bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Human Cell Engraftment and Development of EBV-Related Lymphoproliferative Disorders in Hu-PBL-scidMice
Authors: Wagar Eric J. , Cromwell Mandy A. , Shultz Leonard D. , Woda Bruce A. , Sullivan John L. , Hesselton RuthAnn M. , Greiner Dale L.
Published: April 2014

Human PBMC engraft in mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation (Hu-PBL-scid mice). Hu-PBL-NOD-scid mice generate 5- to 10-fold higher levels of human cells than do Hu-PBL-C.B-17-scid mice, and Hu-PBL-NOD-scid β2-microglobulin-null (NOD-scid-B2mnull) mice support even higher levels of engraftment, particularly CD4+ T cells. The basis for increased engraftment of human PBMC and the functional capabilities of these cells in NOD-scid and NOD-scid-B2mnull mice are unknown. We now report that human cell proliferation in NOD-scid mice increased after in vivo depletion of NK cells. Human cell engraftment depended on CD4+ cells and required CD40-CD154 interaction, but engrafted CD4+ cells rapidly became nonresponsive to anti-CD3 Ab stimulation. Depletion of human CD8+ cells led to increased human CD4+ and CD20+ cell engraftment and increased levels of human Ig. We further document that Hu-PBL-NOD-scid mice are resistant to development of human EBV-related lymphoproliferative disorders. These disorders, however, develop rapidly following depletion of human CD8+ cells and are prevented by re-engraftment of CD8+ T cells. These data demonstrate that 1) murine NK cells regulate human cell engraftment in scid recipients; 2) human CD4+ cells are required for human CD8+ cell engraftment; and 3) once engrafted, human CD8+ cells regulate human CD4+ and CD20+ cell expansion, Ig levels, and outgrowth of EBV-related lymphoproliferative disorders. We propose that the Hu-PBL-NOD-scid model is suitable for the in vivo analysis of immunoregulatory interactions between human CD4+ and CD8+ cells.
Functional Reconstitution of Class II MHC-Restricted T Cell Immunity Mediated by Retroviral Transfer of the αβ TCR Complex
Authors: Fujio Keishi , Misaki Yoshikata , Setoguchi Keigo , Morita Sumiyo , Kawahata Kimito , Kato Ikunoshin , Nosaka Tetsuya , Yamamoto Kazuhiko , Kitamura Toshio
Published: April 2014

Transfer of the αβ TCR genes into T lymphocytes will provide a means to enhance Ag-specific immunity by increasing the frequency of tumor- or pathogen-specific T lymphocytes. We generated an efficient αβ TCR gene transfer system using two independent monocistronic retrovirus vectors harboring either of the class II MHC-restricted α or β TCR genes specific for chicken OVA. The system enabled us to express the clonotypic TCR in 44% of the CD4+ T cells. The transduced cells showed a remarkable response to OVA323–339 peptide in the in vitro culture system, and the response to the Ag was comparable with those of the T lymphocytes derived from transgenic mice harboring OVA-specific TCR. Adoptive transfer of the TCR-transduced cells in mice induced the Ag-specific delayed-type hypersensitivity in response to OVA323–339 challenge. These results indicate that αβ TCR gene transfer into peripheral T lymphocytes can reconstitute Ag-specific immunity. We here propose that this method provides a basis for a new approach to manipulation of immune reactions and immunotherapy.
Heparan Sulfate-Like Proteoglycans Mediate Adhesion of Human Malignant Melanoma A375 Cells to P-Selectin Under Flow
Authors: Ma Yan-Qing , Geng Jian-Guo
Published: April 2014

Selectins, a family of cell adhesion molecules, bind to sialylated and fucosylated carbohydrates, such as sialyl Lewisx (SLex) and its derivatives, as their minimal recognition motif. Here we report that P-selectin bound to human malignant melanoma A375 cells and mediated their adhesion under flow. However, probing with a specific Ab failed to detect any apparent expression of SLex. This finding was bolstered by reduced expression of α-1,3-fucosyltransferase VII mRNA and by absence of the cell surface expression of P-selectin glycoprotein ligand-1. Instead, they expressed heparan sulfate-like proteoglycans on their cell surfaces. Treatment with β-d-xyloside (a proteoglycan biosynthesis inhibitor) or heparinases could reduce the binding of these cells to P-selectin. In the competition assays, heparin, but not other proteoglycans, could abolish the P-selectin recognition. Further, we found that P-selectin could bind specifically to human tongue squamous cancer Tca-8113 cells, which had negative staining of SLex but positive staining of heparan sulfates. Both β-d-xyloside and heparinases could reduce the binding of P-selectin to Tca-8113 cells. Our results thus indicate that heparan sulfate-like proteoglycans can mediate adhesion of certain types of non-blood borne, “epithelial-like” human cancer cells to P-selectin.
Clonal Expansion of Infiltrating T Cells in the Spinal Cords of SJL/J Mice Infected with Theiler’s Virus
Authors: Kang Jeong-Ah , Mohindru Mani , Kang Bong-Su , Park Seol Hee , Kim Byung S.
Published: April 2014

