bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Persistent Viral Infection Prevents Tolerance Induction and Escapes Immune Control Following CD28/CD40 Blockade-Based Regimen
Authors: Williams Matthew A. , Onami Thandi M. , Adams Andrew B. , Durham Megan M. , Pearson Thomas C. , Ahmed Rafi , Larsen Christian P.
Published: April 2014

A continuing concern with CD28 and/or CD40 blockade-based strategies to induce tolerance and mixed chimerism is their potential to disrupt protective immunity to preexisting infections. In this report, we find that preexisting persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13 prevents the induction of tolerance, mixed chimerism, and donor-reactive T cell deletion. Mice continue to be refractory to tolerance induction even after viremia has been resolved and virus is present only at very low levels in peripheral tissues. Conversely, we find that the full tolerance regimen, or costimulation blockade alone, specifically inhibits already ongoing antiviral immune responses, leading to an inability to control viremia. These findings suggest that ongoing T cell responses continue to depend on costimulatory interactions in the setting of a chronic infection and provide insight into potential risks following costimulation blockade posed by chronic or latent viral infections such as hepatitis C, EBV, and CMV.
Cutting Edge: CD4+CD25+ Alloantigen-Specific Immunoregulatory Cells That Can Prevent CD8+ T Cell-Mediated Graft Rejection: Implications for Anti-CD154 Immunotherapy
Authors: Maurik Andre van , Herber Manuela , Wood Kathryn J. , Jones Nick D.
Published: April 2014

Blockade of CD40-CD154 interactions can facilitate long-term allograft acceptance in selected rodent and in primate models, but, due to the ability of CD154-independent CD8+ T cells to initiate graft rejection, this strategy is not always effective. In this work we demonstrate that blockade of the CD40-CD154 pathway at the time of transplantation enables the generation of donor alloantigen-specific CD4+CD25+ regulatory T cells, and that if the regulatory cells are present in sufficient numbers they can suppress allograft rejection mediated by CD154-independent CD8+ T cells.
Cutting Edge: Association of the Motor Protein Nonmuscle Myosin Heavy Chain-IIA with the C Terminus of the Chemokine Receptor CXCR4 in T Lymphocytes
Authors: Rey Mercedes , Vicente-Manzanares Miguel , Viedma Fernando , Yáñez-Mó María , Urzainqui Ana , Barreiro Olga , Vázquez Jesús , Sánchez-Madrid Francisco
Published: April 2014

The binding of chemokines to their receptors guides lymphocyte migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by the actomyosin complex of the cell has not been elucidated. Using biochemical approaches and mass spectrometry analysis, we found an association between the C-termini of CXCR4 and CCR5 and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As expected, myosin L chain was also associated with CXCR4. Confocal microscopy analysis showed that CXCR4 and motor protein nonmuscle myosin H chain-IIA colocalize at the leading edge of migrating T lymphocytes, together with filamentous actin and myosin L chain. These results provide the first evidence of a biochemical association between chemokine receptors and motor proteins, a mechanosignaling mechanism that may have a key role in lymphocyte migration.
TRAIL/Apo-2 Ligand Induces Primary Plasma Cell Apoptosis
Authors: Ursini-Siegel Josie , Zhang Wenli , Altmeyer Anne , Hatada Eunice N. , Do Richard K. G. , Yagita Hideo , Chen-Kiang Selina
Published: April 2014

Apoptosis constitutes the primary mechanism by which noncycling plasma cells are eliminated after the secretion of Ag-specific Abs in a humoral immune response. The underlying mechanism is not known. Here, we demonstrate that the expression of both TRAIL/Apo-2 ligand and the death receptors (DR) DR5 and DR4, but not Fas, are sustained in IL-6-differentiated Ig-secreting human plasma cells as well as primary mouse plasma cells generated in a T-dependent immune response. Plasma cell apoptosis is induced by both endogenous and exogenous TRAIL ex vivo, suggesting that TRAIL-mediated killing may, in part, be plasma cell autonomous. By contrast, resting and activated B cells are resistant to TRAIL killing despite comparable expression of TRAIL and DRs. The preferential killing of plasma cells by TRAIL correlates with decreased expression of CD40 and inactivation of NF-κB. These results provide the first evidence that primary plasma cells synthesize TRAIL and are direct targets of TRAIL-mediated apoptosis, which may relate to the inactivation of the NF-κB survival pathway.
Injection of Soluble Antigen into the Anterior Chamber of the Eye Induces Expansion and Functional Unresponsiveness of Antigen-Specific CD8+ T Cells
Authors: McKenna Kyle C. , Xu Yijun , Kapp Judith A.
Published: April 2014

