bluuebunny/crossref_metadata_2025 huggingface dataset:
Exosomes Derived from IL-10-Treated Dendritic Cells Can Suppress Inflammation and Collagen-Induced Arthritis
Authors: Kim Seon-Hee , Lechman Eric R. , Bianco Nicole , Menon Rajasree , Keravala Annahita , Nash Joan , Mi Zhibao , Watkins Simon C. , Gambotto Andrea , Robbins Paul D.
Published: April 2014

We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular administration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune diseases.
A Critical Role for Sphingosine Kinase in Anaphylatoxin-Induced Neutropenia, Peritonitis, and Cytokine Production in Vivo
Authors: Vlasenko Liudmila Pietrovna , Melendez Alirio J.
Published: April 2014

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-α and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-α and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.
Comparison of Killer Ig-Like Receptor Genotyping and Phenotyping for Selection of Allogeneic Blood Stem Cell Donors
Authors: Leung Wing , Iyengar Rekha , Triplett Brandon , Turner Victoria , Behm Frederick G. , Holladay Marti S. , Houston James , Handgretinger Rupert
Published: April 2014

The repertoire of killer Ig-like receptors (KIRs) can be determined at the level of DNA, RNA, or surface protein expression for selection of blood stem cell donors. We compared genotyping and phenotyping of the four inhibitory KIRs that are important in transplantation for leukemia in 73 unrelated persons. In 5 (7%) of the 68 individuals in whom the KIR2DL1 gene was present and in 10 (15%) of the 67 in whom KIR3DL1 was present, the corresponding receptor was not expressed by NK cells, as determined by flow cytometry analysis. In contrast, one or both allelic forms of KIR2DL2/KIR2DL3 were expressed by a high proportion of NK cells in all 73 individuals. However if both KIR2DL2 and KIR2DL3 genes were present, KIR2DL3 was preferentially expressed, as transcripts of KIR2DL2 was not detectable by RT-PCR in 42% of these individuals. In total, repertoire assessment for the four KIRs by genotyping vs phenotyping was not in complete agreement in 18 (25%) of the 73 individuals. Furthermore, among the samples that tested positive for the expression of a certain KIR gene, the levels of transcripts and surface expression varied considerably as measured by both real-time quantitative PCR and flow cytometry analysis. Extension of this comparative analysis to include all 12 KIR family members showed that KIR2DL3 and KIR3DL2 were the only genes whose transcripts were consistently detectable. These results caution the use of genotyping alone for donor selection or leukemia-relapse prognostication because some KIRs may be expressed at a very low level.
B Cell Heterogeneity in the Teleost Kidney: Evidence for a Maturation Gradient from Anterior to Posterior Kidney
Authors: Zwollo Patty , Cole Suzanne , Bromage Erin , Kaattari Stephen
Published: April 2014

The fish immune system is quite different from the mammalian system because the anterior kidney forms the main site for hematopoiesis in this species. Using transcription factor-specific Abs derived from the murine system, together with anti-trout Ig Abs and Percoll gradient separation, we analyzed B cells from trout kidney sections and compared them to those from spleen and blood. For this study, immune cells were separated by Percoll gradients, and the resulting subpopulations were defined based on expression of B cell-specific transcription factors Pax-5 and B lymphocyte-induced maturation protein-1, as well as proliferative and Ig-secreting properties. Comparison of kidney, blood, and spleen B cell subsets suggest that 1) the anterior kidney contains mostly proliferating B cell precursors and plasma cells; 2) posterior kidney houses significant populations of (partially) activated B cells and plasmablasts; and 3) trout blood contains resting, non-Ig-secreting cells and lacks plasma cells. After LPS induction of resting B cells in vitro, the kidney and spleen have a high capacity for the generation of plasma cells, whereas the blood has virtually none. Our results indicate that trout B cell subsets are profoundly different among blood, anterior kidney, posterior kidney, and spleen. We hypothesize that developing B cells mature in the anterior side of the kidney and then migrate to sites of activation, either the spleen or the posterior kidney. Lastly, our data support the notion that the trout kidney is a complex, multifunctional immune organ with the potential to support both hemopoiesis as well as humoral immune activation.
Rapid Conversion of Effector Mechanisms from NK to T Cells during Virus-Induced Lysis of Allogeneic Implants In Vivo
Authors: Brehm Michael A. , Daniels Keith A. , Ortaldo John R. , Welsh Raymond M.
Published: April 2014

