bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Human Vα24+ CD4+ NKT Cells Activated by α-Glycosylceramide-Pulsed Monocyte-Derived Dendritic Cells
Authors: Takahashi Tsuyoshi , Nieda Mie , Koezuka Yasuhiko , Nicol Andrew , Porcelli Steven A. , Ishikawa Yoshihide , Tadokoro Kenji , Hirai Hisamaru , Juji Takeo
Published: April 2014

Human Vα24+ NKT cells with an invariant TCR (Vα24-JαQ) have been shown to be specifically activated by synthetic glycolipids such as α-galactosylceramide and α-glucosylceramide in a CD1d-restricted and Vα24 TCR-mediated manner. We recently characterized Vα24+ CD4− CD8− double negative (DN) NKT cells using α-galactosylceramide-pulsed monocyte-derived dendritic cells. Here, we compare Vα24+ CD4+ NKT cells with human Vα24+ DN NKT cells from the same donor using α-galactosylceramide-pulsed monocyte-derived dendritic cells. Human Vα24+ CD4+ NKT cells were phenotypically and functionally similar to the human Vα24+ DN NKT cells characterized previously. Both of them use Vα24-JαQ-Vβ11 TCR and express CD161 (NKR-P1A), but not the other NK receptors tested so far. They also produce cytokines such as IL-4 and IFN-γ, and, in regard to IL-4 production, Vα24+ CD4+ NKT cells produce more IL-4 than Vα24+ DN NKT cells. The cells exhibit marked cytotoxic activity against the U937 tumor cell line, but not against the NK target cell line, K562. Although at least some of the factors responsible for the stimulation of Vα24+ NKT cells have been clarified, little is known regarding the killing phase of these cells. Here we show that the cytotoxic activity of Vα24+ NKT cells against U937 cells is mediated mainly through the perforin pathway and that ICAM-1/LFA-1 as well as CD44/hyaluronic acid interactions are important for the effector phase of Vα24+ NKT cell-mediated cytotoxicity against U937 cells.
The Roles of CD28 and CD40 Ligand in T Cell Activation and Tolerance
Authors: Howland Kimberly C. , Ausubel Lara J. , London Cheryl A. , Abbas Abul K.
Published: April 2014

Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28−/− T cells showed a greater reduction in proliferative responses to Ag than did CD40L−/− T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L−/− T cells were defective in Th1 development. In vivo, CD28−/− T cells failed to expand upon immunization, whereas CD40L−/− T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.
The Regulation of CD95 (Fas) Ligand Expression in Primary T Cells: Induction of Promoter Activation in CD95LP-Luc Transgenic Mice
Authors: Norian Lyse A. , Latinis Kevin M. , Eliason Steve L. , Lyson Krzysztof , Yang Chunmei , Ratliff Timothy , Koretzky Gary A.
Published: April 2014

The interaction between CD95 (Fas) and CD95L (Fas ligand) initiates apoptosis in a variety of cell types. Although the regulation of CD95L expression on activated T cells is an area of intense study, knowledge related to the induction of CD95L promoter activity in primary T cells is lacking. In this report we describe the generation of a novel transgenic mouse strain, CD95LP-Luc, in which murine CD95L promoter sequence controls the expression of a luciferase reporter gene. We use these mice to illustrate several important findings related to transcriptional regulation of CD95L in primary T cells. We demonstrate that maximal CD95L promoter activity occurs only after prolonged T cell stimulation and requires costimulation through CD28. We provide evidence that thymocytes express CD95L/luciferase after strong TCR ligation and that inducible CD95L promoter activation is present, but unequal, in both Th1 and Th2 effector cells. We also illustrate that while agonist peptide presentation by APCs generates robust proliferation during a primary T cell response, the same stimulus induces only modest CD95L promoter activity. These results suggest alternate explanations for the well-characterized delay in CD95-mediated activation-induced cell death following initial ligation of the TCR.
Astrocyte-Targeted Expression of IL-12 Induces Active Cellular Immune Responses in the Central Nervous System and Modulates Experimental Allergic Encephalomyelitis
Authors: Pagenstecher Axel , Lassmann Silke , Carson Monica J. , Kincaid Carrie L. , Stalder Anna K. , Campbell Iain L.
Published: April 2014

