−85.0 ± 10.0%; p = 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine elicited NO and ROI production, Ca2+ release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5 ± 0.8-fold; p = 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H2O2 elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with β-actin. H2O2 also led to moderate mitochondrial hyperpolarization; however, Ca2+ influx by ionomycin or Ca2+ release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Δψm elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI- and Ca2+-dependent NO production.
Antigenic Epitopes Fused to Cationic Peptide Bound to Oligonucleotides Facilitate Toll-Like Receptor 9-Dependent, but CD4+ T Cell Help-Independent, Priming of CD8+ T Cells
Authors: Schirmbeck Reinhold , Riedl Petra , Zurbriggen Rinaldo , Akira Shizuo , Reimann Jörg
Published: April 2014

A priority in current vaccine research is the development of adjuvants that support the efficient priming of long-lasting, CD4+ T cell help-independent CD8+ T cell immunity. Oligodeoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I-restricted CD8+ T cell responses to proteins or peptides. We show that the adjuvant effect of ISS+ ODN on CD8+ T cell priming to large, recombinant Ag is enhanced by binding them to short, cationic (arginine-rich) peptides that themselves have no adjuvant activity in CD8+ T cell priming. Fusing antigenic epitopes to cationic (8- to 10-mer) peptides bound to immune-stimulating ISS+ ODN or nonstimulating NSS+ ODN (without CpG-containing sequences) generated immunogens that efficiently primed long-lasting, specific CD8+ T cell immunity of high magnitude. Different MHC class I-binding epitopes fused to short cationic peptides of different origins showed this adjuvant activity. Quantitative ODN binding to cationic peptides strikingly reduced the toxicity of the latter, suggesting that it improves the safety profile of the adjuvant. CD8+ T cell priming supported by this adjuvant was Toll-like receptor 9 dependent, but required no CD4+ T cell help. ODN (with or without CpG-containing sequences) are thus potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of Ag delivery prevailing in many viral nucleocapsids.
Impaired TCR-Mediated Induction of Ki67 by Naive CD4+ T Cells Is Only Occasionally Corrected by Exogenous IL-2 in HIV-1 Infection
Authors: Sieg Scott F. , Bazdar Douglas A. , Lederman Michael M.
Published: April 2014

Perturbations in naive T cell homeostasis and function may play a major role in the immunodeficiency that accompanies HIV infection. By examining naive CD4+ T cell function on a single cell basis, we provide evidence that these cells have significant qualitative defects in HIV disease. Ki67, a molecule expressed during cell cycle progression, is induced less efficiently among naive CD4+ T cells from HIV-infected individuals following activation with anti-TCR Ab. The impairment in Ki67 expression is evident even when a separate function, CD62L down-modulation, is within normal ranges. Moreover, the defects in Ki67 induction are only sometimes corrected by the addition of rIL-2 to cell cultures. An initial assessment of IL-2 unresponsiveness in cells from selected HIV-infected individuals suggests that the defect is not a consequence of impaired IL-2R expression or IL-2R signaling capability. Qualitative defects in naive T cells that cannot be routinely corrected by IL-2 have significant implications for disease pathogenesis and for strategies using IL-2 as a vaccine adjuvant in HIV disease.
EBV-Encoded Latent Membrane Protein 1 Cooperates with BAFF/BLyS and APRIL to Induce T Cell-Independent Ig Heavy Chain Class Switching
Authors: He Bing , Raab-Traub Nancy , Casali Paolo , Cerutti Andrea
Published: April 2014

