bluuebunny/crossref_metadata_2025 huggingface dataset:
Intervention of Thymus and Activation-Regulated Chemokine Attenuates the Development of Allergic Airway Inflammation and Hyperresponsiveness in Mice
Authors: Kawasaki Shin , Takizawa Hajime , Yoneyama Hiroyuki , Nakayama Takashi , Fujisawa Ryuichi , Izumizaki Masahiko , Imai Toshio , Yoshie Osamu , Homma Ikuo , Yamamoto Kazuhiko , Matsushima Kouji
Published: April 2014

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4+) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.
Deficient IL-12(p35) Gene Expression by Dendritic Cells Derived from Neonatal Monocytes
Authors: Goriely Stanislas , Vincart Benoı̂t , Stordeur Patrick , Vekemans Johan , Willems Fabienne , Goldman Michel , De Wit Dominique
Published: April 2014

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-γ to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-γ production by allogenic adult CD4+ T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-γ production by T cells.
Monocytes Are Differentially Activated Through HLA-DR, -DQ, and -DP Molecules Via Mitogen-Activated Protein Kinases
Authors: Matsuoka Takako , Tabata Hiroki , Matsushita Sho
Published: April 2014

When HLA-DR, -DQ, and -DP were cross-linked by solid-phase mAbs, monocytes produced monokines and only anti-DR markedly activated mitogen-activated protein (MAP) kinase extracellular signal-related kinase, whereas anti-DR, anti-DQ, and anti-DP all activated MAP kinase p38. Activation of extracellular signal-related kinase was not inhibited by neutralizing Ab to TNF-α. Anti-DR and DR-restricted T cells stimulated monocytes to produce relatively higher levels of proinflammatory monokines, such as IL-1β, whereas anti-DQ/DP and DQ-/DP-restricted T cells stimulated higher levels of anti-inflammatory monokine IL-10. IL-10 production was abrogated by the p38 inhibitor SB203580, but rather enhanced by the MAP/extracellular signal-related kinase kinase-I-specific inhibitor PD98059, whereas IL-1β was only partially abrogated by SB203580 and PD98059. Furthermore, DR-restricted T cells established from PBMC, which are reactive with mite Ags, purified protein derivative, and random 19-mer peptides, exhibited a higher IFN-γ:IL-4 ratio than did DQ- or DP-restricted T cells. These results indicate that HLA-DR, -DQ, and -DP molecules transmit distinct signals to monocytes via MAP kinases and lead to distinct monokine activation patterns, which may affect T cell responses in vivo. Thus, the need for generation of a multigene family of class II MHC seems apparent.
CD4+ and CD8+ T Cell Priming for Contact Hypersensitivity Occurs Independently of CD40-CD154 Interactions
Authors: Gorbachev Anton V. , Heeger Peter S. , Fairchild Robert L.
Published: April 2014

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-γ-producing CD8+ T cells, whereas CD4+ T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8+ and CD4+ T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154−/− animals. DNFB sensitization of CD154−/− mice primed IFN-γ-producing CD8+ T cells and IL-4-producing CD4+ T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8+, but not CD4+, T cell development and the CHS response to challenge. F(ab′)2 of MR1 failed to inhibit CD8+ T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8+ T cell development and CHS responses in mice depleted of CD4+ T cells or in CD4−/− mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4+ T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-γ-producing CD8+ T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8+ T cell development where anti-CD154 mAb acts indirectly through CD4+ T cells to impair the ability of hpLC to prime CD8+ T cells.
Bacterial Lipopolysaccharide Activates HIV Long Terminal Repeat Through Toll-Like Receptor 4
Authors: Equils Ozlem , Faure Emmanuelle , Thomas Lisa , Bulut Yonca , Trushin Sergey , Arditi Moshe
Published: April 2014