Intracerebral infection of susceptible mice with Theiler’s murine encephalomyelitis virus results in immune-mediated inflammatory demyelination in the white matter and consequent clinical symptoms. This system has been utilized as an important virus model for human multiple sclerosis. Although the potential involvement of virus-specific Th cells has been studied extensively, very little is known about the nature of T cells infiltrating the CNS during viral infection and their role in the development of demyelinating disease. In this study, the clonal nature of T cells in the spinal cord during the disease course was analyzed using size spectratyping and sequencing of the TCR β-chain CDR3 region. These studies clearly indicate that T cells are clonally expanded in the CNS after viral infection, although the overall TCR repertoire appears to be diverse. The clonal expansion appears to be Ag-driven in that it includes Th cells specific for known viral epitopes. Interestingly, such restricted accumulation of T cells was not detectable in the infiltrates of mice with proteolipid protein peptide-induced experimental autoimmune encephalomyelitis. The initial T cell repertoire (7–9 days postinfection) seems to be more diverse than that observed in the later stage (65 days) of virally induced demyelination, despite the more restricted utilization of Vβ subfamilies. These results strongly suggest continuous stimulation and clonal expansion of virus-specific T cells in the CNS of Theiler’s murine encephalomyelitis virus-infected mice during the entire course of demyelinating disease.
Subtle Effects on Myelin Basic Protein-Specific T Cell Responses Can Lead to a Major Reduction in Disease Susceptibility in Experimental Allergic Encephalomyelitis
Authors: Vowles Cheryl , Chan Vera S. F. , Bodmer Helen C.
Published: April 2014

The presence of potentially autoreactive T cells is a necessary, but not sufficient, condition for the development of autoimmune disease. However, the relationship between T cell response and susceptibility to disease is not straightforward. In this report, we use experimental allergic encephalomyelitis as a model to demonstrate that subtle alterations of the T cell response to an encephalitogenic epitope are sufficient to cause a dramatic decrease in disease susceptibility. Transgenic expression of a fusion protein of hen egg lysozyme and an encephalitogenic peptide of myelin basic protein (MBP) residues 84–105, coexpressed with MHC class II, causes profound tolerance to hen egg lysozyme, while maintaining a near normal response to MBP. Detailed analysis of the T cell repertoire of transgenic animals using a panel of T cell hybridomas revealed a highly selective loss of one minor component of the response to the MBP84–104 region. Despite this, transgenic animals were highly resistant to experimental allergic encephalomyelitis induction with the MBP peptide, indicating that minor changes to the T cell repertoire may result in major alterations in disease susceptibility. Possible reasons for this are discussed.
Differential Role of p38 and c-Jun N-Terminal Kinase 1 Mitogen-Activated Protein Kinases in NK Cell Cytotoxicity
Authors: Trotta Rossana , Fettucciari Katia , Azzoni Livio , Abebe Bekele , Puorro Kristin A. , Eisenlohr Laurence C. , Perussia Bice
Published: April 2014

The serine-threonine mitogen-activated protein kinase (MAPK) family includes extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases. In NK cells, spontaneous or Ab-mediated recognition of target cells leads to activation of an ERK-2 MAPK-dependent biochemical pathway(s) involved in the regulation of NK cell effector functions. Here we assessed the roles of p38 and JNK MAPK in NK cell-mediated cytotoxicity. Our data indicate that p38 is activated in primary human NK cells upon stimulation with immune complexes and interaction with NK-sensitive target cells. FcγRIIIA-induced granule exocytosis and both spontaneous and Ab-dependent cytotoxicity were reduced in a dose-dependent manner in cells pretreated with either of two specific inhibitors of this kinase. Target cell-induced IFN-γ and FcγRIIIA-induced TNF-α mRNA accumulation was similarly affected under the same conditions. Lack of inhibition of NK cell cytotoxicity in cells overexpressing an inactive form of JNK1 indicates that this kinase, activated only upon FcγRIIIA ligation, does not play a significant role in cytotoxicity. These data underscore the involvement of p38, but not JNK1, in the molecular mechanisms regulating NK cell cytotoxicity.
Response of Murine γδ T Cells to the Synthetic Polypeptide Poly-Glu50Tyr50 1
Authors: Cady Carol T. , Lahn Michael , Vollmer Michaelann , Tsuji Moriya , Seo Seong Jun , Reardon Christopher L. , O’Brien Rebecca L. , Born Willi K.
Published: April 2014