The role of IL-12 in the evolution of immunoinflammatory responses at a localized tissue level was investigated. Transgenic mice were developed with expression of either both the IL-12 subunits (p35 and p40) or only the IL-12 p40 subunit genes targeted to astrocytes in the mouse CNS. Glial fibrillary acidic protein (GF)-IL-12 mice, bigenic for the p35 and p40 genes, developed neurologic disease which correlated with the levels and sites of transgene-encoded IL-12 expression. In these mice, the brain contained numerous perivascular and parenchymal inflammatory lesions consisting of predominantly CD4+ and CD8+ T cells as well as NK cells. The majority of the infiltrating T cells had an activated phenotype (CD44high, CD45Rblow, CD62Llow, CD69high, VLA-4 high, and CD25+). Functional activation of the cellular immune response was also evident with marked cerebral expression of the IFN-γ, TNF, and IL-1αβ genes. Concomitant with leukocyte infiltration, the CNS expression of immune accessory molecules was induced or up-regulated, including ICAM-1, VCAM-1, and MHC class II and B7-2. Glial fibrillary acidic protein-p40 mice with expression of IL-12 p40 alone remained asymptomatic, with no inflammation evident at any age studied. The effect of local CNS production of IL-12 in the development of experimental autoimmune encephalomyelitis was studied. After immunization with myelin oligodendrocyte glycoprotein-peptides, GF-IL-12 mice had an earlier onset and higher incidence but not more severe disease. We conclude that localized expression of IL-12 by astrocytes can 1) promote the spontaneous development of activated type 1 T cell and NK cellular immunity and cytokine responses in the CNS, and 2) promote more effective Ag-specific T cell dynamics but not activity in experimental autoimmune encephalomyelitis.
Delayed and Separate Costimulation In Vitro Supports the Evidence of a Transient “Excited” State of CD8+ T Cells During Activation
Authors: Pardigon Nathalie , Cambouris Christophe , Bercovici Nadège , Lemaı̂tre Fabrice , Liblau Roland , Kourilsky Philippe
Published: April 2014

Although the two-signal model for T cell activation states that a signal-1 through the TCR and a costimulatory signal-2 are required for optimal stimulation, it is now clear that the requirement for costimulation can be bypassed under certain conditions. We previously reported that this is the case for naive CD8+ T cells in vitro. In the present study we tested the effect of signal-2 when delivered after signal-1 has been disrupted. Naive CD8+ T cells from TCR transgenic mice were stimulated in vitro by using immobilized recombinant single-chain MHC molecules alone as signal-1. This signal was then stopped after different lengths of time, and anti-CD28 mAb as signal-2 was given either immediately or after a time lag. We found that signal-2 can potentiate a short signal-1 when added sequentially. Moreover, a time lag between the two signals does not abolish this potentiation. If the strength of signal-1, but not its duration, is increased, then the time lag between the delivery of signals 1 and 2 can be lengthen without loss of potentiation. Together, our results indicate that the two signals do not need to be delivered concomitantly to get optimal T cell activation. We suggest that the CD8+ T cells can reach a transient “excited” state after being stimulated with signal-1 alone, characterized by the cell’s ability to respond to separate and delayed signal-2.
MHC Class I Antigen Processing of an Adenovirus CTL Epitope Is Linked to the Levels of Immunoproteasomes in Infected Cells
Authors: Sijts Alice J. A. M. , Standera Sybille , Toes René E. M. , Ruppert Thomas , Beekman Nico J. C. M. , van Veelen Peter A. , Ossendorp Ferry A. , Melief Cornelis J. M. , Kloetzel Peter M.
Published: April 2014

Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-γ-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192–200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192–200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-γ-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.
Development of Th1-Inducing Capacity in Myeloid Dendritic Cells Requires Environmental Instruction
Authors: Vieira Pedro L. , de Jong Esther C. , Wierenga Eddy A. , Kapsenberg Martien L. , Kaliński Paweł
Published: April 2014

Dendritic cells (DC) are key initiators of primary immune responses. Myeloid DC can secrete IL-12, a potent Th1-driving factor, and are often viewed as Th1-promoting APC. Here we show that neither a Th1- nor a Th2-inducing function is an intrinsic attribute of human myeloid DC, but both depend on environmental instruction. Uncommitted immature DC require exposure to IFN-γ, at the moment of induction of their maturation or shortly thereafter, to develop the capacity to produce high levels of IL-12p70 upon subsequent contact with naive Th cells. This effect is specific for IFN-γ and is not shared by other IL-12-inducing factors. Type 1-polarized effector DC, matured in the presence of IFN-γ, induce Th1 responses, in contrast to type 2-polarized DC matured in the presence of PGE2 that induce Th2 responses. Type 1-polarized effector DC are resistant to further modulation, which may facilitate their potential use in immunotherapy.
Evidence of Selective Processing of Immunodominant Epitopes in Virally Infected Cells
Authors: Zhang Qian-Jin , Chen Susan S. , Saari Carol-Ann , Massuci Maria G. , Tufaro Frank , Jefferies Wilfred A.
Published: April 2014

Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk− cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.
In Vivo Behavior of Peptide-Specific T Cells During Mucosal Tolerance Induction: Antigen Introduced Through the Mucosa of the Conjunctiva Elicits Prolonged Antigen-Specific T Cell Priming Followed by Anergy
Authors: Egan Rita M. , Yorkey Chris , Black Richard , Loh Wai Khan , Stevens Julia L. , Storozynsky Eugene , Lord Edith M. , Frelinger John G. , Woodward Jerold G.
Published: April 2014

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11.10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.
CD28-Specific Antibody Prevents Graft-Versus-Host Disease in Mice
Authors: Yu Xue-Zhong , Bidwell Sasha J. , Martin Paul J. , Anasetti Claudio
Published: April 2014

The costimulatory molecules B7-1 and B7-2 regulate T cell activation by delivering activation signals through CD28 and inhibitory signals through CTLA4. Graft-vs-host disease (GVHD) is caused by activated donor T cells. Previously, we showed that CD28-deficient donor T cells induced less-severe GVHD than wild-type donor T cells, suggesting that CD28 signals exacerbate GVHD. In this paper we demonstrate that CTLA4 signals attenuate the severity of GVHD. Targeting the CD28 receptor with a specific mAb modulates the receptor in vivo, inhibits donor T cell expansion, and prevents GVHD. CTLA4 signaling was necessary for this effect because treatment with a soluble ligand that blocks binding of B7 to both CD28 and CTLA4 did not prevent GVHD as effectively as anti-CD28 mAb. These results support the current model of T cell costimulation in which CD28 signals amplify GVHD while CTLA4 signals inhibit GVHD, providing evidence that selective targeting of CD28 might be a better therapeutic strategy for inducing immunological tolerance than blocking the ligands for both CD28 and CTLA4.
Faithful Expression of the Human 5q31 Cytokine Cluster in Transgenic Mice
Authors: Lacy Dee A. , Wang Zhi-En , Symula Derek J. , McArthur Clifford J. , Rubin Edward M. , Frazer Kelly A. , Locksley Richard M.
Published: April 2014

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.
A VH11Vκ9 B Cell Antigen Receptor Drives Generation of CD5+ B Cells Both In Vivo and In Vitro
Authors: Chumley Michael J. , Dal Porto Joseph M. , Kawaguchi Susumu , Cambier John C. , Nemazee David , Hardy Richard R.
Published: April 2014

B lymphocytes can be divided into different subpopulations, some with distinctive activation requirements and probably mediating specialized functions, based on surface phenotype and/or anatomical location, but the origins of most of these populations remain poorly understood. B cells constrained by transgenesis to produce an Ag receptor derived from a conventional (B-2) type cell develop a B-2 phenotype, whereas cells from mice carrying a B-1-derived receptor acquire the B-1 phenotype. In this study transgenic enforced expression of a B cell receptor (μ/κ) originally isolated from a CD5+ (B-1a) B cell generates B-1 phenotype cells in bone marrow cultures that show a distinctive B-1 function, survival in culture. Despite their autoreactivity, we find no evidence for receptor editing or that the paucity of B-2 cells is the result of tolerance-induced selection. Finally, Ca2+ mobilization studies reveal a difference between transgenic B-1 cells in spleen and peritoneal cavity, with cells in spleen much more responsive to anti-B cell receptor cross-linking. We discuss these results in terms of specificity vs lineage models for generation of distinctive B cell subpopulations.
Increased DNA Vaccine Delivery and Immunogenicity by Electroporation In Vivo
Authors: Widera Georg , Austin Melissa , Rabussay Dietmar , Goldbeck Cheryl , Barnett Susan W. , Chen Minchao , Leung Louisa , Otten Gillis R. , Thudium Kent , Selby Mark J. , Ulmer Jeffrey B.
Published: April 2014

DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.
Cell Death-Associated Translocation of Plasma Membrane Components Induced by CTL
Authors: Kawasaki Yukishige , Saito Takako , Shirota-Someya Yoshiko , Ikegami Yuko , Komano Hajime , Lee Mi-Heon , Froelich Christopher J. , Shinohara Nobukata , Takayama Hajime
Published: April 2014