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2d) splenocytes but not CBA (H2k) splenocytes, and the rejection was prevented by immunodepletion of NK1.1+ or Ly49D+ NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2k) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.
Identification of Noncanonical Melanoma-Associated T Cell Epitopes for Cancer Immunotherapy
Authors: Bredenbeck Anne , Losch Florian O. , Sharav Tumenjargal , Eichler-Mertens Mathias , Filter Matthias , Givehchi Alireza , Sterry Wolfram , Wrede Paul , Walden Peter
Published: April 2014

The identification of tumor-associated T cell epitopes has contributed significantly to the understanding of the interrelationship of tumor and immune system and is instrumental in the development of therapeutic vaccines for the treatment of cancer. Most of the known epitopes have been identified with prediction algorithms that compute the potential capacity of a peptide to bind to HLA class I molecules. However, naturally expressed T cell epitopes need not necessarily be strong HLA binders. To overcome this limitation of the available prediction algorithms we established a strategy for the identification of T cell epitopes that include suboptimal HLA binders. To this end, an artificial neural network was developed that predicts HLA-binding peptides in protein sequences by taking the entire sequence context into consideration rather than computing the sum of the contribution of the individual amino acids. Using this algorithm, we predicted seven HLA A*0201-restricted potential T cell epitopes from known melanoma-associated Ags that do not conform to the canonical anchor motif for this HLA molecule. All seven epitopes were validated as T cell epitopes and three as naturally processed by melanoma tumor cells. T cells for four of the new epitopes were found at elevated frequencies in the peripheral blood of melanoma patients. Modification of the peptides to the canonical sequence motifs led to improved HLA binding and to improved capacity to stimulate T cells.
CD85j (Leukocyte Ig-Like Receptor-1/Ig-Like Transcript 2) Inhibits Human Osteoclast-Associated Receptor-Mediated Activation of Human Dendritic Cells
Authors: Tenca Claudya , Merlo Andrea , Merck Estelle , Bates Elizabeth E. M. , Saverino Daniele , Simone Rita , Zarcone Daniela , Trinchieri Giorgio , Grossi Carlo E. , Ciccone Ermanno
Published: April 2014

Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated γ-chain, induced Ca2+ flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.
A Defect in Lineage Fate Decision during Fetal Thymic Invariant NKT Cell Development May Regulate Susceptibility to Type 1 Diabetes
Authors: Wagner Melany J. D. , Hussain Shabbir , Mehan Mala , Verdi Joseph M. , Delovitch Terry L.
Published: April 2014

A numerical and functional deficiency in invariant NKT (iNKT) cells detectable by 3 wk of age in the thymus and spleen mediates the pathogenesis of type 1 diabetes in NOD mice, but the stage of T cell development at which this deficiency first occurs is unknown. We report in this study that this deficiency develops after the CD4+CD8+ double-positive stage of thymic T cell development and is due to a lineage-specific depletion of CD4−CD8− double-negative αβ T cells and iNKT cells from the thymus between embryonic day 18 and day 1 after birth. Thus, an inheritable defect in a lineage fate decision that elicits a deficiency in fetal thymic iNKT cell development may predispose to susceptibility to type 1 diabetes.
Regulation of IFN-γ Production by B Effector 1 Cells: Essential Roles for T-bet and the IFN-γ Receptor
Authors: Harris David P. , Goodrich Stephen , Gerth Andrea J. , Peng Stanford L. , Lund Frances E.
Published: April 2014

