bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Morphogenetic Protein 2/4 Signaling Regulates Early Thymocyte Differentiation
Authors: Hager-Theodorides Ariadne L. , Outram Susan V. , Shah Divya K. , Sacedon Rosa , Shrimpton Rachel E. , Vicente Angeles , Varas Alberto , Crompton Tessa
Published: April 2014

Bone morphogenetic protein (BMP)2 and BMP4 are involved in the development of many tissues. In this study, we show that BMP2/4 signaling is involved in thymocyte development. Our data suggest that termination of BMP2/4 signaling is necessary for differentiation of CD44+CD25−CD4−CD8− double negative (DN) cells along the T cell lineage. BMP2 and BMP4 are produced by the thymic stroma and the requisite BMP receptor molecules (BMPR-1A, BMPR-1B, BMPR-II), and signal transduction molecules (Smad-1, -5, -8, and -4) are expressed by DN thymocytes. BMP4 inhibits thymocyte proliferation, enhances thymocyte survival, and arrests thymocyte differentiation at the CD44+CD25− DN stage, before T cell lineage commitment. Neutralization of endogenous BMP2 and BMP4 by treatment with the antagonist Noggin promotes and accelerates thymocyte differentiation, increasing the expression of CD2 and the proportion of CD44−CD25− DN cells and CD4+CD8+ double-positive cells. Our study suggests that the BMP2/4 pathway may function in thymic homeostasis by regulating T cell lineage commitment and differentiation.
Effect of Suppressive DNA on CpG-Induced Immune Activation
Authors: Yamada Hiroshi , Gursel Ihsan , Takeshita Fumihiko , Conover Jackie , Ishii Ken J. , Gursel Mayda , Takeshita Saoko , Klinman Dennis M.
Published: April 2014

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5′→3′ direction, and that a 5′ motif can block recognition of immediately 3′ sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs.
Positional Scanning-Synthetic Peptide Library-Based Analysis of Self- and Pathogen-Derived Peptide Cross-Reactivity with Tumor-Reactive Melan-A-Specific CTL
Authors: Rubio-Godoy Verena , Dutoit Valérie , Zhao Yingdong , Simon Richard , Guillaume Philippe , Houghten Richard , Romero Pedro , Cerottini Jean-Charles , Pinilla Clemencia , Valmori Danila
Published: April 2014

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8+ T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.
CL-46, a Novel Collectin Highly Expressed in Bovine Thymus and Liver
Authors: Hansen Soren , Holm Dorte , Moeller Vivi , Vitved Lars , Bendixen Christian , Reid Kenneth B. M. , Skjoedt Karsten , Holmskov Uffe
Published: April 2014

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via α-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the α-helical neck region and the C-type lectin domain binds preferential N-acetyl-d-glucoseamine and N-acetyl-d-mannoseamine. The gene encoding CL-46 spans ∼10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5′-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.
Induction of HIV-1-Specific Immunity After Vaccination with Apoptotic HIV-1/Murine Leukemia Virus-Infected Cells
Authors: Spetz Anna-Lena , Sörensen Anna Smed , Walther-Jallow Lilian , Wahren Britta , Andersson Jan , Holmgren Lars , Hinkula Jorma
Published: April 2014

Ag-presenting dendritic cells present viral Ags to T cells after uptake of apoptotic bodies derived from virus-infected cells in vitro. However, it is unclear whether apoptotic virus-infected cells are capable of generating immunity in vivo. In this study, we show that inoculation of mice with apoptotic HIV-1/murine leukemia virus (MuLV)-infected cells induces HIV-1-specific immunity. Immunization with apoptotic HIV-1/MuLV-infected syngeneic splenocytes resulted in strong Nef-specific CD8+ T cell proliferation and p24-induced CD4+ and CD8+ T cell proliferation as well as IFN-γ production. In addition, systemic IgG and IgA as well as mucosa-associated IgA responses were generated. Moreover, mice vaccinated with apoptotic HIV-1/MuLV cells were protected against challenge with live HIV-1/MuLV-infected cells, whereas mice vaccinated with apoptotic noninfected or MuLV-infected splenocytes remained susceptible to HIV-1/MuLV. These data show that i.p. immunization with apoptotic HIV-1-infected cells induces high levels of HIV-1-specific systemic immunity, primes for mucosal immunity, and induces protection against challenge with live HIV-1-infected cells in mice. These findings may have implications for the development of therapeutic and prophylactic HIV-1 vaccines.
Resistance to Metastatic Disease in STAT6-Deficient Mice Requires Hemopoietic and Nonhemopoietic Cells and Is IFN-γ Dependent
Authors: Ostrand-Rosenberg Suzanne , Clements Virginia K. , Terabe Masaki , Park Jong Myun , Berzofsky Jay A. , Dissanayake Samudra K.
Published: April 2014