In the very early stages of target cell apoptosis induced by CTL, we found that fluorescence of labeling probes of the target plasma membrane, such as N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridinium dibromide (FM1-43), was translocated into intracellular membrane structures including nuclear envelope and mitochondria. This translocation was associated with the execution of CTL-mediated killing, because neither the CTL-target conjugation alone nor the binding of noncytotoxic Th2 clone with target cell was sufficient to provoke the process. Although FM1-43 translocation was observed in perforin-mediated cytotoxicity, examinations with several other dyes failed to detect the evidence for membrane damages that may cause influx of the dye. Moreover, the translocation was also observed in Fas-dependent apoptosis. These data indicate that the translocation precedes the damage of plasma membrane and intracellular organella in the course of apoptotic cell death and may represent the existence of a membrane trafficking that mediates the translocation of plasma membrane components in the early onset of apoptotic cell death.
IL-6 Receptor Independent Stimulation of Human gp130 by Viral IL-6
Authors: Müllberg Jürgen , Geib Till , Jostock Thomas , Hoischen Susanne H. , Vollmer Petra , Voltz Nicole , Heinz David , Galle Peter R. , Klouche Mariam , Rose-John Stefan
Published: April 2014

The genome of human herpes virus 8, which is associated with Kaposi’s sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.
MHC Class II-Bound Self Peptides from Autoimmune MRL/lprMice Reveal Potential T Cell Epitopes for Autoantibody Production in Murine Systemic Lupus Erythematosus
Authors: Freed John H. , Marrs Amy , VanderWall Jennifer , Cohen Philip L. , Eisenberg Robert A.
Published: April 2014

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The “abnormal” peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice.
DO11.10 and OT-II T Cells Recognize a C-Terminal Ovalbumin 323–339 Epitope
Authors: Robertson Jennifer M. , Jensen Peter E. , Evavold Brian D.
Published: April 2014

The OVA323–339 epitope recognized by DO11.10 (H-2d) and OT-II (H-2b) T cells was investigated using amino- and carboxy-terminal truncations to locate the approximate ends of the epitopes and single amino acid substitutions of OVA323–339 to identify critical TCR contact residues of the OVA323–339 peptide. DO11.10 and OT-II T cells are both specific for a C-terminal epitope whose core encompasses amino acids 329–337. Amino acid 333 was identified as the primary TCR contact residue for both cells, and amino acid 331 was found to be an important secondary TCR contact residue; however, the importance of other secondary TCR contact residues and peptide flanking residues differ between the cells. Additional OVA323–339-specific clones were generated that recognized epitopes found in the N-terminal end or in the center of the peptide. These findings indicate that OVA323–339 can be presented by I-Ad in at least three binding registers. This study highlights some of the complexities of peptide Ags such as OVA323–339, which contain a nested set of overlapping T cell epitopes and MHC binding registers.
Accelerated Proteasomal Degradation of Membrane Ig Heavy Chains
Authors: Ho Siew C. , Chaudhuri Subhra , Bachhawat Anand , McDonald Kenneth , Pillai Shiv
Published: April 2014

Membrane IgG H chains turn over considerably more rapidly than secretory Ig H chains in the 18-81 A2 pre-B cell line. This rapid degradation occurs in proteasomes. N-Glycosylated membrane Ig H chains accumulate in the endoplasmic reticulum in the presence of proteasomal inhibitors, suggesting that retrotranslocation and proteasomal degradation of membrane Ig H chains may be closely coupled processes. Accelerated proteasomal degradation of membrane Ig H chains was also observed in transfected nonlymphoid cells. At steady state, the membrane form of the H chain associates more readily with Bip and calnexin than its secretory counterpart. The preferential recognition of membrane, as opposed to secretory, Ig H chains by some endoplasmic reticulum chaperones, may provide an explanation for the accelerated proteasomal degradation of the former.
RGS Molecule Expression in Murine B Lymphocytes and Ability to Down-Regulate Chemotaxis to Lymphoid Chemokines
Authors: Reif Karin , Cyster Jason G.
Published: April 2014

Ag-mediated changes in B lymphocyte migration are important for normal immune function, yet the mechanisms by which these changes occur are poorly defined. Because chemokines direct many lymphocyte movements, molecules that regulate signaling by G protein-coupled chemokine receptors are likely to participate in Ag receptor-induced changes in cell migration. In this study, we have investigated the expression pattern and activity in murine B cells of members of the regulators of G protein signaling (RGS) family of molecules. We present the sequence of mouse RGS1 and describe a novel short isoform of RGS3 that we term RGS3s. Following in vivo activation by Ag, B cells rapidly up-regulate expression of RGS1 and RGS2 while simultaneously decreasing expression of RGS3 and RGS14. Anergic hen egg lysozyme autoantigen-binding B cells are also shown to have slightly elevated RGS1 and RGS2 expression. CD40 signaling, by contrast, fails to cause rapid up-regulation of RGS1 or RGS2. Using a transient transfection approach in a mature B cell line, 2PK3, we demonstrate that RGS1 and RGS3s are effective inhibitors of chemotaxis toward the lymphoid tissue chemokines stromal cell-derived factor-1, B lymphocyte chemoattractant, and EBV-induced molecule 1 ligand chemokine, whereas RGS2 has a minimal effect on migration to these chemokines. Together these findings support the conclusion that Ag-mediated changes in RGS molecule expression are part of the mechanism by which Ag receptor signaling regulates B cell migration within lymphoid tissues. The findings also suggest important roles for additional G protein-mediated events in B cell activation and tolerance.
Neutralization of Maternal IL-4 Modulates Congenital Protozoal Transmission: Comparison of Innate Versus Acquired Immune Responses
Authors: Long Maureen T. , Baszler Timothy V.
Published: April 2014