Random heterocopolymers of glutamic acid and tyrosine (pEY) evoke strong, genetically controlled immune responses in certain mouse strains. We found that pE50Y50 also stimulated polyclonal proliferation of normal γδ, but not αβ, T cells. Proliferation of γδ T cells did not require prior immunization with this Ag nor the presence of αβ T cells, but was enhanced by IL-2. The γδ T cell response proceeded in the absence of accessory cells, MHC class II, β2-microglobulin, or TAP-1, suggesting that Ag presentation by MHC class I/II molecules and peptide processing are not required. Among normal splenocytes, as with γδ T cell hybridomas, the response was strongest with Vγ1+ γδ T cells, and in comparison with related polypeptides, pE50Y50 provided the strongest stimulus for these cells. TCR gene transfer into a TCR-deficient αβ T cell showed that besides the TCR, no other components unique to γδ T cells are needed. Furthermore, interactions between only the T cells and pE50Y50 were sufficient to bring about the response. Thus, pE50Y50 elicited a response distinct from those of T cells to processed/presented peptides or superantigens, consistent with a mechanism of Ig-like ligand recognition of γδ T cells. Direct stimulation by ligands resembling pE50Y50 may thus selectively evoke contributions of γδ T cells to the host response.
Distinct Molecular Mechanisms of Fas Resistance in Murine B Lymphoma Cells
Authors: Mueller Carolyn M. , Scott David W.
Published: April 2014

A panel of murine B lymphoma cell lines, which express different levels of Fas, was extensively studied for sensitivity to Fas-mediated death signals via an anti-Fas mAb and Fas ligand-bearing cell lines. Expression of the Fas receptor on the B lymphoma cell lines did not correlate with their capacity to undergo Fas-mediated apoptosis. Moreover, Fas-associated death domain protein recruitment to the death-inducing signaling complex (DISC) complex occurred in all cell lines expressing Fas, regardless of whether they were sensitive to Fas-mediated death. Interestingly, the protein synthesis inhibitor, cycloheximide, and protein kinase C inhibitors, such as bisindolylmaleimide, rendered one of the resistant cell lines, CH33, sensitive to signals from the Fas receptor, although the levels of Fas were unchanged. This suggests that constitutive PKC activation plays a role in Fas resistance, perhaps by up-regulating NF-κB or Bcl-2 family members. Interestingly, CH33 demonstrated caspase 8 activity upon engagement of the Fas receptor in the absence of pharmacological manipulation, suggesting that the block in apoptosis is downstream of the DISC complex. In contrast, the fact that Fas-associated death domain protein was recruited to the DISC complex in other resistant lines, such as WEHI-231, with no caspase 8 activation indicates that these cells may be blocked within the DISC complex. Indeed, Western blot analysis showed that WEHI-231 expressed an isoform of FLICE-like inhibitory protein (cFLIPL), an antiapoptotic protein within the DISC. These studies provide evidence that murine B lymphoma cells utilize different molecular mechanisms along the Fas-signaling cascade to block apoptosis.
Opening a Window on Thymic Positive Selection: Developmental Changes in the Influence of Cosignaling by Integrins and CD28 on Selection Events Induced by TCR Engagement
Authors: Lucas Bruno , Germain Ronald N.
Published: April 2014

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.
αβTCR+ Cells Are a Minimal Fraction of Peripheral CD8+ Pool in MHC Class I-Deficient Mice
Authors: Nešić Dragana , Santori Fabio R. , Vukmanović Stanislav
Published: April 2014

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of β2-microglobulin-deficient (β2m−/−) mice are unknown. We here show that very few CD8+ cells in β2m−/− mice coexpress CD8β, a marker of the thymus-derived CD8+ T cells. Most of the CD8α+ cells express CD11c and can be found in β2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8α+CD8β+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8β+ cells in β2m−/− mice. These findings demonstrate that 1) thymic export of CD8+ T cells in β2m−/− mice is reduced more severely than previously thought; 2) non-T cells expressing CD8α become prominent when CD8+ T cells are virtually absent; 3) at least some β2m−/− CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.
Cytolytic Mechanisms and Expression of Activation-Regulating Receptors on Effector-Type CD8+CD45RA+CD27− Human T Cells
Authors: Baars Paul A. , Ribeiro do Couto Laura M. , Leusen Jeanette H. W. , Hooibrink Berend , Kuijpers Taco W. , Lens Susanne M. A. , van Lier René A. W.
Published: April 2014