Mice deficient for the STAT6 gene (STAT6−/− mice) have enhanced immunosurveillance against primary and metastatic tumors. Because STAT6 is a downstream effector of the IL-4R, and IL-13 binds to the type 2 IL-4R, IL-13 has been proposed as an inhibitor that blocks differentiation of tumor-specific CD8+ T cells. Immunity in STAT6−/− mice is unusually effective in that 45–80% of STAT6−/− mice with established, spontaneous metastatic 4T1 mammary carcinoma, whose primary tumors are surgically excised, survive indefinitely, as compared with <10% of STAT+/+ (BALB/c) mice. Surprisingly, STAT6−/− and BALB/c reciprocal bone marrow chimeras do not have increased immunosurveillance, demonstrating that immunity requires STAT6−/− hemopoietic and nonhemopoietic components. Likewise, CD1−/− mice that are NKT deficient and therefore IL-13 deficient also have heightened tumor immunity. However, STAT6−/− and CD1−/− reciprocal bone marrow chimeras do not have increased survival, suggesting that immunity in STAT6−/− and CD1−/− mice is via noncomplementing mechanisms. Metastatic disease is not reduced in BALB/c mice treated with an IL-13 inhibitor, indicating that IL-13 alone is insufficient for negative regulation of 4T1 immunity. Likewise, in vivo depletion of CD4+CD25+ T cells in BALB/c mice does not increase survival, demonstrating that CD4+CD25+ cells do not regulate immunity. Cytokine production and tumor challenges into STAT6−/−IFN-γ−/− mice indicate that IFN-γ is essential for immunity. Therefore, immunosurveillance in STAT6−/− mice facilitates survival against metastatic cancer via an IFN-γ-dependent mechanism involving hemopoietic and nonhemopoietic derived cells, and is not exclusively dependent on counteracting IL-13 or CD4+CD25+ T cells.
Characterization of Nitric Oxide Consumption Pathways by Normal, Chronic Granulomatous Disease and Myeloperoxidase-Deficient Human Neutrophils
Authors: Clark Stephen R. , Coffey Marcus J. , Maclean Rhona M. , Collins Peter W. , Lewis Malcolm J. , Cross Andrew R. , O’Donnell Valerie B.
Published: April 2014

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H2O2 substrate since all superoxide (O2minusdu;) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O2minusdu; generation, significantly faster than expected from 1:1 termination of NO with O2minusdu; Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.
A Role for STAT5 in the Pathogenesis of IL-2-Induced Glucocorticoid Resistance
Authors: Goleva Elena , Kisich Kevin O. , Leung Donald Y. M.
Published: April 2014

Glucocorticoids (GC) are highly effective in the control of diseases associated with T cell activation. However, a subset of individuals is GC insensitive. Previous studies have demonstrated that IL-2 can induce steroid resistance in mouse T cells. However, the mechanism for this phenomenon is unknown. In the current study we found that the murine cell line (HT-2) is steroid resistant when incubated with IL-2, but steroid sensitive when grown in IL-4. Furthermore, when HT-2 cells are treated with IL-2, the glucocorticoid receptor (GCR) does not translocate to the cell nucleus after dexamethasone treatment. In contrast, the GCR in IL-4-stimulated HT-2 cells does translocate into the cell nucleus after dexamethasone treatment. IL-2-induced steroid insensitivity in HT-2 cells appears to be a signaling event as the effects of IL-2 on nuclear translocation of the GCR occurred within 30 min even in the presence of cycloheximide. Indeed, preincubation of HT-2 cells with a Janus-associated kinase 3 inhibitor restored nuclear translocation of the GCR even in the presence of IL-2. Immunoprecipitation experiments revealed that phosphorylated STAT5 and GCR formed immune complexes. This association may lead to retardation of GCR nuclear translocation because IL-2 was not able to induce steroid insensitivity in splenocytes from STAT5 knockout mice. This study demonstrates a novel role for STAT5 in IL-2-induced steroid insensitivity.
In Vivo Role of p38 Mitogen-Activated Protein Kinase in Mediating the Anti-inflammatory Effects of CpG Oligodeoxynucleotide in Murine Asthma
Authors: Choudhury Barun K. , Wild James S. , Alam Rafeul , Klinman Dennis M. , Boldogh Istvan , Dharajiya Nilesh , Mileski William J. , Sur Sanjiv
Published: April 2014