This manuscript systematically identifies the molecular mechanisms that regulate the ability of B cells to produce the critical type 1 cytokine, IFN-γ. B cells produce IFN-γ in response to IL-12 and IL-18 and when primed by Th1 cells. We show that development of IFN-γ-producing B cells by either Th1 cells or IL-12/IL-18 is absolutely dependent on expression of the IFN-γR and the T-box transcription factor, T-bet. Interestingly, although T-bet up-regulation in developing B effector 1 (Be1) cells is controlled by IFN-γR-mediated signals, STAT1-deficient B cells up-regulate T-bet and produce IFN-γ, indicating that additional transcriptional activators must be coupled to the IFN-γR in B cells. Finally, we show that although IL-12/IL-18 or IFN-γ-producing Th1 cells are required to initiate transcription of the IFN-γ gene in B cells, sustained expression of IFN-γ and T-bet by B cells is dependent on an IFN-γ/IFN-γR/T-bet autocrine feedback loop. These findings have significant implications, because they suggest that IFN-γ-producing B cells not only amplify Th1 responses, but also imprint a type 1 phenotype on B cells themselves. In the case of immune responses to bacterial or viral pathogens, this B cell-driven autocrine feedback loop is likely to be beneficial; however, in the case of B cell responses to autoantigens, it may result in amplification of the autoimmune loop and increased pathology.
Soluble MHC-Peptide Complexes Induce Rapid Death of CD8+ CTL
Authors: Cebecauer Marek , Guillaume Philippe , Hozák Pavel , Mark Silke , Everett Helen , Schneider Pascal , Luescher Immanuel F.
Published: April 2014

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8+ CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.
The Role of Protein Kinase A Anchoring via the RIIα Regulatory Subunit in the Murine Immune System
Authors: Schillace Robynn V. , Andrews Sarah F. , Galligan Sarah G. , Burton Kimberly A. , Starks Holly J. , Bouwer H. G. Archie , McKnight G. Stanley , Davey Michael P. , Carr Daniel W.
Published: April 2014

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIα regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIα regulatory subunit of PKA (−/−) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and −/− mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the −/− mice are similar to WT controls. In vitro, splenocytes from −/− mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8+ IFN-γ producing effector cells were stimulated upon infection of WT or −/− mice with Listeria monocytogenes. These data represent the first study of the role of RIIα in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIα −/− mice, suggesting either that RIIα is not required for normal immune function or that other proteins are able to compensate for RIIα function.
Sequential Immune Escape and Shifting of T Cell Responses in a Long-Term Survivor of Melanoma
Authors: Yamshchikov Galina V. , Mullins David W. , Chang Chien-Chung , Ogino Takeshi , Thompson Lee , Presley Jennifer , Galavotti Holly , Aquila William , Deacon Donna , Ross William , Patterson James W. , Engelhard Victor H. , Ferrone Soldano , Slingluff Craig L.
Published: April 2014

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8+ T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient’s spontaneous T cell response adapted, gaining the ability to recognize and to lyse “edited” tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.
CD8+ T Cell Dynamics during Primary Simian Immunodeficiency Virus Infection in Macaques: Relationship of Effector Cell Differentiation with the Extent of Viral Replication
Authors: Monceaux Valérie , Viollet Laurence , Petit Frédéric , Fang Raphaël Ho Tsong , Cumont Marie-Christine , Zaunders John , Hurtrel Bruno , Estaquier Jérôme
Published: April 2014

Immunological and virological events that occur during the earliest stages of HIV-1 infection are now considered to have a major impact on subsequent disease progression. We observed changes in the frequencies of CD8bright T cells expressing different chemokine receptors in the peripheral blood and lymph nodes of rhesus macaques during the acute phase of the pathogenic SIVmac251 infection; the frequency of CD8bright T cells expressing CXCR4 decreased, while the frequency of those expressing CCR5 increased. These reciprocal changes in chemokine receptor expression were associated with changes in the proportion of cycling (Ki67+) CD8bright T cells, and with the pattern of CD8bright T cell differentiation as defined by expression of CCR7 and CD45RA. In contrast, during the primary phase of the attenuated SIVmac251Δnef infection, no major change was observed. Whereas during the acute phase of the infection with pathogenic SIV (2 wk postinfection) no correlate of disease protection was identified, once the viral load set points were established (2 mo postinfection), we found that the levels of cycling and of CCR5- and CXCR4-positive CD8bright T cells were correlated with the extent of viral replication and therefore with SIV-infection outcome. Our data reveal that, during primary SIV infection, despite intense CD8 T cell activation and an increase in CCR5 expression, which are considered as essential for optimal effector function of CD8+ T cells, these changes are associated with a poor prognosis for disease progression to AIDS.
Association of B-1 B Cells with Follicular Dendritic Cells in Spleen
Authors: Wen Lijun , Shinton Susan A. , Hardy Richard R. , Hayakawa Kyoko
Published: April 2014