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8+ CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8+ TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8+ T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8+ T cells.
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Inflammatory Stimuli Up-Regulate Secretion of the Soluble GM-CSF Receptor in Human Monocytes: Evidence for Ectodomain Shedding of the Cell Surface GM-CSF Receptor α Subunit
Authors: Prevost Jay M. , Pelley Jennifer L. , Zhu Weibin , D’Egidio Gianni E. , Beaudry Paul P. , Pihl Carin , Neely Graham G. , Claret Emmanuel , Wijdenes John , Brown Christopher B.
Published: April 2014

Soluble GM-CSF receptor α subunit (sGMRα) is a soluble isoform of the GMRα that is believed to arise exclusively through alternative splicing of the GMRα gene product. The sGMRα mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRα. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRα in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRα arose exclusively through alternative splicing of the GMRα gene product, or whether it could also be generated through ectodomain shedding of GMRα, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRα (Ba/F3.GMRα). The Ba/F3.GMRα cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRα-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRα (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRα. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRα by monocytes, suggesting that shedding of GMRα by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRα is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRα secretion, and that sGMRα arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRα.
Empirical Evaluation of a Dynamic Experiment Design Method for Prediction of MHC Class I-Binding Peptides
Authors: Udaka Keiko , Mamitsuka Hiroshi , Nakaseko Yukinobu , Abe Naoki
Published: April 2014

The ability to predict MHC-binding peptides remains limited despite ever expanding demands for specific immunotherapy against cancers, infectious diseases, and autoimmune disorders. Previous analyses revealed position-specific preference of amino acids but failed to detect sequence patterns. Efforts to use computational analysis to identify sequence patterns have been hampered by the insufficiency of the number/quality of the peptide binding data. We propose here a dynamic experiment design to search for sequence patterns that are common to the MHC class I-binding peptides. The method is based on a committee-based framework of query learning using hidden Markov models as its component algorithm. It enables a comprehensive search of a large variety (209) of peptides with a small number of experiments. The learning was conducted in seven rounds of feedback loops, in which our computational method was used to determine the next set of peptides to be analyzed based on the results of the earlier iterations. After these training cycles, the algorithm enabled a real number prediction of MHC binding peptides with an accuracy surpassing that of the hitherto best performing positional scanning method.
Rejection of Syngeneic Colon Carcinoma by CTLs Expressing Single-Chain Antibody Receptors Codelivering CD28 Costimulation
Authors: Haynes Nicole M. , Trapani Joseph A. , Teng Michele W. L. , Jackson Jacob T. , Cerruti Loretta , Jane Stephen M. , Kershaw Michael H. , Smyth Mark J. , Darcy Phillip K.
Published: April 2014

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-ζ or CD28/TCR-ζ signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA+ tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-ζ chimera had a far greater capacity to control the growth of CEA+ xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-ζ receptor was critically dependent on perforin and the production of IFN-γ. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-ζ and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.
CD8 T Cell-Mediated Killing ofCryptococcus neoformansRequires Granulysin and Is Dependent on CD4 T Cells and IL-15
Authors: Ma Ling Ling , Spurrell Jason C. L. , Wang Jian Fei , Neely Graham G. , Epelman Slava , Krensky Alan M. , Mody Christopher H.
Published: April 2014

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl2, or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.
Nonsecreted Bacterial Proteins Induce Recall CD8 T Cell Responses But Do Not Serve as Protective Antigens
Authors: Zenewicz Lauren A. , Foulds Kathryn E. , Jiang Jiu , Fan Xin , Shen Hao
Published: April 2014