In HIV-infected patients, concurrent infections with bacteria and viruses are known to induce HIV replication as assessed by increases in plasma HIV RNA levels. In the present study, we determined the cell surface receptor and molecular mechanisms of enterobacterial LPS-induced HIV transcription. Human dermal microvessel endothelial cells (HMEC) were transfected with an HIV-long terminal repeat (LTR)-luciferase construct and subsequently stimulated with purified bacterial LPS. Our studies demonstrate that human Toll-like receptor 4 (TLR4) mediates LPS-induced NF-κB and HIV-LTR activation in HMEC through IL-1 signaling molecules, namely myeloid differentiation protein, IL-1R-associated kinase, TNFR-associated factor, and NF-κB-inducing kinase. Cotransfection of HMEC with HIV-LTR-luciferase and TLR4 cDNA from LPS-hyporesponsive C3H/HeJ mice abrogates LPS-induced HIV transcription as does the use of dominant-negative mutants of the IL-1 signaling molecules. Transfection of HMEC with an HIV-LTR-mutant that lacks the NF-κB binding site or pretreatment of cells with chemical inhibitors of the NF-κB pathway also blocked LPS-induced HIV-LTR transactivation. These data support the conclusion that TLR4 mediates enterobacterial LPS-induced HIV transcription via IL-1 signaling molecules and NF-κB activation plays an important role in HIV-LTR transactivation.
Human Peripheral Blood Cells Differentially Recognize and Respond to Two Distinct CpG Motifs
Authors: Verthelyi Daniela , Ishii Ken J. , Gursel Mayda , Takeshita Fumihiko , Klinman Dennis M.
Published: April 2014

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct “clusters” of immunostimulatory CpG ODN. One cluster (“D”) preferentially stimulates IFN-γ production by NK cells, whereas the other (“K”) stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.
Viral IL-10-Induced Immunosuppression Requires Th2 Cytokines and Impairs APC Function Within the Allograft
Authors: Qin Lihui , Ding Yaozhong , Tahara Hideaki , Bromberg Jonathan S.
Published: April 2014

Previous reports demonstrated that retroviral mediated gene transfer of viral IL-10 (vIL-10) prolongs allograft survival by decreasing donor-specific cytotoxic T lymphocyte precursor (CTLp) and IL-2-secreting helper T lymphocyte precursor (HTLp) frequency within graft-infiltrating cells (GIC). This report now shows that vIL-10 efficacy is dependent on CD4+ T cells, suggesting that immunosuppression may involve a switch from a Th1 to a Th2 alloresponse. In support of this, anti-IL-4 or anti-murine IL-10 (anti-mIL-10) mAbs, but not anti-IFN-γ mAb, administered at the time of vIL-10 gene transfer prevents enhanced graft survival. Because Th switching involves APC function, GIC were examined for their ability to present alloantigen. GIC from vIL-10-treated grafts were shown to be mostly of recipient (CBA) origin, yet were unable to elicit alloproliferative responses from donor type (C57BL/6) or third party (BALB/c) responders. The inability of vIL-10-treated GIC to stimulate the MLR was not due to the generation of negative regulatory cells or the production of immunosuppressive cytokines such as IL-4, mIL-10, or TGFβ. Using fractionated GIC subpopulations, the number of recipient type cells in the allografts was modestly reduced by vIL-10 gene transfer, while maintaining both APC phenotype and alloantigen presenting function. Conversely, after vIL-10 gene transfer, donor type GIC were unable to participate in direct alloantigen presentation. Therefore, local immunosuppression induced by vIL-10 gene transfer is CD4 T cell and IL-4 and mIL-10 dependent, and impairs direct alloantigen presentation through an alteration of donor type APC function.
Autologous Regulation of Naive T Cell Homeostasis Within the T Cell Compartment
Authors: Dummer Wolfgang , Ernst Bettina , LeRoy Eric , Lee Dong-Sup , Surh Charles D.
Published: April 2014