DNA containing unmethylated CpG motifs is intrinsically immunostimulatory, inducing the production of a variety of cytokines and chemokines by immune cells. The strong Th1 response triggered by CpG oligodeoxynucleotide (ODN) inhibits the development of Th2-mediated allergic asthma in mice. This work documents that CpG ODN-induced IL-12 production plays a critical role in this process, because intrapulmonary CpG ODN inhibits allergic inflammation in wild-type but not IL-12−/− mice. CpG ODN rapidly localized to alveolar macrophages (AM), thereby triggering the phosphorylation of p38 mitogen-activated protein kinase (MAP kinase). AM cultured with CpG but not control ODN up-regulated IL-12 p40 expression and release, and these effects were blocked by the highly specific p38 MAP kinase inhibitor SB202190. Intrapulmonary administration of this inhibitor blocked the ability of CpG ODN to produce IL-12 in the lungs and reversed the anti-inflammatory effects of CpG ODN on allergic lung inflammation. These findings indicate that IL-12 production by AM is stimulated by intrapulmonary CpG ODN administration through a p38 MAP kinase-dependent process, and IL-12 is a key cytokine that mediates CpG ODN-induced protection against allergic lung inflammation.
The Tissue-Specific Self-Pathogen Is the Protective Self-Antigen: The Case of Uveitis
Authors: Mizrahi Tal , Hauben Ehud , Schwartz Michal
Published: April 2014

Vaccination with peptides derived from interphotoreceptor retinoid-binding protein (a self-Ag that can cause experimental autoimmune uveoretinitis) resulted in protection of retinal ganglion cells from glutamate-induced death or death as a consequence of optic nerve injury. In the case of glutamate insult, no such protection was obtained by vaccination with myelin Ags (self-Ags associated with an autoimmune disease in the brain and spinal cord that evokes a protective immune response against consequences of injury to myelinated axons). We suggest that protective autoimmunity is the body’s defense mechanism against destructive self-compounds, and an autoimmune disease is the outcome of a failure to properly control such a response. Accordingly, the specific self-Ag (although not necessarily its particular epitopes) used by the body for protection against potentially harmful self-compounds (e.g., glutamate) can be inferred from the specificity of the autoimmune disease associated with the site at which the stress occurs (irrespectively of the type of stress) and is in need of help.
Tobacco Reduces Membrane HLA Class I That Is Restored by Transfection with Transporter Associated with Antigen Processing 1 cDNA
Authors: Fine Craig I. , Han C. David , Sun Xuming , Liu Yuexun , McCutcheon Jane A.
Published: April 2014

HLA class I molecules are recognized by CTL that eliminate virally infected and malignantly transformed cells presenting foreign peptide—a process termed immunosurveillance. Many tumors have reduced levels of membrane HLA class I. Tumor cells with mutations that reduce HLA class I avoid immunosurveillance and continue to proliferate. As tobacco use can induce tumors, we examined the effect of tobacco extracts on membrane HLA class I. These studies show that culture of cells in media containing tobacco extracts reduces membrane HLA class I, but not other proteins, on primary keratinocytes and other cell types. Culture in tobacco extracts, but not extracts of other substances, reduces TAP1 protein, but does not reduce expression of HLA class I H chain, L chain, or the housekeeping protein β-actin. The reduction of TAP1 protein occurs within 4 h and is dose-dependent. Culture in tobacco extracts reduces TAP1 protein abundance, but not steady-state mRNA abundance. Tobacco-treated cells show defects in HLA class I biosynthesis similar to those found in TAP1-deficient cell lines. Transfection with TAP1 cDNA restores TAP1 protein abundance, HLA class I biosynthesis, and cell surface expression. Combined, these data show that culture in tobacco extracts reduces TAP1 protein abundance and membrane HLA class I levels. Reduction in membrane HLA class I could permit subsequent malignant transformation of cells to be undetected by the immune system.
Cutting Edge: B7/CD28 Interactions Regulate Cell Cycle Progression Independent of the Strength of TCR Signaling
Authors: Bonnevier Jody L. , Mueller Daniel L.
Published: April 2014