Circulating CD8+ T cells with a CD45RA+CD27− phenotype resemble cytolytic effector cells because they express various cytolytic mediators and are able to execute cytotoxicity without prior stimulation in vitro. We here demonstrate that CD8+CD45RA+CD27− T cells can use both granule exocytosis and Fas/Fas ligand pathways to induce apoptosis in target cells. The availability of these cytolytic mechanisms in circulating T cells suggests that the activity of these cells must be carefully controlled to prevent unwanted tissue damage. For this reason, we analyzed the expression of surface receptors that either enhance or inhibit T cell function. Compared with memory-type cells, effector cells were found to express normal levels of CD3ε and TCRζ and relatively high levels of CD8. CTLA-4 was absent from freshly isolated effector cells, whereas a limited number of unstimulated memory cells expressed this molecule. In line with recent findings on CD8+CD28− T cells, CD45RA+CD27− T cells were unique in the abundant expression of NK cell-inhibitory receptors, both of Ig superfamily and C-type lectin classes. Binding of NK cell-inhibitory receptors to classical and nonclassical MHC class I molecules may inhibit the activation of the cytolytic machinery induced by either Ag receptor-specific or nonspecific signals in CD8+CD45RA+CD27− T cells.
Regulation of Gene Expression of Murine MD-1 Regulates Subsequent T Cell Activation and Cytokine Production
Authors: Gorczynski Reginald M. , Chen Zhigi , Clark David A. , Hu Jiang , Yu Gary , Li Xiarong , Tsang Wendy , Hadidi Sima
Published: April 2014

The immunoadhesin (OX2:Fc) comprising the extracellular domain of murine OX2 linked to IgG2aFc, inhibits production of IL-2 and IFN-γ by activated T cells and increases allograft and xenograft survival in vivo. Increased expression of OX2 on dendritic cells (DC) in vivo following preimmunization via the portal vein is also associated with elevated expression of MD-1. We have used antisense oligodeoxynucleotides (ODNs) to MD-1 to investigate the effect of inhibition of expression of MD-1 by DC on their function as allostimulatory cells. We also investigated by FACS analysis the cell surface expression of OX2, CD80, and CD86 on DC incubated with ODN-1 blocking MD-1 expression. Blocking MD-1 gene expression inhibits surface expression of CD80 and CD86, but not of OX2. DC incubated with ODN-1 to MD-1 did not stimulate IL-2 or IFN-γ production, but generated cells able to suppress, in a second culture of fresh DC plus allogeneic T cells, production of IL-2 and IFN-γ. This inhibition was blocked by anti-OX2 mAb. Infusion of DC preincubated with ODN-1 prolonged renal allograft survival, an effect also reversed by anti-OX2 mAb. By FACS, incubation of DC with anti-MD-1 Ab to promote capping eliminated cell surface expression of MD-1 and CD14 without altering DEC205, DC26, CD80, CD86, or OX2 expression. Thus OX2 and MD-1 are independent surface molecules on DC that may reciprocally regulate T cell stimulation. MD-1 is linked to CD14, a “danger receptor complex,” and activation of this complex can regulate cell surface expression of CD80/CD86, which signal T cells.
Nondepleting Anti-CD4 Has an Immediate Action on Diabetogenic Effector Cells, Halting Their Destruction of Pancreatic β Cells
Authors: Phillips Jenny M. , Harach Silvia Zusman , Parish Nicole M. , Fehervari Zoltan , Haskins Katherine , Cooke Anne
Published: April 2014

The induction of tolerance in a primed immune system is a major aim for therapy in autoimmunity and transplant rejection. In this paper, we investigate the action of the nondepleting anti-CD4 Ab, YTS 177. Although this Ab is nondepleting, we have demonstrated a direct action in vivo on activated effector cells. We show that the Ab inhibits transfer of insulin-dependent diabetes mellitus by the CD4+ Th1 clone BDC2.5 to nonobese diabetic mice. Furthermore, we show that this Ab acts directly on diabetogenic effector cells because it prevented BDC2.5-induced insulin-dependent diabetes mellitus in nonobese diabetic-scid recipients in the absence of other T cells. The Ab halts the diabetic process even when it is administered after the BDC2.5 cells have infiltrated the pancreas and destruction of islets is already underway. This is accompanied by an immediate decrease in proinflammatory cytokine production with cessation of β cell destruction and disappearance of infiltrating cells from the pancreas, leaving any remaining β cells intact. These data suggest that Abs such as this may be effective not only because they induce regulatory T cells but also because they are able to directly prevent effector cell function.
Antitumor Effects of the Mouse Chemokine 6Ckine/SLC Through Angiostatic and Immunological Mechanisms
Authors: Vicari Alain P. , Ait-Yahia Smina , Chemin Karine , Mueller Anja , Zlotnik Albert , Caux Christophe
Published: April 2014