IL-4 levels were modulated in mice to test the hypothesis that induction of a maternal type 1 response would decrease the frequency of congenital Neospora caninum transmission. This hypothesis tested the relationship between IL-4 and both innate and adaptive immunity utilizing two basic experimental designs. In the first, maternal IL-4 was neutralized with mAb during pregnancy in naive mice concomitant with initial, virulent infection. In the second, maternal IL-4 was neutralized before pregnancy concomitant with a priming inoculation consisting of live, avirulent N. caninum tachyzoites followed by virulent challenge during subsequent gestation. In mice that were naive before pregnancy, neutralization of IL-4 during gestational challenge did not result in decreased congenital transmission as measured by PCR performed on 1-day-old neonatal mice. In mice that were primed and modulated before pregnancy, congenital transmission from gestational challenge was significantly decreased compared with control mice. Reduction in transmission constituted a decrease in the numbers of mice transmitting N. caninum and a lower frequency of transmission by individual dams (p < 0.05). Decreased congenital transmission was associated with significantly lower levels of maternal splenocyte IL-4 secretion, lower IL-4 mRNA levels, and higher levels of IFN-γ secretion. Protected mice had significantly decreased Neospora-specific IgG1 compared with nonmodulated mice. These studies define a relationship between maternal Ag-specific immunity and the frequency of congenital transmission and demonstrate that modulation of type 2 cytokine responses can change the frequency of congenital protozoal transmission.
Human Cytomegalovirus Strain-Dependent Changes in NK Cell Recognition of Infected Fibroblasts
Authors: Cerboni Cristina , Mousavi-Jazi Mehrdad , Linde Annika , Söderström Kalle , Brytting Maria , Wahren Britta , Kärre Klas , Carbone Ennio
Published: April 2014

NK cells play a key role in the control of CMV infection in mice, but the mechanism by which NK cells can recognize and kill CMV-infected cells is unclear. In this study, the modulation of NK cell susceptibility of human CMV (hCMV)-infected cells was examined. We used a human lung and a human foreskin fibroblast cell line infected with clinical isolates (4636, 13B, or 109B) or with laboratory strains (AD169, Towne). The results indicate that all three hCMV clinical isolates confer a strong NK resistance, whereas only marginal or variable effects in the NK recognition were found when the laboratory strains were used. The same results were obtained regardless of the conditions of infection, effector cell activation status, cell culture conditions, and/or donor-target cell combinations. The NK cell inhibition did not correlate with HLA class I expression levels on the surface of the target cell and was independent of the leukocyte Ig-like receptor-1, as evaluated in Ab blocking experiments. No relevant changes were detected in the adhesion molecules ICAM-I and LFA-3 expressed on the cell surface of cells infected with hCMV clinical and laboratory strains. We conclude that hCMV possesses other mechanisms, related neither to target cell expression of HLA-I or adhesion molecules nor to NK cell expression of leukocyte Ig-like receptor-1, that confer resistance to NK cell recognition. Such mechanisms may be lost during in vitro passage of the virus. These results emphasize the differences between clinical hCMV isolates compared with laboratory strains.
Regulation and Role of IFN-γ in the Innate Resistance to Infection withChlamydia pneumoniae
Authors: Rottenberg Martín E. , Gigliotti Rothfuchs Antonio , Gigliotti Dulceaydee , Ceausu Monica , Une Clas , Levitsky Victor , Wigzell Hans
Published: April 2014