Secreted or nonsecreted Ag expressed by recombinant Listeria monocytogenes can prime CD8 T cells. However, Ag-specific memory CD8 T cells confer protection against bacteria secreting Ag, but not against bacteria expressing the nonsecreted form of the same Ag. This dichotomy may be explained by a long-standing hypothesis that nonsecreted Ags are less effective than secreted Ags at inducing a protective immune response at the onset of infection. We tested this hypothesis by examining whether these two different forms of Ag induce different primary and secondary CD8 T cell responses. The primary responses to secreted and nonsecreted Ags expanded and contracted almost synchronously, although the responses to nonsecreted Ags were of lower magnitude. These results demonstrate that the kinetics of the CD8 T cell response are similar regardless of whether Ag is accessible to the endogenous MHC class I pathway or can only be presented through cross-presentation. No differences were detected in the CD8 T cell recall response to L. monocytogenes expressing secreted or nonsecreted Ags. Nonsecreted Ags are as effective as secreted Ags at the induction of a rapid recall response by memory CD8 T cells. Thus, the inability of nonsecreted bacterial proteins to serve as protective Ags cannot be attributed to a defective CD8 T cell response.
Inducible Costimulator Protein Controls the Protective T Cell Response Against Listeria monocytogenes
Authors: Mittrücker Hans-Willi , Kursar Mischo , Köhler Anne , Yanagihara Donna , Yoshinaga Steven K. , Kaufmann Stefan H. E.
Published: April 2014

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4+ and CD8+ T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8+ T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8+ T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4+ T cell response revealed that ICOS-Ig treatment also affected the specific CD4+ T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4+ T cells from infected and ICOS-Ig-treated mice responded with IFN-γ production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.
Pulmonary Surfactant Proteins A and D Directly Suppress CD3+/CD4+ Cell Function: Evidence for Two Shared Mechanisms
Authors: Borron Paul J. , Mostaghel Elahe A. , Doyle Carolyn , Walsh Eric S. , McHeyzer-Williams Michael G. , Wright Jo Rae
Published: April 2014

Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3+/CD4+ lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-γ when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca2+ ]i), but not thapsigargin-induced changes in [Ca2+]i. In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca2+]i.
IL-17 Expression in Human Herpetic Stromal Keratitis: Modulatory Effects on Chemokine Production by Corneal Fibroblasts
Authors: Maertzdorf Jeroen , Osterhaus Albert D. M. E. , Verjans Georges M. G. M.
Published: April 2014

Herpetic stromal keratitis (HSK) is an immunopathologic disease triggered by infection of the cornea with HSV. Key events in HSK involve the interaction between cornea-infiltrating inflammatory cells and resident cells. This interaction, in which macrophages, producing IL-1 and TNF-α, and IFN-γ-producing Th1 cells play a crucial role, results in the local secretion of immune-modulatory factors and a major influx of neutrophils causing corneal lesions and blindness. The Th1-derived cytokine IL-17 has been shown to play an important role in several inflammatory diseases characterized by a massive infiltration of neutrophils into inflamed tissue. Here we show that IL-17 is expressed in corneas from patients with HSK and that the IL-17R is constitutively expressed by human corneal fibroblasts (HCF). IL-17 exhibited a strong synergistic effect with TNF-α on the induction of IL-6 and IL-8 secretion by cultured HCF. Secreted IL-8 in these cultures had a strong chemotactic effect on neutrophils. IL-17 also enhanced TNF-α- and IFN-γ-induced secretion of macrophage-inflammatory proteins 1α and 3α, while inhibiting the induced secretion of RANTES. Furthermore, considerable levels of IFN-γ-inducible protein 10 and matrix metalloproteinase 1 were measured in stimulated HCF cultures, while the constitutive secretion of monocyte chemotactic protein 1 remained unaffected. The data presented suggest that IL-17 may play an important role in the induction and/or perpetuation of the immunopathologic processes in human HSK by modulating the secretion of proinflammatory and neutrophil chemotactic factors by corneal resident fibroblasts.
In Vivo Biosynthesis of Endogenous and of Human C1 Inhibitor in Transgenic Mice: Tissue Distribution and Colocalization of Their Expression
Authors: Vinci Giovanna , Lynch Nicholas J. , Duponchel Christiane , Lebastard Thi-May , Milon Geneviève , Stover Cordula , Schwaeble Wilhelm , Tosi Mario
Published: April 2014