By substituting the H chain C region of IgM with that of IgG, IgA, or IgE, class switching enables Abs to acquire new effector functions that are crucial for the neutralization of invading pathogens. Class switching occurs through class switch DNA recombination (CSR) and usually requires engagement of CD40 on B cells by CD40 ligand on Ag-activated CD4+ T cells. CSR must be tightly regulated because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by EBV infection. In this study, we show that EBV induces CD40-independent CSR from Cμ to multiple downstream Cγ, Cα, and Cε genes through latent membrane protein 1 (LMP1), a CD40-like viral protein that signals in a ligand-independent fashion. LMP1-induced CSR is associated with transcriptional activation of germline Cγ, Cα, and Cε genes and triggers the up-regulation of activation-induced cytidine deaminase, a crucial component of the CSR machinery. In addition, LMP1 induces B cells to express B cell-activating factor of the TNF family and a proliferation-inducing ligand, two molecules that mediate B cell survival and T cell-independent Ab production. B cell-activating factor of the TNF family and a proliferation-inducing ligand cooperate with LMP1 to induce Ig class switching because their neutralization by appropriate soluble decoy receptors attenuates CSR in LMP1-expressing B cells. By showing that LMP1 triggers T cell-independent CSR, our findings suggest that EBV could play an important role in the pathogenesis of disorders with aberrant IgG, IgA, and/or IgE production.
Specific Signaling Pathways Triggered by IL-2 in Human Vγ9Vδ2 T Cells: An Amalgamation of NK and αβ T Cell Signaling
Authors: Lafont Virginie , Loisel Séverine , Liautard Janny , Dudal Sherri , Sablé-teychené Magali , Liautard Jean-Pierre , Favero Jean
Published: April 2014

The global immune response can be simplified into two components: the innate and the acquired systems. The innate immune response comprises primarily macrophages and NK cells, while B and T cells orchestrate the acquired response. Human Vγ9Vδ2 T cells represent a minor T cell subpopulation in blood (1–5%) that is activated via the TCR by small nonpeptidic molecules. Their percentage dramatically increases during the early phase of infection by intracellular pathogens, and they display many characteristics of NK cells, which places them at a unique position within the immune system. Our aim was to explore the behavior of these cells when they are activated by a receptor that is common to NK and αβ T cells, and to determine signaling pathways and biological responses induced in these cells through this receptor. Thus, we investigated whether Vγ9Vδ2 T cells behave as NK cells or as αβ T cells. We demonstrated that IL-2 activates not only STAT3, STAT5, the phosphatidylinositol 3-kinase pathway, and extracellular signal-regulated kinase-2 pathway, but also STAT4 as in NK cells, and the p38 mitogen-activated protein kinase pathway as in αβ T cells. Moreover, IL-2 induces the production of IFN-γ in Vγ9Vδ2 T cells as observed in NK cells. Due to their double profiles, Vγ9Vδ2 T cells are at the interface of the innate and the acquired immune response and may therefore not only modulate the activity of innate cells, but also influence Th1/Th2 differentiation.
Timing of IFN-β Exposure during Human Dendritic Cell Maturation and Naive Th Cell Stimulation Has Contrasting Effects on Th1 Subset Generation: A Role for IFN-β-Mediated Regulation of IL-12 Family Cytokines and IL-18 in Naive Th Cell Differentiation
Authors: Nagai Taro , Devergne Odile , Mueller Thomas F. , Perkins David L. , van Seventer Jean Maguire , van Seventer Gijs A.
Published: April 2014

Type I IFNs, IFN-α and IFN-β, are early effectors of innate immune responses against microbes that can also regulate subsequent adaptive immunity by promoting antimicrobial Th1-type responses. In contrast, the ability of IFN-β to inhibit autoimmune Th1 responses is thought to account for some of the beneficial effects of IFN-β therapy in the treatment of relapsing remitting multiple sclerosis. To understand the basis of the paradoxical effects of IFN-β on the expression of Th1-type immune responses, we developed an in vitro model of monocyte-derived dendritic cell (DC)-dependent, human naive Th cell differentiation, in which one can observe both positive and negative effects of IFN-β on the generation of Th1 cells. In this model we found that the timing of IFN-β exposure determines whether IFN-β will have a positive or a negative effect on naive Th cell differentiation into Th1 cells. Specifically, the presence of IFN-β during TNF-α-induced DC maturation strongly augments the capacity of DC to promote the generation of IFN-γ-secreting Th1 cells. In contrast, exposure to IFN-β during mature DC-mediated primary stimulation of naive Th cells has the opposite effect, in that it inhibits Th1 cell polarization and promotes the generation of an IL-10-secreting T cell subset. Studies with blocking mAbs and recombinant cytokines indicate that the mechanism by which IFN-β mediates these contrasting effects on Th1 cell generation is at least in part by differentially regulating DC expression of IL-12 family cytokines (IL-12 and/or IL-23, and IL-27) and IL-18.
Role of Complement-Binding CD21/CD19/CD81 in Enhancing Human B Cell Protection from Fas-Mediated Apoptosis
Authors: Mongini Patricia K. A. , Jackson Anna E. , Tolani Sonia , Fattah Rasem J. , Inman John K.
Published: April 2014