By using mice genomically lacking IFN-γR, IL-12, perforin, and recombination-activating gene-1 (RAG-1), we analyzed the regulation and importance of IFN-γ in the control of infection with Chlamydia pneumoniae. IL-12 participates in resistance of mice to C. pneumoniae, probably by regulating the protective levels of IFN-γ mRNA. In turn, IFN-γ is necessary for the increased IL-12p40 mRNA accumulation that occurs in lungs during infection with C. pneumoniae, suggesting a positive feedback regulation between these two cytokines. In experiments including RAG-1−/−/IFN-γR−/− mice we showed that IFN-γ produced by innate cells controls the bacterial load and is necessary for the increased accumulation of transcripts for enzymes controlling high output NO release (inducible NO synthase), superoxide production (gp-91 NADPH oxidase), and catalyzis of tryptophan (indoleamine 2,3-dioxygenase (IDO)), mechanisms probably related to bacterial killing. Adaptive immune reponses diminish the levels of IFN-γ and IL-12 mRNA and thereby the levels of inducible NO synthase, IDO, and gp91 NADPH oxidase trancripts. By using RAG-1−/−/perforin−/− mice, we excluded the overt participation of NK cell cytotoxicity in the control of C. pneumoniae. However, NK cells and probably other innate immune cells release IFN-γ during the bacterial infection.
Ley/H: An Endothelial-Selective, Cytokine-Inducible, Angiogenic Mediator
Authors: Halloran Margaret M. , Carley William W. , Polverini Peter J. , Haskell Catherine J. , Phan Stacie , Anderson Byron J. , Woods James M. , Campbell Phillip L. , Volin Michael V. , Bäcker Annika E. , Koch Alisa E.
Published: April 2014

Endothelial cells (ECs) are key participants in angiogenic processes that characterize tumor growth, wound repair, and inflammatory diseases, such as human rheumatoid arthritis (RA). We and others have shown that EC molecules, such as soluble E-selectin, mediate angiogenesis. Here we describe an EC molecule, Lewisy-6/H-5-2 glycoconjugate (Ley/H), that shares some structural features with the soluble E-selectin ligand, sialyl Lewisx (sialyl Lex). One of the main previously recognized functions of Lewisy is as a blood group glycoconjugate. Here we show that Ley/H is rapidly cytokine inducible, up-regulated in RA synovial tissue, where it is cell-bound, and up-regulated in the soluble form in angiogenic RA compared with nonangiogenic osteoarthritic joint fluid. Soluble Ley/H also has a novel function, for it is a potent angiogenic mediator in both in vitro and in vivo bioassays. These results suggest a novel paradigm of soluble blood group Ags as mediators of angiogenic responses and suggest new targets for therapy of diseases, such as RA, that are characterized by persistent neovascularization.
IL-6 Is Required for the Development of Th1 Cell-Mediated Murine Colitis
Authors: Yamamoto Mitsunari , Yoshizaki Kazuyuki , Kishimoto Tadamitsu , Ito Hiroaki
Published: April 2014

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis of Crohn’s disease. Among those cytokines, strong expression of IL-6 has been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn’s disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL-6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 expression were analyzed by immunohistochemistry. Colonic cytokine expression was determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN-γ, TNF-α, and IL-1β mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn’s disease.
The T Cell Repertoire Selected In Vitro Against EBV: Diversity, Specificity, and Improved Purification Through Early IL-2 Receptor α-Chain (CD25)-Positive Selection
Authors: Ibisch Catherine , Saulquin Xavier , Gallot Géraldine , Vivien Régine , Ferrand Christophe , Tiberghien Pierre , Houssaint Elisabeth , Vié Henri
Published: April 2014

Polyclonal T cell lines specific for EBV proteins have proved efficient in preventing EBV-related immunoblastic lymphoma after allogeneic bone marrow transplantation. To gain insight into the composition of the EBV-specific T cell repertoire that ensured patient protection, we performed for the first time an extensive characterization of eight cytotoxic T cell lines selected in vitro against EBV-transformed autologous lymphoblastoid cell lines (BLCL). These T cell lines consist of 50–100 distinct T cell clones, of which 32–96% are specific for autologous BLCL. Moreover, we demonstrate that reactivities against only five EBV proteins (BZLF1, BMLF1, EBNA-3A, EBNA-3C, and LMP2) cover 86% (32/37) of the specificities detected. In addition, we describe an improved method of T cell harvesting using a CD25 selection procedure which reduces the time required to obtain specific T cells and improves the purity of EBV-specific T cells, thus showing promise for use in adoptive transfer protocols.
Baseline Airway Hyperreactivity in A/J Mice Is not Mediated by Cells of the Adaptive Immune System
Authors: Hadeiba Husein , Corry David B. , Locksley Richard M.
Published: April 2014