We have produced transgenic mice expressing human C1 inhibitor mRNA and protein under the control of the human promoter and regulatory elements. The transgene was generated using a minigene construct in which most of the human C1 inhibitor gene (C1NH) was replaced by C1 inhibitor cDNA. The construct retained the promoter region extending 1.18 kb upstream of the transcription start site, introns 1 and 2 as well as a stretch of 2.5 kb downstream of the polyadenylation site, and therefore carried all known elements involved in transcriptional regulation of the C1NH gene. Mice with high serum levels of human C1 inhibitor, resulting from multiple tandem integrations of the C1 inhibitor transgene, were selected. Immunohistochemistry in combination with in situ hybridization was applied to localize the sites of C1 inhibitor biosynthesis and to demonstrate its local production in brain, spleen, liver, heart, kidney, and lung. The distribution of human C1 inhibitor-expressing cells was qualitatively indistinguishable from that of its mouse counterpart, but expression levels of the transgene were significantly higher. In the spleen, production of C1 inhibitor was colocalized with that of a specific marker for white pulp follicular dendritic cells. This study demonstrates a stringently regulated expression of both the endogenous and the transgenic human C1 inhibitor gene and reveals local biosynthesis of C1 inhibitor at multiple sites in which the components of the macromolecular C1 complex are also produced.
Defective Production of Functional 98-kDa Form of Elf-1 Is Responsible for the Decreased Expression of TCR ζ-Chain in Patients with Systemic Lupus Erythematosus
Authors: Juang Yuang-Taung , Tenbrock Klaus , Nambiar Madhusoodana P. , Gourley Mark F. , Tsokos G. C.
Published: April 2014

Systemic lupus erythematosus (SLE), the prototypic autoimmune disease, is characterized by defective expression of TCR ζ-chain. Elf-1 (E-74-like factor) is a member of the Ets (E-26-specific) family and is crucial for the basal transcription of TCR ζ-chain in Jurkat cells. We previously demonstrated that Elf-1 exists in the cytoplasm mainly as 80-kDa form and after phosphorylation and O-glycosylation it moves to the nucleus as a 98-kDa which binds DNA. We now demonstrate that Elf-1 is crucial for the transactivation of TCR ζ-chain promoter in normal and SLE T cells. Defective expression of TCR ζ-chain in SLE T cells is associated with two distinct molecular defects in the generation of the 98-kDa DNA binding Elf-1 form. In the first, the levels of the 98-kDa form were either decreased or absent. In the second, the apparent levels of the nuclear Elf-1 form were normal but included only two of the three bands into which the nuclear Elf-1 form separated in isoelectric focusing gels. Because both the transcription and the translation processes of Elf-1 gene are normal in SLE T cells, our data demonstrate that abnormal posttranslational mechanisms of the Elf-1 protein result in defective expression of functional Elf-1, and consequently, the transcriptional defect of TCR ζ-chain in patients of SLE.
Cutting Edge: Differential Chemokine Production by Myeloid and Plasmacytoid Dendritic Cells
Authors: Penna Giuseppe , Vulcano Marisa , Roncari Andrea , Facchetti Fabio , Sozzani Silvano , Adorini Luciano
Published: April 2014

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.
Cutting Edge: Selection of B Lymphocyte Subsets Is Regulated by Natural IgM
Authors: Baker Nicole , Ehrenstein Michael R.
Published: April 2014

Natural IgM has a wide range of actions in the immune system. Here we demonstrate that mice lacking serum IgM have an expansion in splenic marginal zone B cells with a proportionately smaller reduction in follicular B cells. The increase in the marginal zone-follicular B cell ratio (and an expansion in peritoneal B1a cells) is fully reversed by administration of polyclonal IgM, but not by two IgM monoclonals. Mice engineered to have a secreted oligoclonal IgM repertoire with an endogenous membrane IgM also exhibited a similar expansion of marginal zone B cells. We propose that natural IgM, by virtue of its polyreactivity, enhances Ag-driven signaling through the B cell receptor and promotes the formation of follicular B cells. These results demonstrate that natural IgM regulates the selection of B lymphocyte subsets.
Cutting Edge: Dynamic Redistribution of Tetraspanin CD81 at the Central Zone of the Immune Synapse in Both T Lymphocytes and APC
Authors: Mittelbrunn María , Yáñez-Mó María , Sancho David , Ursa Ángeles , Sánchez-Madrid Francisco
Published: April 2014