Mouse 6Ckine/SLC (secondary lymphoid tissue chemokine) is a chemotactic factor for dendritic cells, T cells, and NK cells in vitro. In addition, mouse 6Ckine/SLC interacts with the chemokine receptor CXCR3, as do several chemokines with antiangiogenic properties. These dual properties of mouse 6Ckine/SLC were tested for the induction of an antitumor response by transducing the C26 colon carcinoma tumor cell line with a cDNA encoding mouse 6Ckine/SLC. The C26-6CK-transduced cells showed reduced tumorigenicity in immunocompetent or in nude mice. Part of this effect was likely due to angiostatic mechanisms as shown by immunohistochemistry and Matrigel assay. C26-6CK tumors were also heavily infiltrated with leukocytes, including granulocytes, dendritic cells, and CD8+ T cells. In vivo, anti-CD8 treatment increased the tumorigenicity of the C26-6CK tumor cells, and tumor-infiltrating CD8+ T cells had the phenotype of memory effector cells, suggesting the induction of cytotoxic tumor-specific T lymphocytes. On the other hand, anti-asialo-GM1 depletion also increased the tumorigenicity of C26-6CK cells, supporting the participation of NK cells. Finally, tumor-infiltrating dendritic cells had the phenotype and functional features of immature dendritic cells. Overall, these results suggest that mouse 6Ckine/SLC has strong antitumor effects by inducing both angiostatic, CD8+ T cell-mediated, and possibly NK-mediated tumor resistance mechanisms.
Identification of T Cell Ligands in a Library of Peptides Covalently Attached to HLA-DR4
Authors: Boen Eric , Crownover Angie R. , McIlhaney Mary , Korman Alan J. , Bill Jerry
Published: April 2014

While T cells have been clearly implicated in a number of disease processes including autoimmunity, graft rejection, and atypical immune responses, the precise Ags recognized by the pathogenic T cells have often been difficult to identify. This has particularly been true for MHC class II-restricted CD4+ T cells. Although such cells can be demonstrated to have undergone clonal expansion at sites of pathology, they are frequently difficult to establish as stable T cell clones. Furthermore, in general, larger peptides in higher concentrations are required to stimulate CD4+ T cells than CD8+ T cells, which makes some of the techniques developed to identify CD8+ T cell Ags impractical. To circumvent some of these problems, we developed a model system consisting of two parts. The first part involves the construction of an indicator T cell hybridoma expressing a chimeric TCR comprised of murine constant regions and human variable regions specific for influenza hemagglutinin 307–319 presented by DR4. The second part consists of a library of fibroblasts each expressing multiple peptides as amino terminal covalent extensions of the β-chain of HLA-DR4 (DRA1*0101, DRB1*0401). Using this model system, we screened ∼100,000 peptides and identified three novel peptides stimulatory for the HA1.7 TCR. While there is some convergence at residues known to be important for T cell recognition, all three peptides differ markedly from each other and bear little resemblance to wild-type hemagglutinin 307–319.
Essential Role of CD8 Palmitoylation in CD8 Coreceptor Function
Authors: Arcaro Alexandre , Grégoire Claude , Boucheron Nicole , Stotz Sabine , Palmer Ed , Malissen Bernard , Luescher Immanuel F.
Published: April 2014

To investigate the molecular basis that makes heterodimeric CD8αβ a more efficient coreceptor than homodimeric CD8αα, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252–260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8β and that this allows partitioning of CD8αβ, but not of CD8αα, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8β abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3ζ in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8β is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.
Recombinant Attenuated Toxoplasma gondii Expressing the Plasmodium yoelii Circumsporozoite Protein Provides Highly Effective Priming for CD8+ T Cell-Dependent Protective Immunity Against Malaria
Authors: Charest Hugues , Sedegah Martha , Yap George S. , Gazzinelli Ricardo T. , Caspar Patricia , Hoffman Stephen L. , Sher Alan
Published: April 2014