Although CD5+ B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied VH11/DH/JH gene-targeted mice, VH11t, that develop elevated numbers of CD5+ VH11/Vk9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgDhigh follicular B cells in adults. Among neonatal B cells, CD5+ B-1 cells in both normal and VH11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.
Cross-Desensitization among CXCR1, CXCR2, and CCR5: Role of Protein Kinase C-ε
Authors: Nasser Mohd W. , Marjoram Robin J. , Brown Stephan L. , Richardson Ricardo M.
Published: April 2014

The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKCα, -β1, and -β2 to the membrane, but only CXCR1 activated PKCε. CXCL8 also failed to activate PKCε in RBL-2H3 cells stably expressing CXCR2. ΔCXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKCε as well as CXCR1. Expression of the PKCε inhibitor peptide εV1 in RBL-2H3 cells blocked PKCε translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization. εV1 also inhibited CXCR1-, CCR5-, and ΔCXCR2-mediated cross-regulatory signals for GTPase activity, Ca2+ mobilization, and internalization. Peritoneal macrophages from PKCε-deficient mice (PKCε−/−) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca2+ mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKCε to mediate cross-inhibitory signals. Inhibition or deletion of PKCε decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization, suggesting that cross-regulation is a Ca2+-independent process. Because ΔCXCR2, but not CXCR2, activates PKCε and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.
The Antiapoptotic Protein Bcl-xL Is Dispensable for the Development of Effector and Memory T Lymphocytes
Authors: Zhang Nu , He You-Wen
Published: April 2014

The antiapoptotic protein Bcl-xL is induced in activated T lymphocytes upon costimulation through CD28, 4-1BB, and OX40. Bcl-xL is also highly enriched in memory T lymphocytes. Based on this body of evidence, it was thought that Bcl-xL plays an essential role in the generation of effector and memory T lymphocytes. We report that mice with a conditional deletion of Bcl-x in T lymphocytes develop a normal CD8+ T cell response to Listeria monocytogenes infection. Furthermore, Bcl-x conditional knockout mice exhibit normal T-dependent humoral immune responses. These results indicate that Bcl-x is dispensable for the generation of effector and memory T lymphocytes and suggest that costimulation of T lymphocytes promotes their survival through a Bcl-xL independent mechanism.
C5a-Mediated Leukotriene B4-Amplified Neutrophil Chemotaxis Is Essential in Tumor Immunotherapy Facilitated by Anti-Tumor Monoclonal Antibody and β-Glucan
Authors: Allendorf Daniel J. , Yan Jun , Ross Gordon D. , Hansen Richard D. , Baran Jarek T. , Subbarao Krishnaprasad , Wang Li , Haribabu Bodduluri
Published: April 2014

Intravenous and orally administered β-glucans promote tumor regression and survival by priming granulocyte and macrophage C receptor 3 (CR3, iC3bR and CD11b/CD18) to trigger the cytotoxicity of tumor cells opsonized with iC3b via anti-tumor Abs. Despite evidence for priming of macrophage CR3 by oral β-glucan in vivo, the current study in C57BL/6 and BALB/c mice showed that granulocytes were the essential killer cells in mAb- and oral β-glucan-mediated tumor regression, because responses were absent in granulocyte-depleted mice. Among granulocytes, neutrophils were the major effector cells, because tumor regression did not occur when C5a-dependent chemotaxis was blocked with a C5aR antagonist, whereas tumor regression was normal in C3aR−/− mice. Neutrophil recruitment by C5a in vivo required amplification via leukotriene B4, because both C5a-mediated leukocyte recruitment into the peritoneal cavity and tumor regression were suppressed in leukotriene B4R-deficient (BLT-1−/−) mice.
Reduction of Spontaneous Metastases through Induction of Carbohydrate Cross-Reactive Apoptotic Antibodies
Authors: Monzavi-Karbassi Behjatolah , Artaud Cecile , Jousheghany Fariba , Hennings Leah , Carcel-Trullols Jaime , Shaaf Saeid , Korourian Soheila , Kieber-Emmons Thomas
Published: April 2014