Naive T cells undergo spontaneous slow proliferation on adoptive transfer into syngeneic T cell (T)-deficient hosts. Recent work has shown that such “homeostatic” T cell proliferation is driven by MHC molecules loaded with self-peptides rather than foreign peptides. Because naive T cells in normal T-sufficient hosts remain in interphase despite continuous contact with self-MHC/peptide ligands, T cells apparently inhibit homeostatic proliferation of neighboring T cells. To address this, we have investigated the requirements necessary for “bystander” T cells to inhibit homeostatic proliferation of other T cells. Three key findings are reported. First, homeostatic proliferation of T cells only occurs in specific microenvironments, namely the T cell compartment of the secondary lymphoid tissues. Second, direct entry into T cell compartments is also required for bystander inhibition of homeostatic proliferation. Third, bystander inhibition is mediated largely by naive rather than activated/memory T cells and does not require proliferation or TCR ligation. These findings suggest that homeostasis of naive T cells is unlikely to be regulated through competition for systemic soluble factors or for specific stimulatory self-MHC/peptide ligands. Rather, the data favor mechanisms that involve competition for local non-MHC stimulatory factors or direct cell-to-cell interactions between the T cells themselves within the T cell compartment.
Acquisition of CD80 (B7-1) by T Cells
Authors: Sabzevari Helen , Kantor Judy , Jaigirdar Adnan , Tagaya Yutaka , Naramura Mayumi , Hodge James W. , Bernon John , Schlom Jeffrey
Published: April 2014

Activation of T cells usually requires two signals. Signal 1 is mediated via a peptide-MHC on the APC; signal 2 is mediated via a costimulatory molecule on the APC surface. We demonstrate here that naive CD4+ T cells actually acquire the costimulatory molecule CD80 (B7-1) from syngeneic APCs after activation. This phenomenon was demonstrated showing acquisition of CD80 by T cells from CD80/CD86 (B7-2) knockout mice, and by treating T cells with cyclohexamide to further rule out endogenous expression of CD80 by T cells. Moreover, no CD80 mRNA could be detected in T cells that had acquired CD80. The amount of acquisition of CD80 by T cells was shown to be directly related to both the strength of signal 1 and the amount of CD80 on the APC. Specificity of this acquisition was also shown by the lack of acquisition by T cells from CD28 knockout mice (implicating CD28 in this process), the lack of acquisition of CD40 (another molecule on the APC surface) by T cells, and confocal microscopy studies. We demonstrate for the first time that 1) naive T cells, following acquisition of CD80 from APCs, were themselves shown to be capable of acting as APCs; and 2) memory T cells that have acquired CD80 from APCs undergo apoptosis in the presence of increased levels of signal 1. Thus we demonstrate both immunostimulatory and immunoregulatory functions as a result of CD80 acquisition by different T cell populations.
Ordered and Coordinated Rearrangement of the TCR α Locus: Role of Secondary Rearrangement in Thymic Selection
Authors: Huang Ching-Yu , Kanagawa Osami
Published: April 2014

The Ag receptor of the T lymphocyte is composed of an αβ heterodimer. Both α- and β-chains are products of the somatic rearrangement of V(D)J segments encoded on the respective loci. During T cell development, β-chain rearrangement precedes α-chain rearrangement. The mechanism of allelic exclusion ensures the expression of a single β-chain in each T cell, whereas a large number of T cells express two functional α-chains. Here we demonstrate evidence that TCR α rearrangement is initiated by rearranging a 3′ Vα segment and a 5′ Jα segment on both chromosomes. Rearrangement then proceeds by using upstream Vα and downstream Jα segments until it is terminated by successful positive selection. This ordered and coordinated rearrangement allows a single thymocyte to sequentially express multiple TCRs with different specificities to optimize the efficiency of positive selection. Thus, the lack of allelic exclusion and TCR α secondary rearrangement play a key role in the formation of a functional T cell repertoire.
Reduction in DNA Binding Activity of the Transcription Factor Pax-5a in B Lymphocytes of Aged Mice
Authors: Anspach Jillian , Poulsen Gail , Kaattari Ilsa , Pollock Roberta , Zwollo Patty
Published: April 2014