The protozoan parasite Toxoplasma gondii elicits strong cell-mediated immunity against itself as well as nonspecific resistance against other pathogens and tumors. For this reason, we asked whether recombinant Toxoplasma could be utilized as an effective vaccine vehicle for inducing immunity against heterologous microbial infections. The circumsporozoite protein (PyCSP) of Plasmodium yoelii was engineered into a T. gondii temperature-sensitive strain (ts-4), a mutant that induces complete protection against virulent Toxoplasma challenge. When administered to mice in a single dose, a recombinant ts-4 (CSC3) that both secretes and expresses surface PyCSP induced strong anti-CSP Ab responses, with an isotype distribution pattern similar to that stimulated by the T. gondii carrier. When challenged with P. yoelii sporozoites during the first month after CSC3 vaccination, these animals displayed substantial levels of nonspecific resistance attributable entirely to the T. gondii carrier. Nevertheless, after the nonspecific protection had waned, high levels (up to 79%) of specific immunity against sporozoite challenge were achieved by boosting the animals with recombinant vaccinia virus expressing PyCSP. These CSC3-primed PyCSP-vaccinia-boosted mice displayed high frequencies of splenic PyCSP-specific IFN-γ-producing cells, as well as CD8+ T cell-dependent cytolytic activity. In vivo depletion of CD8+ lymphocytes at the time of challenge completely ablated protective immunity in the T. gondii-primed/vaccinia-boosted animals, while neutralization of IFN-γ or IL-12 caused a partial but significant reduction in resistance. Together these findings establish the efficacy of recombinant attenuated Toxoplasma as a vaccine vehicle for priming CD8+-dependent cell-mediated immunity.
Distinct Pathways of Mannan-Binding Lectin (MBL)- and C1-Complex Autoactivation Revealed by Reconstitution of MBL with Recombinant MBL-Associated Serine Protease-2
Authors: Vorup-Jensen Thomas , Petersen Steen V. , Hansen Annette G. , Poulsen Knud , Schwaeble Wilhelm , Sim Robert B. , Reid Kenneth B. M. , Davis Simon J. , Thiel Steffen , Jensenius Jens C.
Published: April 2014

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.
Expression of Intracellular IFN-γ in HSV-1-Specific CD8+ T Cells Identifies Distinct Responding Subpopulations During the Primary Response to Infection
Authors: Andersen Hanne , Dempsey Deborah , Chervenak Robert , Jennings Stephen R.
Published: April 2014

Cutaneous infection in the footpads of C57BL/6 mice with HSV-1 results in an accumulation of activated (CD44high CD25+) CD8+ T cells within the draining popliteal lymph node (PLN). These studies were undertaken to evaluate the frequency and phenotype of the CD8+ T cell population within the PLN, recognizing the single immunodominant HSV-1 epitope derived from the viral envelope glycoprotein, glycoprotein B (gB), using an intracellular IFN-γ-staining assay. It revealed that ∼6% of the CD8+ T cells were specific for the gB epitope. Phenotypic analysis of the IFN-γ-producing gB-specific CD8+ T cells generated in the PLN during the course of the acute infection expressed the CD44high CD25+ phenotype on days 3–5 postinfection. Surprisingly, IFN-γ-producing CD8+ T cells expressed the CD44high CD25− phenotype on days 5–8 postinfection, in contrast to expectations for a CD8+ effector T cell. IFN-γ-producing CD25− CD8+ T cells were detected in the PLN on day 21 postinfection, long after infectious virus had been cleared. Throughout the response, the spleen was found to be the major reservoir of gB-specific CD8+ T cells, even during the peak of the response. In contrast to the gB-specific CD8+ T cell population within the PLN, the entire gB-specific CD8+ T cell population within the spleen was CD25−. Collectively, these results suggest the generation of subpopulations of virus-specific CD8+ T cells, distinguished by the expression of CD25, during the acute phase of the primary response to a localized viral infection.
Regulation of Neutrophil Adhesion by Pituitary Growth Hormone Accompanies Tyrosine Phosphorylation of Jak2, p125FAK, and Paxillin
Authors: Ryu Hoon , Lee Jung-Hee , Kim Kwon Seop , Jeong Seong-Min , Kim Pyeung-Hyeun , Chung Hun-Taeg
Published: April 2014