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIPL. This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.
Differential Responses to IFN-α Subtypes in Human T Cells and Dendritic Cells
Authors: Hilkens Catharien M. U. , Schlaak Jörg F. , Kerr Ian M.
Published: April 2014

Type I IFNs (IFN-αβ) constitute a family of cytokines that have important antiviral and immunoregulatory properties and have been successfully used in the treatment of a wide variety of diseases. There are 12 functional human IFN-α subtypes and one IFN-β subtype that signal through the common cell surface IFN-αβR. To date, virtually no information is available on the specificity of IFN-α responses in immune cells. In this study, Janus kinase/STAT signaling and transcriptional responses to selected IFN-α subtypes in human T cells and dendritic cells were analyzed. Evidence for IFN-α subtype and cell type specificity was found. Also, differences between kinetics of expression of IFN-stimulated genes (ISGs) and in the requirements of individual ISGs for additional signaling pathways were observed. In particular, IFN-γ-inducible protein-10 (IP-10), a key chemokine in Th1-type inflammatory diseases, was differentially regulated. In dendritic cells, it was highly induced by IFN-α2 and IFN-α21 but much less efficiently by IFN-α1. It was only marginally induced by these subtypes in T cells. In marked contrast to other ISGs analyzed, optimum induction of IP-10 was dependent on activation of p38 kinase(s). The observed variations (subtype-, cell type-, and ISG-related differentials) provide further insight into the complexity and plasticity of the IFN-αβ response. Furthermore, the novel observation that IFN-α1 poorly induces IP-10 is potentially of clinical importance, because this subtype may be more beneficial in cases where Th1-mediated side effects (e.g., exacerbation of autoimmune diseases) are not desirable.
HLA-F Surface Expression on B Cell and Monocyte Cell Lines Is Partially Independent from Tapasin and Completely Independent from TAP
Authors: Lee Ni , Geraghty Daniel E.
Published: April 2014

In this study we examined HLA-F expression in normal cells and cell lines, with a particular focus on identifying cells that express surface protein. While HLA-F protein was expressed in a number of diverse tissues and cell lines, including bladder, skin, and liver cell lines, no surface expression could be detected in the majority of them. However, surface expression was observed on EBV-transformed lymphoblastoid cell lines and on some monocyte cell lines. Expression on B lymphoblastoid cell lines was observed, while no surface expression on normal B cells or on any peripheral blood lymphocytes could be detected. Surface expression correlated with the presence of a limited amount of endoglycosidase H (Endo H)-resistant HLA-F. However, clearly not all surface-expressed HLA-F was fully glycosylated. We further examined the requirement of HLA-F surface expression for functional TAP and tapasin molecules and identified a clear departure from the dependence shown by other class I molecules on TAP. In contrast, of the two surface glycosylation forms expressed, an Endo H-sensitive form was tapasin independent, while an Endo H-resistant form was clearly tapasin dependent. Finally, we tested whether HLA-F could be stabilized for surface expression without peptide by using the classical cold treatment for surface stabilization of empty class I. Of several cell lines tested, only MHC deletion mutant 721.221 demonstrated a typical class I phenotype, indicating that control of surface stabilization may have a genetic basis resident in the MHC.
Separate Functional Domains of Human MD-2 Mediate Toll-Like Receptor 4-Binding and Lipopolysaccharide Responsiveness
Authors: Re Fabio , Strominger Jack L.
Published: April 2014

Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys95 and Cys105, which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.z
Human CD94 Gene Expression: Dual Promoters Differing in Responsiveness to IL-2 or IL-15
Authors: Lieto Louis D. , Borrego Francisco , You Chi-hyun , Coligan John E.
Published: April 2014