Aging has been associated with intrinsic changes of the humoral immune response, which may lead to an increased occurrence of autoimmune disorders and pathogenic susceptibility. The transcription factor Pax-5 is a key regulator of B cell development. Pax-5a/B cell-specific activator protein and an alternatively spliced isoform, Pax-5d, may have opposing functions in transcriptional regulation due to the lack of a transactivation domain in Pax-5d. To study B cell-specific changes that occur during the aging process, we investigated expression patterns of Pax-5a and 5d in mature B cells of young and aged mice. RNase protection assays showed a similar transcriptional pattern for both age groups that indicates that aging has no affect on transcription initiation or alternative splicing for either isoform. In contrast, a significant reduction in the DNA binding activity of Pax-5a but not Pax-5d protein was observed in aged B cells in vitro, while Western blot analyses showed that similar levels of Pax-5a and 5d proteins were present in both age groups. The observed decrease in Pax-5a binding activity correlated with changes in expression of two Pax-5 target genes in aged B cells. Expression of the Ig J chain and the secreted form of Ig μ, which are both known to be suppressed by Pax-5a in mature B cells, were increased in B cells of aged mice. Together, our studies suggest that changes associated with the aging phenotype cause posttranslational modification(s) of Pax-5a but not Pax-5d, which may lead to an abnormal B cell phenotype in aged mice, associated with elevated levels of J chain, and secretion of IgM.
IL-10 Is a Central Regulator of Cyclooxygenase-2 Expression and Prostaglandin Production
Authors: Berg Daniel J. , Zhang Juan , Lauricella Dina M. , Moore Steven A.
Published: April 2014

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10−/− mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10−/− mice. LPS-induced PGE2 production from IL-10−/− spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10−/− spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10−/− mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10−/− mice. Neutralization of IFN-γ, TNF-α, or IL-12 markedly decreased the induction of COX-2 in IL-10−/− spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10−/− mice. Treatment of IL-10−/− mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10’s central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.
NF-κB Regulation by IκB Kinase-2 in Rheumatoid Arthritis Synoviocytes
Authors: Aupperle Karlfried R. , Bennett Brydon L. , Han Zuoning , Boyle David L. , Manning Anthony M. , Firestein Gary S.
Published: April 2014

IκB kinase-1 and IκB kinase-2 (IKK1 and IKK2; also called IKKα and IKKβ, respectively) are part of the signal complex that regulates NF-κB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-κB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-κB pathway revealed that cytokine-induced IKK activation, IκB degradation, and NF-κB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-κB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-κB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-κB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.
Serum Amyloid A (apoSAA) Expression Is Up-Regulated in Rheumatoid Arthritis and Induces Transcription of Matrix Metalloproteinases
Authors: Vallon Rüdiger , Freuler Felix , Desta-Tsedu Netsanet , Robeva Anna , Dawson Janet , Wenner Peter , Engelhardt Petra , Boes Ludwig , Schnyder Jörg , Tschopp Claude , Urfer Roman , Baumann Götz
Published: April 2014

The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1β induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1β induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases’ transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium. A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.
Cutting Edge: The Class II Transactivator Prevents Activation-Induced Cell Death by Inhibiting Fas Ligand Gene Expression
Authors: Gourley Tania S. , Chang Cheong-Hee
Published: April 2014

The Fas:Fas ligand pathway is critical in regulating immune homeostasis by eliminating activated T cells that proliferated in response to an infection. Here, we show that the MHC class II transactivator (CIITA) can suppress this pathway by inhibiting transcription of the Fas ligand gene. CIITA can effectively repress transcription from the Fas ligand promoter in both T cell lines as well as primary cells. The repression appears to be at least partly due to interference of NFAT-mediated induction of Fas ligand gene transcription. T cells that express CIITA constitutively do not up-regulate Fas ligand on the cell surface after activation via the TCR. Consequently, these cells lack the ability to undergo activation-induced cell death, and to kill Fas-bearing target cells.
Fas-Mediated Apoptosis Causes Elimination of Virus-Specific Cytotoxic T Cells in the Virus-Infected Liver
Authors: Liu Zhang-Xu , Govindarajan Sugantha , Okamoto Shigefumi , Dennert Gunther
Published: April 2014

Immunity to allogeneic MHC Ags is weak in rodent livers, raising questions as to the mechanisms that might control responses in this organ. Infection with an adenovirus vector reveals that T cell-mediated immunity to nonself-Ags in the liver is self-limiting. Virus-induced liver injury decreases and coincides with disappearance of virus-specific CTL, concomitant to an increase of apoptotic T cells early after infection. But whereas death in CD4 cells is independent of Fas, perforin, and TNF-α, that of CD8 cells requires Fas and not perforin or TNF-α pathways. Fas ligand is expressed on liver-infiltrating cells, pointing to death by fratricide that causes almost complete disappearance of virus-specific CTL 4 wk after infection. CTL elimination is virus dose dependent, and high doses induced high alanine aminotransferase values, elevated expression of Fas ligand on CD8 cells, and increased CD8 cell migration into the infected liver.
Phenotypic Distinction and Functional Characterization of Pro-B Cells in Adult Mouse Bone Marrow
Authors: Mojica Mariluz P. , Perry S. Scott , Searles A. Elena , Elenitoba-Johnson Kojo S. J. , Pierce L. Jeanne , Wiesmann Anne , Slayton William B. , Spangrude Gerald J.
Published: April 2014