Neutrophil adhesion is fundamentally important during the onset of inflammatory responses. The adhesion signaling pathways control neutrophil arrest and extravasation and influence neutrophil shape and function at sites of inflammation. In the present study the intracellular signaling pathways for the adhesion of human neutrophils by pituitary growth hormone (GH) were examined. Pituitary GH triggered the tyrosine phosphorylation of Janus kinase 2 (Jak2) and STAT3 in neutrophils. In addition, pituitary GH treatment resulted in the morphological changes and the tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Preincubation with genistein, a tyrosine kinase inhibitor, blocked the GH-stimulated adhesion and Jak2, STAT3, p125FAK, and paxillin phosphorylation. Confocal microscopy revealed that pituitary GH stimulates the focal localization of p125FAK, paxillin, phosphotyrosine, and filamentous actin filament into the membrane rufflings and uropods of human neutrophils. Immunoprecipitation experiments revealed a physical association of Jak2 with p125FAK via STAT3 in vivo. Also an in vitro kinase assay showed an augmentation of p125FAK autophosphorylation as a result of pituitary GH treatment. These results suggest that pituitary GH modulates neutrophil adhesion through tyrosine phosphorylation of Jak2, p125FAK, and paxillin and actin polymerization.
The Involvement of Tyrosine Kinases, Cyclic AMP/Protein Kinase A, and p38 Mitogen-Activated Protein Kinase in IL-13-Mediated Arginase I Induction in Macrophages: Its Implications in IL-13-Inhibited Nitric Oxide Production
Authors: Chang Chiung-I , Zoghi Behyar , Liao James C. , Kuo Lih
Published: April 2014

In macrophages, l-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, l-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.
TNF-Induced Shedding of TNF Receptors in Human Polymorphonuclear Leukocytes: Role of the 55-kDa TNF Receptor and Involvement of a Membrane-Bound and Non-Matrix Metalloproteinase
Authors: Dri Pietro , Gasparini Chiara , Menegazzi Renzo , Cramer Rita , Albéri Lavinia , Presani Gianni , Garbisa Spiridione , Patriarca Pierluigi
Published: April 2014

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.
Requirements for C5a Receptor-Mediated IL-4 and IL-13 Production and Leukotriene C4 Generation in Human Basophils
Authors: Eglite Santa , Plüss Katharina , Dahinden Clemens A.
Published: April 2014

Anaphylatoxin derived from the fifth complement component (C5a) in the presence of IL-3 induces continuous leukotriene C4 generation and IL-4 and IL-13 expression in human basophils for a period of 16–18 h. This indicates that the G protein-coupled C5a receptor (C5aR) can induce long-lasting cellular responses. Using anti-N-terminal C5aR Abs, C-terminal C5a hexapeptide analogs, and pertussis toxin, we demonstrate that the putative activation site of the C5aR is both necessary and sufficient for these late cellular responses. Furthermore, continuous pertussis toxin-sensitive G protein-coupled receptor activation and receptor-ligand interaction is ongoing and required during the entire period of product release. However, the late basophil responses have a more stringent requirement for optimal receptor activation. Leukotriene C4 generation appears to be influenced mostly by the way the receptor is activated, because the most active hexapeptide is a superagonist for this response. By contrast, C5adesarg, lacking the C-terminal arginine, induces minimal lipid mediator formation but is fully active to induce IL-4 production and is even a superagonist for IL-13 release. Nevertheless, IL-4/IL-13 synthesis in response to C5adesarg could be blocked by both C-terminal antagonistic peptide as well as anti-N-terminal C5aR Abs, indicating only minor differences of ligand-receptor interactions between C5a and C5adesarg. Taken together, our data demonstrate that long-lasting and continuous signaling occurs through a limited activation domain of the C5aR, which can differentially promote separate basophil functions.
The Differential Role of Extracellular Signal-Regulated Kinases and p38 Mitogen-Activated Protein Kinase in Eosinophil Functions
Authors: Adachi Tetsuya , Choudhury Barun K. , Stafford Susan , Sur Sanjiv , Alam Rafeul
Published: April 2014

The activation of eosinophils by cytokines is a major event in the pathogenesis of allergic diseases. We have investigated the activation of mitogen-activated protein (MAP) kinases and their functional relevance in eosinophil differentiation, survival, degranulation, and cytokine production. IL-5 induced phosphorylation and activation of extracellular signal-regulated kinases (ERK) and p38 MAP kinases in eosinophils. PD98059, a MAP/ERK kinase inhibitor, blocked phosphorylation of ERK1/2 in a dose-dependent manner. SB202190, a p38 inhibitor, blocked p38-dependent phosphorylation of activating transcription factor-2. To study the importance of the MAP kinases on eosinophil differentiation, we cultured mouse bone marrow cells with IL-3 and IL-5 in the presence of the inhibitors. SB202190 dramatically inhibited eosinophil differentiation by 71%. PD98059 was less potent and reduced eosinophil differentiation by 28%. Both inhibitors marginally inhibited eosinophil survival only at the highest doses. Prolonged incubation of eosinophils with IL-5 induced significant eosinophil-derived neurotoxin release. Both PD98059 and SB202190 nearly completely inhibited (87% and 100% inhibition, respectively) IL-5-stimulated eosinophil-derived neurotoxin release in a dose-dependent manner. Next, we examined the effect of the MAP kinase inhibitors on eosinophil production of the cytokine macrophage-inflammatory protein (MIP)-1α. PD98059 blocked C5a- but not ionomycin-induced MIP-1α production (59% inhibition at 50 μM concentration). In contrast, SB202190 nearly completely inhibited (99%) C5a-induced MIP-1α production. Further, it blocked ionomycin-stimulated production by 66%. Our results suggest that both p38 and ERK1/2 MAP kinases play an important role in eosinophil differentiation, cytokine production, and degranulation. The p38 MAP kinase plays a greater role than ERK1/2 in eosinophil differentiation and cytokine production.
Proinflammatory and Th2-Derived Cytokines Modulate CD40-Mediated Expression of Inflammatory Mediators in Airway Epithelia: Implications for the Role of Epithelial CD40 in Airway Inflammation
Authors: Propst Stacie M. , Denson Raquia , Rothstein Emily , Estell Kim , Schwiebert Lisa M.
Published: April 2014