The role of B7/CD28 signals in Ag-induced cell cycle progression of CD4+ T cells was examined using the technique of CFSE dye dilution and flow cytometry. In wild-type T cells, proliferation was directly related to the concentration of Ag available to the APC. Consistent with this, the rate of G0→G1 cell cycle progression varied with the concentration of Ag. However, cell division by T cell blasts occurred at a constant rate, independent of Ag concentration. G0→G1 phase progression by CD28-deficient CD4+ T cells or wild-type T cells cultured in the presence of neutralizing anti-B7 mAbs was slowed, confirming that a synergy does exist between TCR and CD28 signaling in the initial activation of the T cells. However, unlike the TCR, the strength of CD28 stimulation was also shown to play a unique role in controlling the rate of cell division by T cell blasts.
Cutting Edge: Distal Regulatory Elements Are Required to Achieve Selective Expression of IFN-γ in Th1/Tc1 Effector Cells
Authors: Soutto Mohammed , Zhou Weisong , Aune Thomas M.
Published: April 2014

Using a transgenic approach, we analyzed the contribution of introns located within the IFN-γ gene and distal regulatory regions to IFN-γ gene expression. Intron 1 and 3 from the IFN-γ gene displayed strong enhancer activity. This activity appeared to be dependent upon integration into the genome but resulted in a loss of Th1 selectivity. We also found that distal regulatory elements are not required for high level expression of the human IFN-γ gene, but rather for cell lineage-specific expression. An 8.6-kb human IFN-γ transgene was sufficient to yield high level expression but a 191-kb IFN-γ transgene with ∼90 kb of flanking 5′ and 3′ sequence was necessary to achieve both high level and Th1 selective expression of human IFN-γ.
Murine CD4 T Cells Selected in a Highly Disparate Xenogeneic Porcine Thymus Graft Do Not Show Rapid Decay in the Absence of Selecting MHC in the Periphery
Authors: Rodriguez-Barbosa Jose-Ignacio , Zhao Yong , Zhao Guiling , Ezquerra Angel , Sykes Megan
Published: April 2014

CD4 repopulation can be achieved in T cell-depleted, thymectomized mice grafted with xenogeneic porcine thymus tissue. These CD4 T cells are specifically tolerant of the xenogeneic porcine thymus donor and the recipient, but are positively selected only by porcine MHC. Recent studies suggest that optimal peripheral survival of naive CD4 T cells requires the presence of the same class II MHC in the periphery as that of the thymus in which they were selected. These observations would suggest that T cells selected on porcine thymic MHC would die rapidly in the periphery, where porcine MHC is absent. Persistent CD4 reconstitution achieved in mice grafted with fetal porcine thymus might be due to increased thymic output to compensate for rapid death of T cells in the periphery. Comparison of CD4 T cell decay after removal of porcine or murine thymic grafts ruled out this possibility. No measurable role for peripheral murine class II MHC in maintaining the naive CD4 pool originating in thymic grafts was demonstrable. However, mouse class II MHC supported the conversion to, survival, and/or proliferation of memory-type CD4 cells selected in fetal porcine thymus. Thus, the same MHC as that mediating positive selection in the thymus is not critical for maintenance of the memory CD4 cell pool in the periphery. Our results support the interpretation that xenogeneic thymic transplantation is a feasible strategy to reconstitute CD4 T cells and render recipients tolerant of a xenogeneic donor.
The Role of the MHC Class II Transactivator in Class II Expression and Antigen Presentation by Astrocytes and in Susceptibility to Central Nervous System Autoimmune Disease
Authors: Stüve Olaf , Youssef Sawsan , Slavin Anthony J. , King Chelsea L. , Patarroyo Juan Carlos , Hirschberg David L. , Brickey W. June , Soos Jeanne M. , Piskurich Janet F. , Chapman Harold A. , Zamvil Scott S.
Published: April 2014

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4+ T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4+ Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-γ-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-γ activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.
Porcine Endothelial Cells, Unlike Human Endothelial Cells, Can Be Killed by Human CTL Via Fas Ligand and Cannot Be Protected by Bcl-2
Authors: Zheng Lian , Ben Li-Hong , Pober Jordan S. , Bothwell Alfred L. M.
Published: April 2014