Human asthma is characterized by increased airway hyperreactivity to a variety of bronchoconstricting agents. Aberrant type 2 immune responses in the lung have been associated with airway hyperreactivity in both human asthma and in murine models of allergic airways disease. Despite their intrinsically elevated basal airway reactivity to smooth muscle constricting agents, A/J mice demonstrated no inherent inflammatory cell infiltration nor elevation of type 2 cytokines in the lung. Crossed bone marrow reconstitution experiments between A/J and MHC congenic B10.A mice revealed enhanced airway reactivity only in A/J recipients, irrespective of whether they had been reconstituted with A/J or B10.A hemopoietic cells. Further, A/J-derived bone marrow cells did not affect the reactivity of B10.A recipients. Although mice on RAG-deficient and IL-4-deficient backgrounds demonstrate substantial abrogation of allergen-induced airway hyperreactivity, these gene deletions had no impact on the elevated baseline reactivity when backcrossed onto A/J mice. Thus, in these mice, basal airway hyperreactivity is maintained independently of type 2 immunity induced by allergens.
T Cell Activity After Dendritic Cell Vaccination Is Dependent on Both the Type of Antigen and the Mode of Delivery
Authors: Serody Jonathan S. , Collins Edward J. , Tisch Roland M. , Kuhns Jennifer J. , Frelinger Jeffrey A.
Published: April 2014

Previous work in both human and animal models has shown that CTL responses can be generated against proteins derived from tumors using either peptide-pulsed dendritic cells (DCs) or nucleic acids from the tumor transfected into autologous DCs. Despite the efficacy of this approach for vaccine therapy, many questions remain regarding whether the route of administration, the frequency of administration, or the type of Ag is critical to generating T cell responses to these Ags. We have investigated methods to enhance CTL responses to a peptide derived from the human proto-oncogene HER-2/neu using mice containing a chimeric HLA A2 and H2Kb allele. Changes in amino acids in the anchor positions of the peptide enhanced the binding of the peptide to HLA-A2 in vitro, but did not enhance the immunogenicity of the peptide in vivo. In contrast, when autologous DCs presented peptides, significant CTL activity was induced with the altered, but not the wild-type, peptide. We found that the route of administration affected the anatomic site and the time to onset of CTL activity, but did not impact on the magnitude of the response. To our surprise, we observed that weekly administration of peptide-pulsed DCs led to diminishing CTL activity after 6 wk of treatment. This was not found in animals injected with DCs every 3 wk for six treatments or in animals initially given DCs weekly and then injected weekly with peptide-pulsed C1R-A2 transfectants.
Cutting Edge: OFF Cycling of TNF Production by Antigen-Specific CD8+ T Cells Is Antigen Independent
Authors: Badovinac Vladimir P. , Corbin Gail A. , Harty John T.
Published: April 2014

Although they are known for their capacity to kill infected cells, Ag-specific CD8+ T cells elaborate other effector mechanisms, including TNF and IFN-γ, that contribute to defense against infection. Ag-specific CD8+ T cells rapidly turn ON and turn OFF IFN-γ production in direct response to Ag contact, presumably to minimize the potential immunopathology that could result from inappropriate secretion of this inflammatory mediator. In this study, we show, using in vitro propagated and directly ex vivo-analyzed Ag-specific CD8+ T cells, that in contrast to Ag-dependent ON/OFF cycling of IFN-γ production, the cessation of TNF production by the same IFN-γ producing cells is rapid and Ag independent.
Cutting Edge: TLR2-Deficient and MyD88-Deficient Mice Are Highly Susceptible toStaphylococcus aureusInfection
Authors: Takeuchi Osamu , Hoshino Katsuaki , Akira Shizuo
Published: April 2014

Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns. We previously showed that TLR2 recognizes Gram-positive bacterial components whereas TLR4 recognizes LPS, a component of Gram-negative bacteria. MyD88 is shown to be an adaptor molecule essential for TLR family signaling. To investigate the role of TLR family in host defense against Gram-positive bacteria, we infected TLR2- and MyD88-deficient mice with Staphylococcus aureus. Both TLR2- and MyD88-deficient mice were highly susceptible to S. aureus infection, with more enhanced susceptibility in MyD88-deficient mice. Peritoneal macrophages from MyD88-deficient mice did not produce any detectable levels of cytokines in response to S. aureus. In contrast, TLR2-deficient macrophages produced reduced, but significant, levels of the cytokines, and TLR4-deficient macrophages produced the same amounts as wild-type cells, indicating that S. aureus is recognized not only by TLR2, but also by other TLR family members except for TLR4.
Cutting Edge: An Essential Role for Notch-1 in the Development of Both Thymus-Independent and -Dependent T Cells in the Gut
Authors: Wilson Anne , Ferrero Isabel , MacDonald H. Robson , Radtke Freddy
Published: April 2014