Cytokines produced by activated macrophages and Th2 cells within the lung play a key role in asthma-associated airway inflammation. Additionally, recent studies suggest that the molecule CD40 modulates lung immune responses. Because airway epithelial cells can act as immune effector cells through the expression of inflammatory mediators, the epithelium is now considered important in the generation of asthma-associated inflammation. Therefore, the goal of the present study was to examine the effects of proinflammatory and Th2-derived cytokines on the function of CD40 in airway epithelia. The results show that airway epithelial cells express CD40 and that engagement of epithelial CD40 induces a significant increase in expression of the chemokines RANTES, monocyte chemoattractant protein (MCP-1), and IL-8 and the adhesion molecule ICAM-1. Cross-linking epithelial CD40 had no effect on expression of the adhesion molecule VCAM-1. The proinflammatory cytokines TNF-α and IL-1β and the Th2-derived cytokines IL-4 and IL-13 modulated the positive effects of CD40 engagement on inflammatory mediator expression in airway epithelial cells. Importantly, CD40 ligation enhanced the sensitivity of airway epithelial cells to the effects of TNF-α and/or IL-1β on expression of RANTES, MCP-1, IL-8, and VCAM-1. In contrast, neither IL-4 nor IL-13 modified the effects of CD40 engagement on the expression of RANTES, MCP-1, IL-8, or VCAM-1; however, both IL-4 and IL-13 attenuated the effects of CD40 cross-linking on ICAM-1 expression. Together, these findings suggest that interactions between CD40-responsive airway epithelial cells and CD40 ligand+ leukocytes, such as activated T cells, eosinophils, and mast cells, modulate asthma-associated airway inflammation.
Inhibition of Keratinocyte Apoptosis by IL-15: A New Parameter in the Pathogenesis of Psoriasis?
Authors: Rückert René , Asadullah Khusru , Seifert Martina , Budagian Vadim M. , Arnold Ralf , Trombotto Claudia , Paus Ralf , Bulfone-Paus Silvia
Published: April 2014

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Rα protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.
Status of Activation of Circulating Vaccine-Elicited CD8+ T Cells
Authors: Nielsen Mai-Britt , Monsurro Vladia , Migueles Stephen A. , Wang Ena , Perez-Diez Ainhoa , Lee Kang-Hun , Kammula Udai , Rosenberg Steven A. , Marincola Francesco M.
Published: April 2014

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-γ expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-γ expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.
Cutting Edge: DC-SIGN; a Related Gene, DC-SIGNR; and CD23 Form a Cluster on 19p13
Authors: Soilleux Elizabeth J. , Barten Roland , Trowsdale John
Published: April 2014

DC-SIGN is a C-type lectin, expressed on a dendritic cell subset. It is able to bind ICAM3 and HIV gp120 in a calcium-dependent manner. Here we report the genomic organization of DC-SIGN and map it to chromosome 19p13 adjacent to the C-type lectin CD23 (FcεRII). We also report a novel, closely linked gene, DC-SIGNR, which shows 73% identity to DC-SIGN at the nucleic acid level and a similar genomic organization. Proteins encoded by both genes have tracts of repeats of 23 aa, predicted to form a coiled coil neck region. They also possess motifs that are known to bind mannose in a calcium-dependent fashion. We show concomitant expression of the two genes in endometrium, placenta, and stimulated KG1 cells (phenotypically similar to monocyte-derived dendritic cells). The existence of a DC-SIGN-related gene calls for reinterpretation of the HIV data to consider possible DC-SIGN/DC-SIGNR hetero-oligomerization.