In clinical transplantation host CTL are major effectors of acute rejection, and graft endothelial cells (EC) are major targets of the CTL response. It is unclear what roles CTL will play in pig-into-human xenotransplantation. We compared the mechanisms of killing used by human CTL (huCTL) vs allogeneic and pig xenogeneic EC targets. Both responses show MHC class I restriction of target cell recognition. A granzyme B inhibitor peptide completely blocks anti-human and partially blocks anti-pig responses, while inhibitory Fas ligand Ab only blocks killing of porcine cells despite similar levels of Fas expression in both target cell types. Transduction of Bcl-2 completely protects human EC from huCTL, but has no effect on huCTL-mediated killing of porcine EC despite its efficacy vs drug-induced apoptosis. Bcl-2 effectively protects human EC rendered sensitive to Fas ligand by overexpressing Fas from huCTL, yet fails to protect porcine aortic endothelial cells from huCTL in the presence of anti-Fas ligand Ab. These data reveal differences in the susceptibility of human and porcine targets to huCTL.
The Activation and Subsequent Regulatory Roles of Lyn and CD19 After B Cell Receptor Ligation are Independent
Authors: Xu Yuekang , Beavitt Sarah-Jane E. , Harder Kenneth W. , Hibbs Margaret L. , Tarlinton David M.
Published: April 2014

The cell surface glycoprotein CD19 and the Src-related protein tyrosine kinase Lyn are key mediators of, respectively, positive and negative signaling in B cells. Despite the apparent opposition of their regulatory functions, a recent model of the biochemical events after B cell receptor (BCR) ligation intimately links the activation of Lyn and CD19. We examined the biochemical consequences of BCR ligation in mouse B cells lacking either Lyn or CD19 for evidence of interaction or codependence. In contrast to published results, we found CD19 phosphorylation after BCR ligation to be unaffected by the absence of Lyn, yet dependent on Src family protein tyrosine kinases as it was inhibited fully by PP2, an Src family-specific inhibitor. Consistent with normal CD19 phosphorylation in lyn−/− B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and extracellular signal-regulated kinase 1/2 activation after coligation with the BCRs were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. CD19−/− B cells were normal for increased Lyn kinase activity after BCR ligation, inhibition of BCR-mediated calcium flux after CD22 coligation, and inhibition of extracellular signal-regulated kinase phosporylation after FcγRIIB coligation. Collectively, these data show that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. We conclude that the roles of Lyn and CD19 after BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory.
Repression ofrac2mRNA Expression byAnaplasma phagocytophilaIs Essential to the Inhibition of Superoxide Production and Bacterial Proliferation
Authors: Carlyon Jason A. , Chan Wai-Tsing , Galán Jorge , Roos Dirk , Fikrig Erol
Published: April 2014

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O2−), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91phox, an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91phox from the CMV immediate early promoter maintained the ability to generate O2− 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91phox transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.
Use of Single Point Mutations in Domain I of β2-Glycoprotein I to Determine Fine Antigenic Specificity of Antiphospholipid Autoantibodies
Authors: Iverson G. Michael , Reddel Stephen , Victoria Edward J. , Cockerill Keith A. , Wang Ying-Xia , Marti-Renom Marc A. , Sali Andrej , Marquis David M. , Krilis Steven A. , Linnik Matthew D.
Published: April 2014

Autoantibodies against β2-glycoprotein I (β2GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in β2GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole β2GPI containing single point mutations in the first domain. The binding to β2GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40–43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-β2GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-β2GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40–43 contributes to a dominant target epitope.
APCs in the Liver and Spleen Recruit Activated Allogeneic CD8+ T Cells to Elicit Hepatic Graft-Versus-Host Disease
Authors: Zhang Yi , Shlomchik Warren D. , Joe Gerard , Louboutin Jean-Pierre , Zhu Jiang , Rivera Adam , Giannola Diane , Emerson Stephen G.
Published: April 2014

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8+ T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8+ T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8+ T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8+ T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.
Inhibition of Allergic Inflammation in the Airways Using Aerosolized Antisense to Syk Kinase
Authors: Stenton Grant R. , Ulanova Marina , Déry René E. , Merani Shaheed , Kim Moo-Kyung , Gilchrist Mark , Puttagunta Lakshmi , Musat-Marcu Sorin , James Deborah , Schreiber Alan D. , Befus A. Dean
Published: April 2014