Whereas most T cells arise in the thymus, a distinct lineage of extrathymically derived T cells is present in the gut mucosa. The developmental origin of extrathymic T cells is poorly understood. We show here that Notch-1, a transmembrane receptor involved in T cell fate specification of bipotential T/B precursors in the thymus, is absolutely required for the development of extrathymic (as well as thymus-derived) mature T cells in the intestinal epithelium. In the absence of Notch-1, CD117+ T cell precursors are relatively more abundant in the gut than the thymus, whereas immature B cells accumulate in the thymus but not the gut. Collectively, these data demonstrate that Notch-1 is essential for both thymic and extrathymic T cell fate specification and further suggest that bipotential T/B precursors that do not receive a Notch-1 signal adopt a B cell fate in the thymus but become developmentally arrested in the gut.
Cutting Edge: Targeted Disruption of the C3a Receptor Gene Demonstrates a Novel Protective Anti-Inflammatory Role for C3a in Endotoxin-Shock
Authors: Kildsgaard Jens , Hollmann Travis J. , Matthews Kirstin W. , Bian Ka , Murad Ferid , Wetsel Rick A.
Published: April 2014

The complement anaphylatoxin C3a, on binding the C3aR, mediates numerous proinflammatory activities. In addition, recent in vitro studies with C3a have implicated C3aR as a possible anti-inflammatory receptor. Because of its possible dual role in modulating the inflammatory response, it is uncertain whether C3aR contributes to the pathogenesis of endotoxin shock. Here, the targeted-disruption of the C3aR in mice is reported. These mice exhibit an enhanced lethality to endotoxin shock with a pronounced gene dosage effect. In addition, the plasma concentration of IL-1β was significantly elevated in the C3aR−/− mice compared with their littermates following LPS challenge. These findings demonstrate an important protective role for the C3aR in endotoxin shock and indicate that, in addition to its traditionally accepted functions in mediating inflammation, the C3aR also acts in vivo as an anti-inflammatory receptor by attenuating LPS-induced proinflammatory cytokine production.
Lymphocytes Lacking IκB-α Develop Normally, But Have Selective Defects in Proliferation and Function
Authors: Chen Chih-Li , Singh Nagendra , Yull Fiona E. , Strayhorn David , Van Kaer Luc , Kerr Lawrence D.
Published: April 2014

NF-κB has been implicated in the development, activation, and function of B and T lymphocytes. We have evaluated the in vivo effects of deletion of IκB-α, a major inhibitor of NF-κB, on lymphocyte development, proliferation, and function. To elucidate the long term role of IκB-α in lymphocytes, fetal liver cells of 14.5-day-old IκB-α−/− or wild-type embryos were transplanted into irradiated recombinase-activating gene-2-deficient mice. Within 4 wk, the IκB-α−/− fetal liver cells reconstitute mature B and T cell populations in the recipients comparable to those produced by wild-type fetal liver cells. However, the proliferative responses of IκB-α−/− B cells are enhanced, whereas those of IκB-α−/− T cells are reduced. The levels of IgG1, IgG2a, IgA, and IgE produced by IκB-α−/− B cells are elevated relative to those produced by IκB-α+/+ or IκB-α+/−. Moreover, the specific immune responses to OVA and the generation of germinal centers are impaired in recipients of IκB-α−/− fetal liver cells. These results indicate that IκB-α plays a vital role in signal transduction pathways regulating lymphocyte proliferation and also in the production of specific Ig isotypes.
Dermal Microvascular Endothelial Cells Express the 180-kDa Macrophage Mannose Receptor In Situ and In Vitro
Authors: Gröger Marion , Holnthoner Wolfgang , Maurer Dieter , Lechleitner Sonja , Wolff Klaus , Mayr Bettina Beate , Lubitz Werner , Petzelbauer Peter
Published: April 2014

Expression of the 180-kDa mannose receptor (MR) is mainly found on cells of the macrophage lineage. MR mediates the uptake of micro-organisms and host-derived glycoproteins. We demonstrate that endothelium of the human skin in situ and dermal microvascular endothelial cells (DMEC) in vitro expressed MR at both the protein and mRNA levels. In contrast, HUVEC were consistently negative for MR expression. DMEC internalized dextran as well as Escherichia coli by the way of MR into acidic phagosomes, only a few of which fused with CD63- and lysosomal-associated membrane glycoprotein-2-positive lysosomes. This contrasts with the situation in monocyte-derived dendritic cells, where almost all of the MR-Ag complexes reached CD63- and lysosomal-associated membrane glycoprotein-2-positive compartments, indicating differences in the phagolysosomal fusion rate between DMEC and dendritic cells. In conclusion, DMEC express functional MR, a finding that corroborates a role of skin endothelium in Ag capture/clearing.