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1negSca-1posc-KitlowLinneg. These cells were CD43posCD24pos on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.
The Roles of MHC Class II, CD40, and B7 Costimulation in CTL Induction by Plasmid DNA
Authors: Chan Kee , Lee Delphine J. , Schubert Amy , Tang Chih Min , Crain Brian , Schoenberger Stephen P. , Corr Maripat
Published: April 2014

DNA-based vaccines generate potent CTL responses. The mechanism of T cell stimulation has been attributed to plasmid-transfected dendritic cells. These cells have also been shown to express plasmid-encoded proteins and to become activated by surface marker up-regulation. However, the increased surface expression of CD40 and B7 on these dendritic cells is insufficient to overcome the need for MHC class II-restricted CD4+ T cell help in the priming of a CTL response. In this study, MHC class II−/− mice were unable to generate a CTL response following DNA immunization. This deficit in CTL stimulation by MHC class II-deficient mice was only modestly restored with CD40-activating Ab, suggesting that there were other elements provided by MHC class II-restricted T cell help for CTL induction. CTL activity was also augmented by coinjection with a vector encoding the costimulatory ligand B7.1, but not B7.2. These data indicate that dendritic cells in plasmid DNA-injected mice require conditioning signals from MHC class II-restricted T cells that are both CD40 dependent and independent and that there are different roles for costimulatory molecules that may be involved in inducing optimal CTL activity.
The Production of IFN-γ by IL-12/IL-18-Activated Macrophages Requires STAT4 Signaling and Is Inhibited by IL-4
Authors: Schindler Heike , Lutz Manfred B. , Röllinghoff Martin , Bogdan Christian
Published: April 2014

Macrophages release IFN-γ on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-γ by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-γ production in NK cells, was not required for IFN-γ production by these macrophages. IL-12 alone already induced the expression of IFN-γ mRNA, but nuclear translocation of STAT4, the release of IFN-γ protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-κB, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-β) also suppressed macrophage IFN-γ production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-γ by macrophages and point to a diversity in the signals required for IFN-γ production by various cell types.
Proteasomes Modulate Balance Among Proapoptotic and Antiapoptotic Bcl-2 Family Members and Compromise Functioning of the Electron Transport Chain in Leukemic Cells
Authors: Marshansky Vladimir , Wang Xin , Bertrand Richard , Luo Hongyu , Duguid William , Chinnadurai G. , Kanaan Nada , Vu Minh Diem , Wu Jiangping
Published: April 2014

The mechanism underlying apoptosis induced by proteasome inhibition in leukemic Jurkat and Namalwa cells was investigated in this study. The proteasome inhibitor lactacystin differentially regulated the protein levels of proapoptotic Bcl-2 family members and Bik was accumulated at the mitochondria. Bik overexpression sufficed to induce apoptosis in these cells. Detailed examination along the respiration chain showed that lactacystin compromised a step after complex III, and exogenous cytochrome c could overcome this compromise. Probably as a result, the succinate-stimulated generation of mitochondrial membrane potential was significantly diminished. Bcl-xL interacted with Bik in the cells, and Bcl-xL overexpression prevented cytochrome c leakage out of the mitochondria, corrected the mitochondrial membrane potential defect, and protected the cells from apoptosis. These results show that proteasomes can modulate apoptosis of lymphocytes by affecting the half-life of Bcl-2 family members, Bik being one of them.
Regulation and Function of T1/ST2 Expression on CD4+ T Cells: Induction of Type 2 Cytokine Production by T1/ST2 Cross-Linking
Authors: Meisel Christian , Bonhagen Kerstin , Löhning Max , Coyle Anthony J. , Gutierrez-Ramos Jose-Carlos , Radbruch Andreas , Kamradt Thomas
Published: April 2014