The selective targeting of tumor-associated carbohydrate Ags by the induction of serum Abs that trigger apoptosis of tumor cells as a means to reduce circulating tumor cells and micrometastases would be an advantage in cancer vaccine development. Some plant lectins like Griffonia simplicifolia lectin I and wheat germ agglutinin mediate the apoptosis of tumor cells. We investigated the possibility of using these lectins as templates to select peptide mimotopes of tumor-associated carbohydrate Ags as immunogens to generate cross-reactive Abs capable of mediating apoptosis of tumor cells. In this study, we show that immunization with a mimotope selected based on its reactivity with Griffonia simplicifolia lectin I and wheat germ agglutinin induced serum IgM Abs in mice that mediated the apoptosis of murine 4T1 and human MCF7 cell lines in vitro, paralleling the apoptotic activity of the lectins. Vaccine-induced anti-carbohydrate Abs reduced the outgrowth of micrometastases in the 4T1 spontaneous tumor model, significantly increasing survival time of tumor-bearing animals. This finding parallels suggestions that carbohydrate-reactive IgM with apoptotic activity may have merit in the adjuvant setting if the right carbohydrate-associated targets are identified.
In Silico Identification of Supertypes for Class II MHCs
Authors: Doytchinova Irini A. , Flower Darren R.
Published: April 2014

The development of epitope-based vaccines, which have wide population coverage, is greatly complicated by MHC polymorphism. The grouping of alleles into supertypes, on the basis of common structural and functional features, addresses this problem directly. In the present study we applied a combined bioinformatics approach, based on analysis of both protein sequence and structure, to identify similarities in the peptide binding sites of 2225 human class II MHC molecules, and thus define supertypes and supertype fingerprints. Two chemometric techniques were used: hierarchical clustering using three-dimensional Comparative Similarity Indices Analysis fields and nonhierarchical k-means clustering using sequence-based z-descriptors. An average consensus of 84% was achieved, i.e., 1872 of 2225 class II molecules were classified in the same supertype by both techniques. Twelve class II supertypes were defined: five DRs, three DQs, and four DPs. The HLA class II supertypes and their fingerprints given in parenthesis are DR1 (Trp9β), DR3 (Glu9β, Gln70β, and Gln/Arg74β), DR4 (Glu9β, Gln/Arg70β, and Glu/Ala74β), DR5 (Glu9β, Asp70β), and DR9 (Lys/Gln9β); DQ1 (Ala/Gly86β), DQ2 (Glu86β, Lys71β), and DQ3 (Glu86β, Thr/Asp71β); DPw1 (Asp84β and Lys69β), DPw2 (Gly/Val84β and Glu69β), DPw4 (Gly/Val84β and Lys69β), and DPw6 (Asp84β and Glu69β). Apart from the good agreement between known binding motifs and our classification, several new supertypes, and corresponding thematic binding motifs, were also defined.
Influence of Maternal-Fetal Histocompatibility and MHC Zygosity on Maternal Microchimerism
Authors: Kaplan Joseph , Land Susan
Published: April 2014

To investigate the relationship between maternal-fetal histocompatibility and maternal microchimerism, we developed a sensitive quantitative PCR assay for the neomycin resistance gene (neoR), and, in a mouse model system, used neoR as a noninherited maternal allele marker of maternal cells to detect and quantitate maternal microchimerism in tissues of neoR−/− N2 backcross progeny of (neoR+/−)F1 females mated with neoR−/− males. Using this approach, we obtained evidence for the presence of chimeric maternal cells in the brain, spleen, and thymus of all weanling and adult mice so tested. The numbers of chimeric maternal cells present in the spleen did not differ significantly from those in the thymus regardless of age or maternal-fetal histocompatibility. At all ages, brain tissue had higher level of maternal microchimerism than lymphoid tissue in mice MHC identical with their mothers, but the levels were similar in mice MHC disparate with their mothers. The levels of chimeric maternal cells in both brain and lymphoid tissue of mice with homozygous syngenicity and maternal allogenicity were similar, and tended to be higher than tissue-specific levels in mice with either combined maternal-fetal allogenicity or heterozygous syngenicity. Thus, MHC homozygous progeny had higher levels of maternal microchimerism than MHC heterozygous progeny. We conclude that normal mice possess small numbers of maternal cells in spleen, thymus, brain, and probably most other tissues, and that maternal-fetal histocompatibility influences the levels of these cells by mechanisms related to MHC zygosity of the progeny.
Adoptive Transfer of In Vitro-Stimulated CD4+CD25+ Regulatory T Cells Increases Bacterial Clearance and Improves Survival in Polymicrobial Sepsis
Authors: Heuer Josef G. , Zhang Tonghai , Zhao Jingyong , Ding Chunjin , Cramer Martin , Justen Kathy L. , Vonderfecht Steven L. , Na Songqing
Published: April 2014