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of FcγR and FcεR, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of β2 integrins, α4 integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.
Critical Role for CXCR3 Chemokine Biology in the Pathogenesis of Bronchiolitis Obliterans Syndrome
Authors: Belperio John A. , Keane Michael P. , Burdick Marie D. , Lynch Joseph P. , Xue Ying Ying , Li Kewang , Ross David J. , Strieter Robert M.
Published: April 2014

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival post-lung transplantation and is characterized by a persistent peribronchiolar inflammation that eventually gives way to airway fibrosis/obliteration. Acute rejection is the main risk factor for the development of BOS and is characterized by a perivascular/bronchiolar leukocyte infiltration. The specific mechanism(s) by which these leukocytes are recruited have not been elucidated. The CXC chemokines (monokine induced by IFN-γ (MIG)/CXC chemokine ligand (CXCL)9, IP-10/CXCL10, and IFN-inducible T cell α chemoattractant (ITAC)/CXCL11) act through their shared receptor, CXCR3. Because they are potent leukocyte chemoattractants and are involved in other inflammation/fibroproliferative diseases, we hypothesized that the expression of these chemokines during an allogeneic response promotes the persistent recruitment of mononuclear cells, leading to chronic lung rejection. We found that elevated levels of MIG/CXCL9, IFN-inducible protein 10 (IP-10)/CXCL10, and ITAC/CXCL11 in human bronchoalveolar lavage fluid were associated with the continuum from acute to chronic rejection. Translational studies in a murine model demonstrated increased expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 paralleling the recruitment of CXCR3-expressing mononuclear cells. In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated BOS. This supports the notion that ligand/CXCR3 biology plays an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of BOS.
Prevention and Induction of Autoimmune Exocrinopathy Is Dependent on Pathogenic Autoantigen Cleavage in Murine Sjögren’s Syndrome
Authors: Saegusa Kaoru , Ishimaru Naozumi , Yanagi Kumiko , Mishima Kenji , Arakaki Rieko , Suda Takashi , Saito Ichiro , Hayashi Yoshio
Published: April 2014

The in vivo role of autoantigen cleavage during apoptosis in autoimmune diseases remains unclear. Previously, we found a cleavage product of 120-kDa α-fodrin as an important autoantigen in the pathogenesis of primary Sjögren’s syndrome (SS). In the murine primary SS model, tissue-infiltrating CD4+ T cells purified from the salivary glands bear a large proportion of Fas ligand, and the salivary gland duct cells constitutively possess Fas. Infiltrating CD4+ T cells, but not CD8+ T cells, identified significant 51Cr release against mouse salivary gland cells. In vitro studies demonstrated that apoptotic mouse salivary gland cells result in a specific α-fodrin cleavage into 120 kDa and that preincubation with caspase inhibitor peptides blocked α-fodrin cleavage. In vivo treatment with caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and N-acetyl-Asp-Glu-Val-Asp-al-CHO into the murine model results in dramatic inhibitory effects on the development of autoimmune lesions and in restoration of sicca syndrome. Furthermore, we found that immunization with recombinant α-fodrin protein identical with an autoantigen into normal recipients induced autoimmune lesions similar to SS. These data indicate that prevention and induction of autoimmune exocrinopathy is dependent on autoantigen cleavage via caspase cascade and that caspase inhibitors might provide a new therapeutic option directed at reducing tissue damage in the murine model for SS.
“Superhumanized” Antibodies: Reduction of Immunogenic Potential by Complementarity-Determining Region Grafting with Human Germline Sequences: Application to an Anti-CD28
Authors: Tan Philip , Mitchell David A. , Buss Timothy N. , Holmes Margaret A. , Anasetti Claudio , Foote Jefferson
Published: April 2014

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.
Cutting Edge: CCR7+ and CCR7− Memory T Cells Do Not Differ in Immediate Effector Cell Function
Authors: Unsoeld Heike , Krautwald Stefan , Voehringer David , Kunzendorf Ulrich , Pircher Hanspeter
Published: April 2014