The orphan receptor T1/ST2, a member of the IL-1R family, is preferentially expressed on the surface of murine Th2 cells. In this study, we analyzed the kinetics and function of T1/ST2 expression on Th2 cells in vitro. Whereas naive CD4+ cells did not express T1/ST2, most CD4+ cells became T1/ST2+ upon repeated antigenic stimulation under Th2-polarizing conditions. Flow cytometric analyses revealed that the kinetics of T1/ST2 expression on Th2 cells was delayed compared with the kinetics of type 2 cytokine production. Exogenous IL-6, IL-5, IL-1, and TNF-α enhanced the expression of T1/ST2 on Th2 cells, and IL-6 was by far most effective in this regard. However, the expression of T1/ST2 did not depend on the presence of IL-6 and was also detected in IL-6-deficient mice. Most important, cross-linking of T1/ST2 provided a costimulatory signal for Th2 but not Th1 cells and directly induced proliferation and type 2 cytokine production. Thus, T1/ST2 is not only a Th2 cell marker but also plays an important role in the activation of Th2 cells.
Single-Cell Analyses Reveal Two Defects in Peptide-Specific Activation of Naive T Cells from Aged Mice
Authors: Garcia Gonzalo G. , Miller Richard A.
Published: April 2014

Confocal fluorescent microscopy was used to study redistribution of membrane-associated proteins in naive T cells from young and old mice from a transgenic stock whose T cells express a TCR specific for a peptide derived from pigeon cytochrome C. About 50% of the T cells from young mice that formed conjugates with peptide-pulsed APC were found to form complexes, at the site of binding to the APC, containing CD3ε, linker for activation of T cells (LAT), and Zap-70 in a central area and c-Cbl, p95vav, Grb-2, PLCγ, Fyn, and Lck distributed more uniformly across the interface area. Two-color staining showed that those cells that were able to relocalize c-Cbl, LAT, CD3ε, or PLCγ typically relocalized all four of these components of the activation complex. About 75% of conjugates that rearranged LAT, c-Cbl, or PLCγ also exhibited cytoplasmic NF-AT migration to the T cell nucleus. Aging had two effects. First, it led to a diminution of ∼2-fold in the proportion of T cell/APC conjugates that could relocalize any of the nine tested proteins to the immune synapse. Second, aging diminished by ∼2-fold the frequency of cytoplasmic NF-AT migration among cells that could generate immune synapses containing LAT, c-Cbl, or PLCγ. Thus naive CD4 T cells from old mice exhibit at least two separable defects in the earliest stages of activation induced by peptide/MHC complexes.
The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites
Authors: Bladergroen Bellinda A. , Strik Merel C. M. , Bovenschen Niels , van Berkum Oskar , Scheffer George L. , Meijer Chris J. L. M. , Hack C. Erik , Kummer J. Alain
Published: April 2014

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.
CD95 Ligand-Expressing Tumors Are Rejected in Anti-Tumor TCR Transgenic Perforin Knockout Mice
Authors: Behrens Christian K. , Igney Frederik H. , Arnold Bernd , Möller Peter , Krammer Peter H.
Published: April 2014