CD94 is a C-type lectin required for the dimerization of the CD94/NKG2 family of receptors, which are expressed on NK cells and T cell subsets. Little is known about CD94 gene expression and the elements that regulate CD94 transcription. In this study, we report that CD94 gene expression is regulated by distal and proximal promoters that transcribe unique initial exons specific to each promoter. This results in two species of transcripts; the previously described CD94 mRNA and a novel CD94C mRNA. All NK cells and CD94+, CD8+ αβ T cells transcribe CD94 mRNA. Stimulation of NK and CD8+ αβ T cells with IL-2 or IL-15 induced the transcription of CD94C mRNA. The distal and proximal promoters both contain elements with IFN-γ-activated and Ets binding sites, known as GAS/EBS. Additionally, an unknown element, termed site A, was identified in the proximal promoter. EMSA analyses showed that constitutive factors could bind to oligonucleotide probes containing each element. After treatment of primary NK cells with IL-2 or IL-15, separate inducible complexes could be detected with oligonucleotide probes containing either the proximal or distal GAS/EBS elements. These elements are highly conserved between mice and humans, which suggests that both species regulate CD94 gene expression via mechanisms that predate their evolutionary divergence.
Tapasin Is a Facilitator, Not an Editor, of Class I MHC Peptide Binding
Authors: Zarling Angela L. , Luckey Chance John , Marto Jarrod A. , White Forest M. , Brame Cynthia J. , Evans Anne M. , Lehner Paul J. , Cresswell Peter , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H.
Published: April 2014

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin+ and tapasin− cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin− cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.
V(D)J Recombination Frequencies Can Be Profoundly Affected by Changes in the Spacer Sequence
Authors: Montalbano Alina , Ogwaro Kisani M. , Tang Alan , Matthews Adam G. W. , Larijani Mani , Oettinger Marjorie A. , Feeney Ann J.
Published: April 2014

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two VHS107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is ∼9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.
Two Novel Fully Functional Isoforms of CX3CR1 Are Potent HIV Coreceptors
Authors: Garin Alexandre , Tarantino Nadine , Faure Sophie , Daoudi Mehdi , Lécureuil Cédric , Bourdais Anne , Debré Patrice , Deterre Philippe , Combadiere Christophe
Published: April 2014

We identified two novel isoforms of the human chemokine receptor CX3CR1, produced by alternative splicing and with N-terminal regions extended by 7 and 32 aa. Expression of the messengers coding these isoforms, compared with that of previously described V28 messengers, is lower in monocytes and NK cells, but higher in CD4+ T lymphocytes. CX3CR1 and its extended isoforms were expressed in HEK-293 cells and compared for expression, ligand binding, and cellular responses. In steady state experiments, all three CX3CR1 isoforms bound CX3CL1 with similar affinity. In kinetic binding studies, however, kon and koff were significantly greater for the extended CX3CR1 isoforms, thereby suggesting that the N-terminal extensions may alter the functions induced by CX3CL1. In signaling studies, all three CX3CR1 isoforms mediated agonist-dependent calcium mobilization, but the EC50 was lower for the extended than for the standard isoforms. In addition, chemotactic responses for these extended isoforms shifted left, also indicating a more sensitive response. Finally, the longer variants appeared to be more potent HIV coreceptors when tested in fusion and infection assays. In conclusion, we identified and characterized functionally two novel isoforms of CX3CR1 that respond more sensitively to CX3CL1 and HIV viral envelopes. These data reveal new complexity in CX3CR1 cell activation and confirm the critical role of the N-terminal domain of the chemokine receptors in ligand recognition and cellular response.
STAT-1α and IFN-γ as Modulators of TNF-α Signaling in Macrophages: Regulation and Functional Implications of the TNF Receptor 1:STAT-1α Complex
Authors: Wesemann Duane R. , Benveniste Etty N.
Published: April 2014