Regulatory CD4+CD25+ T cells (Tregs) suppress autoimmune and inflammatory diseases through mechanisms that are only partly understood. Previous studies suggest that Tregs can suppress bacterially triggered intestinal inflammation and respond to LPS through TLRs with enhanced suppressive activity. In this study, we have used murine cecal ligation and puncture as a model of polymicrobial sepsis to explore the effects of adoptive transfer of Tregs on septic outcome. Adoptive transfer of in vitro-stimulated Tregs in both prevention and therapeutic modes significantly improved survival of cecal ligation and puncture mice. Furthermore, the effect was dependent on both the number of Tregs adoptively transferred and the presence of host T cells. Animals that received stimulated Tregs had significantly increased peritoneal mast cells and peritoneal TNF-α production. More importantly, adoptive transfer of in vitro-stimulated Tregs significantly improved bacterial clearance, which resulted in improved survival. Our results suggest a novel role for Tregs in sepsis.
IL-4-Transfected Tumor Cell Vaccines Activate Tumor-Infiltrating Dendritic Cells and Promote Type-1 Immunity
Authors: Eguchi Junichi , Kuwashima Naruo , Hatano Manabu , Nishimura Fumihiko , Dusak Jill E. , Storkus Walter J. , Okada Hideho
Published: April 2014

We previously demonstrated that IL-4 gene-transfected glioma cell vaccines induce effective therapeutic immunity in preclinical glioma models, and have initiated phase I trials of these vaccines in patients with malignant gliomas. To gain additional mechanistic insight into the efficacy of this approach, we have treated mice bearing the MCA205 (H-2b) or CMS-4 (H-2d) sarcomas. IL-12/23 p40−/− and IFN-γ−/− mice, which were able to reject the initial inoculation of IL-4 expressing tumors, failed to mount a sustained systemic response against parental (nontransfected) tumor cells. Paracrine production of IL-4 in vaccine sites promoted the accumulation and maturation of IL-12p70-secreting tumor-infiltrating dendritic cells (TIDCs). Adoptive transfer of TIDCs isolated from vaccinated wild-type, but not IL-12/23 p40−/−, mice were capable of promoting tumor-specific CTL responses in syngeneic recipient animals. Interestingly, both STAT4−/− and STAT6−/− mice failed to reject IL-4-transfected tumors in concert with the reduced capacity of TIDCs to produce IL-12p70 and to promote specific antitumor CTL reactivity. These results suggest that vaccines consisting of tumor cells engineered to produce the type 2 cytokine, IL-4, critically depend on type 1 immunity for their observed therapeutic efficacy.
Overexpression of Stat3C in Pulmonary Epithelium Protects against Hyperoxic Lung Injury
Authors: Lian Xuemei , Qin Yulin , Hossain Shaikh Abu , Yang Li , White Amanda , Xu Huan , Shipley J. Michael , Li Tingyu , Senior Robert M. , Du Hong , Yan Cong
Published: April 2014