It has been proposed that expression of the chemokine receptor CCR7 represents a defining factor for nonpolarized central (CCR7+) and polarized effector memory (CCR7−) T cells. In this study, we have tested this hypothesis using in vivo-activated T cells from P14 and SMARTA TCR-transgenic (tg) mice specific for MHC class I- and II-restricted epitopes of the lymphocytic choriomeningitis virus (LCMV) glycoprotein. CCR7 cell surface expression on TCR-tg cells was monitored with a CC chemokine ligand 19-Ig fusion protein. CC chemokine ligand 19-Ig staining separated TCR-tg cells activated by LCMV infection into CCR7− and CCR7+ effector/memory T cell populations. Nonetheless, both T cell populations isolated from spleen and liver produced identical amounts of IFN-γ after short-term Ag stimulation. Furthermore, CCR7+ and CCR7− CD8 TCR-tg cells from LCMV-infected mice exhibited similar lytic activity against LCMV peptide-coated target cells. These results question the proposed concept of differential effector cell function of CCR7+ and CCR7− memory T cells.
Cutting Edge: Th2 Cell Trafficking into the Allergic Lung Is Dependent on Chemoattractant Receptor Signaling
Authors: Mathew Anuja , Medoff Benjamin D. , Carafone Andrew D. , Luster Andrew D.
Published: April 2014

Th2 cells are recruited to the lung where they mediate the asthma phenotype. Since the molecular mechanisms regulating Th2 cell trafficking remain unknown, we sought to determine whether trafficking of Th2 cells into the lung is mediated by Gαi-coupled chemoattractant receptors. We show here that in contrast to untreated Th2 cells, pertussis toxin-treated Th2 cells were unable to traffic into the lung, airways, or lymph nodes following Ag challenge and therefore were unable to induce allergic inflammation in vivo. Pertussis toxin-treated Th2 cells were functional cells, however, and when directly instilled into the airways of mice, bypassing their need to traffic to the lung, were able to induce airway eosinophilic inflammation. These studies conclusively demonstrate that trafficking of Th2 cells into the lung is an active process dependent on chemoattractant receptors.
Endothelial Cell Apoptosis Induced by Antibodies Against Dengue Virus Nonstructural Protein 1 Via Production of Nitric Oxide
Authors: Lin Chiou-Feng , Lei Huan-Yao , Shiau Ai-Li , Liu Hsiao-Sheng , Yeh Trai-Ming , Chen Shun-Hua , Liu Ching-Chuan , Chiu Shu-Chen , Lin Yee-Shin
Published: April 2014

The onset of vascular leakage and hemorrhagic diathesis is one of the life-threatening complications occurring in dengue patients, yet the pathogenic mechanisms are not well understood. In this study, we demonstrated that Abs against dengue virus nonstructural protein 1 (NS1) generated in mice cross-reacted with human endothelial cells and mouse vessel endothelium. After binding, mouse anti-NS1 Abs induced endothelial cell apoptosis in a caspase-dependent manner. Inducible NO synthase expression could be observed; it showed a time- and dose-dependent correlation with NO production. Endothelial cell apoptosis, characterized by exposure of phosphatidylserine on the cell surface and nuclear DNA fragmentation, was blocked by treatment with the NO synthase inhibitor Nω-nitro-l-arginine methyl ester. Further studies demonstrated that the expression of Bcl-2 and Bcl-xL decreased in both mRNA and protein levels, whereas p53 and Bax increased after anti-NS1 treatment. Cytochrome c release was also observed. All of these effects could be inhibited by Nω-nitro-l-arginine methyl ester. Taken together, anti-NS1 Abs act as autoantibodies that cross-react with noninfected endothelial cells and trigger the intracellular signaling leading to the production of NO and to apoptosis. Endothelial cell damage may cause vascular leakage that contributes to the pathogenesis of dengue disease.
Platelet Factor 4 Inhibits Proliferation and Cytokine Release of Activated Human T Cells
Authors: Fleischer Jens , Grage-Griebenow Evelin , Kasper Brigitte , Heine Holger , Ernst Martin , Brandt Ernst , Flad Hans-Dieter , Petersen Frank
Published: April 2014