TNF-α and IFN-γ cooperate in the activation of macrophages. TNF-α-dependent activation of NF-κB is stronger in the presence of IFN-γ. STAT-1α associates with TNFR1 in TNF-α-treated cells, and this association attenuates TNF-α-mediated NF-κB activation. We hypothesized that nuclear localization of STAT-1α due to IFN-γ signaling would preclude it from being recruited to the TNFR1 and therefore enhance TNF-α-induced NF-κB activation. In the RAW264.7 macrophage cell line, TNF-α treatment indeed recruits STAT-1α to the TNFR1, and this association is abrogated when cells are exposed to IFN-γ. TNF-α treatment induces a more robust activation of NF-κB in STAT-1α-deficient cells, and restoration of STAT-1α inhibits TNF-α-dependent NF-κB activation. Our results suggest that a receptor-proximal level of cross-talk exists between these two cytokine pathways: IFN-γ limits STAT-1α availability to the TNFR1 by depleting STAT-1α from the cytoplasm, thus allowing for optimal NF-κB activation upon TNF-α ligation.
Neuronal Calcium Sensor-1 and Phosphatidylinositol 4-Kinase β Regulate IgE Receptor-Triggered Exocytosis in Cultured Mast Cells
Authors: Kapp-Barnea Yaara , Melnikov Semyon , Shefler Irit , Jeromin Andreas , Sagi-Eisenberg Ronit
Published: April 2014

We examined the possible occurrence and function of neuronal Ca2+ sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase β (PI4Kβ) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kβ, increases IgE receptor (FcεRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and stimulates FcεRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kβ reduces PI4Kβ activity, decreases FcεRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcεRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kβ, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P2 that serves as substrate for the generation of lipid-derived second messengers in FcεRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kβ and positive regulation of its activity.
Identification of Novel Genes Regulated by IL-12, IL-4, or TGF-β during the Early Polarization of CD4+ Lymphocytes
Authors: Lund Riikka , Aittokallio Tero , Nevalainen Olli , Lahesmaa Riitta
Published: April 2014

Th1 and Th2 cells arise from a common precursor cell in response to triggering through the TCR and cytokine receptors for IL-12 or IL-4. This leads to activation of complex signaling pathways, which are not known in detail. Disturbances in the balance between type 1 and type 2 responses can lead to certain immune-mediated diseases. Thus, it is important to understand how Th1 and Th2 cells are generated. To clarify the mechanisms as to how IL-12 and IL-4 induce Th1 and Th2 differentiation and how TGF-β can inhibit this process, we have used oligonucleotide arrays to examine the early polarization of Th1 and Th2 cells in the presence and absence of TGF-β. In addition to genes previously implicated in the process, we have identified 20 genes with various known and unknown functions not previously associated with Th1/2 polarization. We have also further determined which genes are targets of IL-12, IL-4, and TGF-β regulation in the cells induced to polarize to Th1 and Th2 directions. Interestingly, a subset of the genes was coregulated by IL-12 or IL-4 and TGF-β. Among these genes are candidates that may modulate the balance between Th1 and Th2 responses.
Reciprocal Transfer of Class I MHC Allele Specificity between Activating Ly-49P and Ly-49W Receptors by Exchange of β4–β5 Loop Residues
Authors: Ma Brian J. , Silver Elizabeth T. , Hazes Bart , Kane Kevin P.
Published: April 2014

Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2Dd, or H-2Dd and Dk, respectively. In this report, we demonstrate that specificity for H-2Dk can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the β4–β5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2Dk recognition while still preserving H-2Dd recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the β4–β5 loop, in determining class I MHC allele specificity.
Mast Cell Degranulation RequiresN-Ethylmaleimide-Sensitive Factor-Mediated SNARE Disassembly
Authors: Puri Niti , Kruhlak Michael J. , Whiteheart Sidney W. , Roche Paul A.
Published: April 2014

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.
Protective and Memory Immunity toHistoplasma capsulatumin the Absence of IL-10
Authors: Deepe George S. , Gibbons Reta S.
Published: April 2014

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10−/− mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-α or IFN-γ, but not GM-CSF, abrogated protection in naive IL-10−/− mice; mAb toTNF-α, but not IFN-γ or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10−/− mice was altered in those given mAb to IFN-γ or TNF-α. More Gr-1+ and Mac-3+ cells were present in lungs of IL-10−/− mice given mAb to IFN-γ, and treatment with mAb to TNF-α sharply reduced the number of CD8+ cells in lungs of IL-10−/− mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3+, CD44high, CD62low, and IFN-γ+ cells in IL-10−/− mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10−/− mice were required to mediate protection upon adoptive transfer into infected TCR αβ−/− mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-α and/or IFN-γ. Memory cell generation was transiently increased in IL-10−/− mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.
Myeloid C3 Determines Induction of Humoral Responses to Peripheral Herpes Simplex Virus Infection
Authors: Verschoor Admar , Brockman Mark A. , Gadjeva Mihaela , Knipe David M. , Carroll Michael C.
Published: April 2014