Acute lung injury is a side effect of therapy with a high concentration of inspired oxygen in patients. The molecular mechanism underlining this effect is poorly understood. In this study, we report that overexpression of Stat3C, a constitutive active form of STAT3, in respiratory epithelial cells of a doxycycline-controlled double-transgenic mouse system protects lung from inflammation and injury caused by hyperoxia. In this mouse line, >50% of transgenic mice survived exposure to 95% oxygen at day 7, compared with 0% survival of wild-type mice. Overexpression of STAT3C delays acute capillary leakage and neutrophil infiltration into the alveolar region. This protection is mediated at least partially through inhibition of hyperoxia-induced synthesis and release of matrix metalloproteinase (MMP)-9 and MMP-12 by neutrophils and alveolar resident cells. In some MMP-9−/− mice, prolonged survival was observed under hyperoxic condition. The finding supports a concept that activation of the Stat3 pathway plays a role to prevent hyperoxia-induced inflammation and injury in the lung.
Intrinsic Resistance of Hepatocytes to Complement-Mediated Injury
Authors: Koch Cody A. , Kanazawa Akiyoshi , Nishitai Ryuta , Knudsen Bruce E. , Ogata Kiyoshi , Plummer Timothy B. , Butters Kim , Platt Jeffrey L.
Published: April 2014

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.
Respiratory Syncytial Virus Infection of Human Lung Endothelial Cells Enhances Selectively Intercellular Adhesion Molecule-1 Expression
Authors: Arnold Ralf , König Wolfgang
Published: April 2014

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1α, IL-1β, IL-6, and TNF-α, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.
Flt3 Ligand Treatment Reverses Endotoxin Tolerance-Related Immunoparalysis
Authors: Wysocka Maria , Montaner Luis J. , Karp Christopher L.
Published: April 2014

Endotoxin tolerance, the secondary blunting of a subset of microbial product-driven responses, is presumed to provide protection from pathological hyperactivation of the innate immune system during infection. However, endotoxin tolerance can itself be harmful. A significant percentage of sepsis survivors exhibit the phenotype of systemic endotoxin tolerance, a state termed immunoparalysis. Similar immune hyporeactivity, associated with an elevated risk of succumbing to bacterial superinfection, is also seen in the aftermath of major trauma, surgery, and burns. We recently demonstrated that in vivo endotoxin tolerance in murine models involves dendritic cell loss as well as alterations in the responsiveness of macrophages and remaining dendritic cells. Furthermore, the kinetics of recovery from immunoparalysis-associated inhibition of proinflammatory and immunoregulatory cytokine production directly parallels the kinetics of dendritic cell repopulation in these models. Given this, we examined whether recovery from immunoparalysis could be accelerated therapeutically with flt3 ligand, a growth factor that stimulates the differentiation and mobilization of dendritic cells. Notably, administration of flt3 ligand rapidly reverses immunoparalysis in vivo, accelerating and amplifying repopulation of tissues with proinflammatory and immunoregulatory cytokine-producing dendritic cells.
Role of Fibrinogen-Like Protein 2 Prothrombinase/Fibroleukin in Experimental and Human Allograft Rejection
Authors: Ning Qin , Sun Yi , Han Meifang , Zhang Li , Zhu Chuanglong , Zhang Weijie , Guo Hui , Li Jinwen , Yan Weiming , Gong Feili , Chen Zhonghua , He William , Koscik Cheryl , Smith Robert , Gorczynski Reginald , Levy Gary , Luo Xiaoping
Published: April 2014

Immune coagulation is a major contributor to the pathogenesis of xenograft rejection, viral-induced hepatocellular injury and cytokine-induced fetal loss syndrome. In this study, we investigated the contribution of the novel gene product, fibrinogen-like protein 2 (fgl2) prothrombinase, in mediating immune injury in experimental and human acute allograft rejection. Using a mouse heterotopic cardiac transplant model, mouse fgl2(mfgl2)/fibroleukin mRNA transcripts and protein were highly expressed in macrophages, CD4- and CD8-positive T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Although mfgl2-deficient mice rejected allografts at similar rates to littermate controls, survival of grafts from mfgl2-deficient mice were prolonged and deposition of intravascular fibrin was diminished. Treatment of wild-type mice with a neutralizing anti-fgl2 Ab ameliorated histological evidence for allorejection and intravascular fibrin deposition, and resulted in an increase in graft survival. To address further the relevance of fgl2 in acute allograft rejection, we examined kidney biopsies from patients who had undergone renal transplantation. Human fgl2 mRNA transcripts and protein were markedly expressed mainly in renal tubule cells, infiltrating lymphoid cells including macrophages, CD8+ T cells, mature B cells (plasma cells), and endothelial cells. Dual staining showed fibrin deposition was localized mainly to blood vessels, in the glomerulus and interstitium and the lumen of tubules, and occurred in association with human fgl2 expression. These data collectively suggest that fgl2 accounts for the fibrin deposition seen in both experimental and human allograft rejection and provide a rationale for targeting fgl2 as adjunctive therapy to treat allograft rejection.
Modular Transcriptional Activity Characterizes the Initiation and Progression of Autoimmune Encephalomyelitis
Authors: Baranzini Sergio E. , Bernard Claude C. A. , Oksenberg Jorge R.
Published: April 2014