The tetraspanin CD81 has been involved in T-dependent B cell-mediated immune responses. However, the behavior of CD81 during immune synapse (IS) formation has not been elucidated. We determined herein that CD81 redistributed to the contact area of T cell-B cell and T cell-dendritic cell conjugates in an Ag-dependent manner. Confocal microscopy showed that CD81 colocalized with CD3 at the central supramolecular activation complex. Videomicroscopy studies with APC or T cells transiently expressing CD81-green fluorescent protein (GFP) revealed that in both cells CD81 redistributed toward the central supramolecular activation complex. In T lymphocytes, CD81-GFP rapidly redistributed to the IS, whereas, in the APC, CD81-GFP formed a large accumulation in the contact area that later concentrated in a discrete cluster and waves of CD81 accumulated at the IS periphery. These results suggest a relevant role for CD81 in the topography of the IS that would explain its functional implication in T cell-B cell collaboration.
Murine Plasmacytoid Pre-Dendritic Cells Generated from Flt3 Ligand-Supplemented Bone Marrow Cultures Are Immature APCs
Authors: Brawand Pierre , Fitzpatrick David R. , Greenfield Brad W. , Brasel Kenneth , Maliszewski Charles R. , De Smedt Thibaut
Published: April 2014

The putative counterparts of human plasmacytoid pre-dendritic cells (pDCs) have been described in vivo in mouse models and very recently in an in vitro culture system. In this study, we report that large numbers of bone marrow-derived murine CD11c+B220+ pDCs can be generated with Flt3 ligand (FL) as the sole exogenous differentiation/growth factor and that pDC generation is regulated in vivo by FL because FL-deficient mice showed a major reduction in splenic pDC numbers. We extensively analyzed bone marrow-derived CD11c+B220+ pDCs and described their immature APC phenotype based on MHC class II, activation markers, and chemokine receptor level of expression. CD11c+B220+ pDCs showed a nonoverlapping Toll-like receptor pattern of expression distinct from that of classical CD11c+B220− dendritic cells and were poor T cell stimulators. Stimulation of CD11c+B220+ pDCs with oligodeoxynucleotides containing certain CpG motifs plus CD40 ligand plus GM-CSF led to increased MHC class II, CD80, CD86, and CD8α expression levels, to a switch in chemokine receptor expression that affected their migration, to IFN-α and IL-12 secretion, and to the acquisition of priming capacities for both CD4+ and CD8+ OVA-specific TCR-transgenic naive T cells. Thus, the in vitro generation of murine pDCs may serve as a useful tool to further investigate pDC biology as well as the potential role of these cells in viral immunity and other settings.
Cross-Regulation of T Cell Growth Factor Expression by p53 and the Tax Oncogene
Authors: Chaudhry Sohail , Freebern Wendy J. , Smith James L. , Butscher Wayne G. , Haggerty Cynthia M. , Gardner Kevin
Published: April 2014

In this study, we demonstrate that p53 directly inhibits expression of the T cell growth factor (IL-2) in activated T cells. This repression is independent of the intrinsic transcriptional activity of p53 and is mediated by the Tax-responsive CD28RE-3′-12-O-tetradecanoylphorbol-13-acetate response element (AP1) element of the IL-2 promoter. Coexpression of the Tax oncogene causes full reversal of this repression through coordinate targeting of p300, CREB, and the NF-κB pathways. Paradoxically, IL-2 repression by p53 is not reversed by mdm2. Instead, mdm2 represses the IL-2 promoter by a mechanism that is synergistic with p53 and resistant to Tax reversal. The p300 structure-function studies show that these effects are linked to competitive associations among p53, Tax, and mdm2 with multiple domains of p300. The functional outcome of these antagonistic associations is revealed further by the observation that Tax and p53 induce apoptosis in activated T cells through separate and mutually exclusive pathways. Interestingly, both pathways are abrogated by mdm2. These results provide evidence that a dynamic interplay, between Tax and specific elements of the p53 network, mediates growth factor expression and programmed cell death in activated T cells.
The Role of T Cell Antagonism and Original Antigenic Sin in Genetic Immunization
Authors: Singh Rana A. K. , Rodgers John R. , Barry Michael A.
Published: April 2014

To counter highly mutable pathogens like HIV-1, a number of vaccines are being developed to deliver multiple mutant forms of viral Ags to provoke multivalent antiviral CTLs. However, it is uncertain whether such multiple mutant epitope vaccines will generate the diverse CTL responses desired or will instead create immune interference. To characterize the role of immune interference by mutant epitopes in this process, we have tested a “worst case” scenario in which the immunodominant epitope of OVA (SIINFEKL) and its in vitro TCR antagonist (SIINFEDL) have been used to genetically immunize C57BL/6 mice. We demonstrate here that sequential delivery of these mutant epitopes provokes original antigenic sin in CD8 T cells as demonstrated by attenuation of CTLs, intracellular IFN-γ production, and MHC I peptide-tetramer staining. By contrast, simultaneous exposure of the immune system to this agonist/antagonist pair not only fails to generate T cell antagonism in vivo, but also avoids original antigenic sin. These observations suggest that simultaneous immunization with vaccines containing mutant epitopes, even T cell antagonists, can indeed generate a diverse array of T cell responses and that at least some immune interference can be avoided by delivering mutant Ags to the immune system simultaneously.
Protection Against Helicobacter pylori Infection Following Immunization Is IL-12-Dependent and Mediated by Th1 Cells
Authors: Akhiani Ali A. , Pappo Jacques , Kabok Zita , Schön Karin , Gao Wei , Franzén Lennart E. , Lycke Nils
Published: April 2014