Platelet factor 4 (PF-4), a platelet-derived CXC chemokine, has been shown to induce the differentiation of monocytes into a subset of macrophages that lack the expression of HLA-DR Ag. This suggests a potential role for PF-4 in the modulation of monocyte-dependent T cell activation. Using an Ag-specific stimulation model in which T cells were cocultured with monocytes in the presence of recall Ags, we could show that under these conditions PF-4-treatment caused a strong decrease of T cell proliferation as well as of IFN-γ release. However, inhibition of T cell functions such as proliferation, IL-2 release, and IL-2 mRNA production did also occur when isolated T cells were activated in the absence of monocytes with immobilized Abs directed against CD3 in combination with cross-linked anti-CD28 Abs. The effect could be reversed when low concentrations of exogenous IL-2 instead of anti-CD28 were used as a costimulus in combination with anti-CD3 Abs. Further evidence for direct modulation of T cell function by PF-4 was obtained by the detection of specific binding sites for the chemokine on the surface of these cells. Taken together, our results show that specific binding of PF-4, resulting in the down-regulation of the IL-2-release correlates with the inhibition of functions in activated T cells.
Inhibition of Antigen-Specific T Cell Proliferation and Cytokine Production by Protein Kinase A Type I
Authors: Aandahl Einar Martin , Moretto Walter J. , Haslett Patrick A. , Vang Torkel , Bryn Tone , Tasken Kjetil , Nixon Douglas F.
Published: April 2014

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4+ and CD8+ T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.
Immobilized Lactoferrin Is a Stimulus for Eosinophil Activation
Authors: Thomas Larry L. , Xu Wei , Ardon Tamir T.
Published: April 2014

Eosinophils are strongly implicated in the pathogenesis of asthma, particularly in damage to the airway epithelial lining. We examined the potential for lactoferrin, a multifunctional glycoprotein present in the airway surface liquid, to activate eosinophils. Incubating eosinophils in tissue culture wells pretreated with 1–100 μg/ml human lactoferrin stimulated concentration-dependent superoxide production by eosinophils. The same concentrations of immobilized transferrin were without effect. The potency of immobilized lactoferrin was approximately one-third that of immobilized secretory IgA in the same experiments. In contrast, immobilized lactoferrin did not stimulate neutrophil superoxide production. Eosinophils bound lactoferrin as determined by flow cytometry and by binding of 125I-labeled lactoferrin. Transferrin did not block binding of 125I-labeled lactoferrin. Soluble lactoferrin, however, did not activate the eosinophils and did not block superoxide production stimulated by immobilized lactoferrin. Immobilized lactoferrin also stimulated release of eosinophil-derived neurotoxin and low levels of leukotriene C4 production; the latter was significantly enhanced in the presence of 100 pg/ml GM-CSF. GM-CSF also enhanced superoxide production and eosinophil-derived neurotoxin release stimulated by the lower concentrations of immobilized lactoferrin. Pretreatment of the lactoferrin with peptide N-glycosidase F or addition of heparin or chondroitin sulfate to the incubation contents had no or only a minimal effect on the activity of immobilized lactoferrin. These results demonstrate that lactoferrin adherent to the surface epithelium may contribute to the activation of eosinophils that infiltrate the airway lumen in eosinophil-associated disorders such as asthma.
Cutting Edge: Fyn Is Essential for Tyrosine Phosphorylation of Csk-Binding Protein/Phosphoprotein Associated with Glycolipid-Enriched Microdomains in Lipid Rafts in Resting T Cells
Authors: Yasuda Koubun , Nagafuku Masakazu , Shima Takaki , Okada Masato , Yagi Takeshi , Yamada Takenao , Minaki Yasuko , Kato Akiko , Tani-ichi Shizue , Hamaoka Toshiyuki , Kosugi Atsushi
Published: April 2014

In resting T cells, Csk is constitutively localized in lipid rafts by virtue of interaction with a phosphorylated adaptor protein, Csk-binding protein (Cbp)/phosphoprotein associated with glycolipid-enriched microdomains, and sets an activation threshold in TCR signaling. In this study, we examined a kinase responsible for Cbp phosphorylation in T cell membrane rafts. By analyzing T cells from Fyn−/− mice, we clearly demonstrated that Fyn, but not Lck, has its kinase activity in membrane rafts, and plays a critical role in Cbp phosphorylation, Cbp-Csk interaction, and Csk kinase activity. Naive CD44lowCD62 ligandhigh T cells were substantially reduced in Fyn−/− mice, presumably due to the inhibition of Cbp phosphorylation. Thus, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts by phosphorylating Cbp. Csk recruited to rafts would then be activated and inhibit the kinase activity of Lck to keep resting T cells in a quiescent state. Our results elucidate a negative regulatory role for Fyn in proximal TCR signaling in lipid rafts.