CD95 (APO-/Fas) ligand (CD95L) is a member of the TNF family predominantly expressed by activated T and NK cells but also by tumors of diverse cellular origin. CD95L trimerizes surface CD95 expressed by target cells that subsequently undergo apoptosis. The role of the CD95/CD95L system in the down-regulation of an immune response (activation-induced cell death) is established. However, it is so far unclear why tumors express CD95L. To investigate whether tumors use the CD95L to down-regulate an anti-tumor immune response, we established a transgenic (tg) mouse model consisting of 1) apoptosis-resistant tumor cells, designated LKC-CD95L, which express functional CD95L and the model tumor Ag Kb; and 2) perforin knockout (PKO) anti-Kb TCR tg mice. L1210-Fas antisense expressing Kb, crmA, and CD95L (LKC-CD95L) killed CD95+ unrelated tumor targets and Con A-activated splenocytes from anti-Kb TCR tg PKO mice by a CD95L-dependent mechanism in vitro. However, we could not detect any cytotoxic activity against anti-tumor (anti-Kb) T cells in vivo. We also observed reduced growth of LKC-CD95L in nude mice and rapid rejection in anti-Kb TCR tg PKO mice. Because the tumor cells are resistant to CD95L-, TNF-α-, and TNF-related apoptosis-inducing ligand-induced apoptosis and the mice used are perforin-deficient, the involvement of these four cytotoxicity mechanisms in tumor rejection can be excluded. The histological examination of tumors grown in nude mice showed infiltration of LKC-CD95L tumors by neutrophils, whereas L1210-Fas antisense expressing Kb and crmA (LKC) tumor tissue was neutrophil-free. Chemotaxis experiments revealed that CD95L has no direct neutrophil-attractive activity. Therefore, we conclude that LKC-CD95L cells used an indirect mechanism to attract neutrophils that may cause tumor rejection.
MHC Class I-Related Neonatal Fc Receptor for IgG Is Functionally Expressed in Monocytes, Intestinal Macrophages, and Dendritic Cells
Authors: Zhu Xiaoping , Meng Gang , Dickinson Bonny L. , Li Xiaotong , Mizoguchi Emiko , Miao Lili , Wang Yuansheng , Robert Caroline , Wu Benyan , Smith Phillip D. , Lencer Wayne I. , Blumberg Richard S.
Published: April 2014

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the β2-microglobulin (β2m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical FcγRs: FcγRI, FcγRII, and FcγRIII.
Association of Mouse Mammary Tumor Virus Superantigen with MHC Class II During Biosynthesis
Authors: Hsu Ping-Ning , Bryant Paula Wolf , Sutkowski Natalie , McLellan Brian , Ploegh Hidde L. , Huber Brigitte T.
Published: April 2014

Mouse mammary tumor viruses encode superantigens that interact with MHC class II proteins and stimulate T cells. We show here that presentation of mouse mammary tumor virus superantigen does not require DM. Furthermore, we have identified a strong class II peptide binding motif in the Mtv-7 superantigen, and we show that this motif is necessary for association with class II molecules in in vitro translation and in vivo functional assays. Our results suggest that endogenously synthesized viral superantigen can bind to MHC class II heterodimers during biosynthesis in the endoplasmic reticulum in a manner analogous to that used by the class II-associated invariant chain.
Early Recruitment of Neutrophils Determines Subsequent T1/T2 Host Responses in a Murine Model ofLegionella pneumophilaPneumonia
Authors: Tateda Kazuhiro , Moore Thomas A. , Deng Jane C. , Newstead Michael W. , Zeng Xianying , Matsukawa Akihiro , Swanson Michele S. , Yamaguchi Keizo , Standiford Theodore J.
Published: April 2014

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice ∼100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-γ and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-γ and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-γ drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.
Hypercholesterolemia Exacerbates Virus-Induced Immunopathologic Liver Disease Via Suppression of Antiviral Cytotoxic T Cell Responses
Authors: Ludewig Burkhard , Jäggi Martin , Dumrese Tilman , Brduscha-Riem Karin , Odermatt Bernhard , Hengartner Hans , Zinkernagel Rolf M.
Published: April 2014