Murine experimental autoimmune encephalomyelitis is a well-established model that recapitulates many clinical and physiopathological aspects of multiple sclerosis (MS). An important conceptual development in the understanding of both experimental autoimmune encephalomyelitis and MS pathogenesis has been the compartmentalization of the mechanistic process into two distinct but overlapping and connected phases, inflammatory and neurodegenerative. However, the dynamics of CNS transcriptional changes that underlie the development and regression of the phenotype are not well understood. Our report presents the first high frequency longitudinal study looking at the earliest transcriptional changes in the CNS of NOD mice immunized with myelin oligodendrocyte glycoprotein 35–55 in CFA. Microarray-based gene expression profiling and histopathological analysis were performed from spinal cord samples obtained at 13 time points around the first clinical symptom (every other day until day 11 and every day onward until day 19 postimmunization). Advanced statistics and data-mining algorithms were used to identify expression signatures that correlated with disease stage and histological profiles. Discrete phases of neuroinflammation were accompanied by distinctive expression signatures, in which altered immune to neural gene expression ratios were observed. By using high frequency gene expression analysis we captured expression profiles that were characteristic of the transition from innate to adaptive immune response in this experimental paradigm between days 11 and 12 postimmunization. Our study demonstrates the utility of large-scale transcriptional studies and advanced data mining to decipher complex biological processes such as those involved in MS and other neurodegenerative disoders.
Infections and Autoimmunity—Good or Bad?
Authors: Christen Urs , von Herrath Matthias G.
Published: April 2014

The relationship between infections and autoimmunity is complex. Current evidence indicates that microbes can initiate, enhance, or, conversely, abrogate autoimmunity. In this paper, we will review experimental examples illustrating mechanisms involved in these three scenarios. Microbial infections can act as environmental triggers inducing or promoting autoimmunity resulting in clinical manifestations of autoimmune disease in genetically predisposed individuals. However, increasing evidence suggests the opposite outcome, which is the prevention or amelioration of autoimmune processes following microbial encounters. These latter observations support conceptually the “hygiene hypothesis,” suggesting that cleaner living conditions will lead to enhanced incidence of autoimmune disorders, asthma, and allergies. Because proof of concept in humans is difficult to obtain, we will discuss relevant animal model data in context with likely or proven human associations. Knowledge of mechanisms that underlie either positive or negative effects of infections on autoimmunity will facilitate exploration of molecular details for prospective clinical studies in the future.
Cutting Edge: CD4+CD25+ Regulatory T Cells Impaired for Intestinal Homing Can Prevent Colitis
Authors: Denning Timothy L. , Kim Gisen , Kronenberg Mitchell
Published: April 2014

Transfer of CD4+CD45RBhigh T cells into RAG−/− mice causes colitis, which can be prevented by CD4+CD25+ regulatory T cells (Treg). Colitis induction by CD4+CD45RBhigh T cells requires β7 integrin-dependant intestinal localization, but the importance of β7 integrins for Treg function is unknown. In this study, we show that β7−/− Treg were effective in preventing colitis. Treg expanded in vivo to the same extent as CD4+CD45RBhigh T cells after transfer and they did not inhibit CD4+CD45RBhigh T cell expansion in lymphoid tissues, although they prevented the accumulation of Th1 effector cells in the intestine. β7−/− Treg were significantly reduced in the large intestine, however, compared with wild-type Treg, and regulatory activity could not be recovered from the intestine of recipients of β7−/− Treg. These data demonstrate that Treg can prevent colitis by inhibiting the accumulation of tissue-seeking effector cells and that Treg accumulation in the intestine is dispensable for colitis suppression.