The complement system, in addition to its role in innate immunity, is an important regulator of the B cell response. Complement exists predominantly in the circulation and although the primary source is hepatic, multiple additional cellular sources have been described that can contribute substantially to the complement pool. To date, however, complement produced by these secondary sources has been deemed redundant to that secreted by the liver. In contrast, using a bone marrow chimeric model, we observed that C3 synthesis by myeloid cells, a relatively minor source of complement, provided a critical function during the induction of humoral responses to peripheral HSV infection. Anti-viral Ab, as generated in an efficient humoral response, has been associated with protection from severe consequences of HSV dissemination. This report offers insight into the generation of the adaptive immune response in the periphery and describes a unique role for a nonhepatic complement source.
Epitope Escape Mutation and Decay of Human Immunodeficiency Virus Type 1-Specific CTL Responses
Authors: Jamieson Beth D. , Yang Otto O. , Hultin Lance , Hausner Mary Ann , Hultin Patricia , Matud Jose , Kunstman Kevin , Killian Scott , Altman John , Kommander Kristina , Korber Bette , Giorgi Janis , Wolinsky Steven
Published: April 2014

To investigate possible mechanisms behind HIV-1 escape from CTL, we performed detailed longitudinal analysis of Gag (SLYNTVATL)- and RT (ILKEPVHGV)-specific CTL responses and plasma epitope sequences in five individuals. Among those with CTL against consensus epitope sequences, epitope mutations developed over several years, invariably followed by decay of the CTL targeting the consensus epitopes. The maturation state of the CTL varied among individuals and appeared to affect the rate of epitope mutation and CTL decay, despite similar IFN-γ production. Escape mutations were oligoclonal, suggesting fitness constraints. The timing of escape indicated that the net selective advantage of escape mutants was slight, further underscoring the importance of understanding factors determining selective pressure and viral fitness in vivo. Our data show surprisingly consistent decay of CTL responses after epitope escape mutation and provide insight into potential mechanisms for both immune failure and shifting CTL specificities.
Alveolar Macrophage Apoptosis Contributes to Pneumococcal Clearance in a Resolving Model of Pulmonary Infection
Authors: Dockrell David H. , Marriott Helen M. , Prince Lynne R. , Ridger Victoria C. , Ince Paul G. , Hellewell Paul G. , Whyte Moira K. B.
Published: April 2014

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.
Fibrinogen Depletion Attenuates Staphyloccocus aureus Infection by Preventing Density-Dependent Virulence Gene Up-Regulation
Authors: Rothfork Jacob M. , Dessus-Babus Sophie , Van Wamel Willem J. B. , Cheung Ambrose L. , Gresham Hattie D.
Published: April 2014

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including α hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.
Activation of a Subset of Human NK Cells upon Contact with Plasmodium falciparum-Infected Erythrocytes
Authors: Artavanis-Tsakonas Katerina , Eleme Konstantina , McQueen Karina L. , Cheng Nathalie W. , Parham Peter , Davis Daniel M. , Riley Eleanor M.
Published: April 2014

Human NK cells are the earliest source of the protective cytokine IFN-γ when PBMC from nonimmune donors are exposed to Plasmodium falciparum-infected RBC (iRBC) in vitro. In this study, we show that human NK cells form stable conjugates with iRBC but not with uninfected RBC and that induction of IFN-γ synthesis is dependent on direct contact between the NK cell and the iRBC. NK cells respond to iRBC only in the presence of a source of IL-12/IL-18 and the subset of NK cells that preferentially respond to iRBC express high levels of the lectin-like receptor CD94/NKG2A. There is heterogeneity between donors in their ability to respond to iRBC. DNA analysis has revealed considerable heterogeneity of killer Ig-like receptor (KIR) genotype among the donor population and has identified 21 new KIR allelic variants in the donors of African and Asian descent. Importantly, we find evidence for significant associations between KIR genotype and NK responsiveness to iRBC. This emphasizes the need for large-scale population-based studies to address associations between KIR genotype and susceptibility to malaria.