The immune system has to be optimally balanced to be highly effective against infections with cytopathic microbial pathogens and must guarantee efficient destruction of cells infected with noncytopathic agents while leaving the integrity of noninfected cells largely unaltered. We describe here the effects of genetically induced hypercholesterolemia on cellular immunity in apolipoprotein E (ApoE−/−) and low density lipoprotein receptor-deficient (LDLR−/−) mice during infection with the hepatotropic lymphocytic choriomeningitis virus WE strain. In both ApoE−/− and LDLR−/− mice hypercholesterolemia aggravated virus-induced immunopathologic liver disease. ApoE−/− mice exhibited a higher susceptibility to virus-induced immunopathology than LDLR−/− mice and usually succumbed to immunopathologic disease when infected with high doses of virus. Initial virus spread was not influenced by the hypercholesterolemia, whereas clearance of the virus from spleen and nonlymphoid organs, including liver, was delayed. Activation of antiviral CTL, measured by ex vivo cytotoxicity and IFN-γ production, and recruitment of specific CTL into blood and liver were impaired in hypercholesterolemic mice, indicating that hypercholesterolemia had a significant suppressive effect on cellular immunity. Taken together, these data provide evidence that hypercholesterolemia suppresses antiviral immune responses, thereby changing the host-virus balance, and can increase susceptibility to acute or chronic and potentially lethal virus-induced immunopathologic disease. These findings impinge on our understanding of hypercholesterolemia as a disease parameter and may explain aspects of the frequent association of persistent pathogens with hypercholesterolemia-induced diseases, such as atherosclerosis.
C5a and C5adesArg Enhance the Susceptibility of Monocyte-Derived Macrophages to HIV Infection
Authors: Kacani Laco , Bánki Zoltán , Zwirner Jörg , Schennach Harald , Bajtay Zsuzsa , Erdei Anna , Stoiber Heribert , Dierich Manfred P.
Published: April 2014

Mononuclear phagocytes, which include circulating blood monocytes and differentiated tissue macrophages, are believed to play a central role in the sexual transmission of HIV infection. The ability of HIV to productively infect these cells may be influenced by action of exogenous or host-derived substances at the site of viral entry. Given the potent capacities of inflammatory mediators to stimulate anaphylatoxic and immunomodulatory functions in mucosa, the effects of complement-derived anaphylatoxins on the susceptibility of monocytes and monocyte-derived macrophages (MDM) to HIV-1 infection were examined. In our in vitro system, the susceptibility to infection was up to 40 times increased in MDM that had been exposed to C5a or C5adesArg, but not to C3a or C3adesArg, for 2 days before adding of virus. By contrast, the treatment with complement anaphylatoxins did not affect HIV replication in fresh monocytes. Stimulatory effect of C5a and its desArg derivative on HIV infection correlated with the increase of TNF-α and IL-6 secretion from MDM. All these functional effects of C5a and C5adesArg were reversible by treatment of cells with the mAb that functionally blocks C5aR. Taken together, these results indicate that C5a and C5adesArg may increase the susceptibility of MDM to HIV infection through stimulation of TNF-α and IL-6 secretion from these cells.
Characterization of Hepatitis C Virus Core-Specific Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine
Authors: Polakos Noelle K. , Drane Debbie , Cox John , Ng Philip , Selby Mark J. , Chien David , O’Hagan Derek T. , Houghton Michael , Paliard Xavier
Published: April 2014

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8+ CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in ∼1-μm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4+ and CD8+ T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30–0.71 and 0.32–2.21% of the circulating CD8+ and CD4+ T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.
γδ T Cells Are Needed for Ocular Immune Privilege and Corneal Graft Survival
Authors: Skelsey Molly E. , Mellon Jessamee , Niederkorn Jerry Y.
Published: April 2014

It has been recognized for over a century that the anterior chamber of the eye is endowed with a remarkable immune privilege. One contributing component is the Ag-specific down-regulation of systemic delayed-type hypersensitivity (DTH) that is induced when Ags are introduced into the anterior chamber. This phenomenon, termed anterior chamber-associated immune deviation (ACAID), culminates in the generation of regulatory cells that inhibit the induction (afferent suppression) and expression (efferent suppression) of DTH. Since γδ T cells play a major role in other forms of immune regulation, we suspected they might contribute to the induction and expression of ACAID. Mice treated with anti-γδ Ab failed to develop ACAID following anterior chamber injection of either soluble Ag (OVA) or alloantigens (spleen cells). Additional experiments with knockout mice confirmed that mice lacking functional γδ T cells also fail to develop ACAID. Using a local adoptive transfer of DTH assay, we found that γδ T cells were required for the generation of regulatory T cells, but did not function as the efferent regulatory cells of ACAID. The importance of γδ T cells in corneal allograft survival was confirmed by blocking γδ T cells with GL3 Ab before corneal transplantation. While in vivo treatment with normal hamster serum had no effect on corneal graft survival, infusion of anti-γδ Ab resulted in a profound increase in corneal allograft rejection. Thus, γδ T cells are needed for sustaining at least one aspect of ocular immune privilege and for promoting corneal allograft survival.