The regulatory roles of Th1 and Th2 cells in immune protection against Helicobacter infection are not clearly understood. In this study, we report that a primary H. pylori infection can be established in the absence of IL-12 or IFN-γ. However, IFN-γ, but not IL-12, was involved in the development of gastritis because IFN-γ−/− (GKO) mice exhibited significantly less inflammation as compared with IL-12−/− or wild-type (WT) mice. Both IL-12−/− and GKO mice failed to develop protection following oral immunization with H. pylori lysate and cholera toxin adjuvant. By contrast, Th2-deficient, IL-4−/−, and WT mice were equally well protected. Mucosal immunization in the presence of coadministered rIL-12 in WT mice increased Ag-specific IFN-γ-producing T cells by 5-fold and gave an additional 4-fold reduction in colonizing bacteria, confirming a key role of Th1 cells in protection. Importantly, only protected IL-4−/− and WT mice demonstrated substantial influx of CD4+ T cells in the gastric mucosa. The extent of inflammation in challenged IL-12−/− and GKO mice was much reduced compared with that in WT mice, indicating that IFN-γ/Th1 cells also play a major role in postimmunization gastritis. Of note, postimmunization gastritis in IL-4−/− mice was significantly milder than WT mice, despite a similar level of protection, indicating that immune protection is not directly linked to the degree of gastric inflammation. Only protected mice had T cells that produced high levels of IFN-γ to recall Ag, whereas both protected and unprotected mice produced high levels of IL-13. We conclude that IL-12 and Th1 responses are crucial for H. pylori-specific protective immunity.
Cell Surface Peptidase CD26/Dipeptidylpeptidase IV Regulates CXCL12/Stromal Cell-Derived Factor-1α-Mediated Chemotaxis of Human Cord Blood CD34+ Progenitor Cells
Authors: Christopherson Kent W. , Hangoc Giao , Broxmeyer Hal E.
Published: April 2014

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1α (SDF-1α) at its position two proline. CXCL12/SDF-1α induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34+ hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34+ hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1α lacks the ability to induce the migration of CD34+ cord blood cells and acts to inhibit normal CXCL12/SDF-1α-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34+ cells enhances the migratory response of these cells to CXCL12/SDF-1α. This process of CXCL12/SDF-1α cleavage by CD26/DPPIV on a subpopulation of CD34+ cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.
Resistance to Experimental Autoimmune Myasthenia Gravis in IL-6-Deficient Mice Is Associated with Reduced Germinal Center Formation and C3 Production
Authors: Deng Caishu , Goluszko Elzbieta , Tüzün Erdem , Yang Huan , Christadoss Premkumar
Published: April 2014

To provide direct genetic evidence for a role of IL-6 in experimental autoimmune myasthenia gravis (EAMG), IL-6 gene KO (IL-6−/−) mice in the C57BL/6 background were immunized with Torpedo californica acetylcholine receptor (AChR) and evaluated for EAMG. Only 25% of AChR-immunized IL-6−/− mice developed clinical EAMG compared to 83% of C57BL/6 (wild-type) mice. A significant reduction in the secondary anti-AChR Ab of IgG, IgG2b, and IgG2c, but not the primary or secondary IgM response was observed in AChR-immunized IL-6−/− mice, suggesting a possible defect in T cell help and class switching to anti-AChR IgG2 isotype. The AChR-specific lymphocyte proliferative response, IFN-γ, and IL-10 production were suppressed in AChR-immunized IL-6−/− mice. EAMG resistance in IL-6−/− mice was associated with a significant reduction in germinal center formation and decreased serum complement C3 levels. The data provide the first direct genetic evidence for a key role of IL-6 in the autoimmune response to AChR and in EAMG pathogenesis.
Late Pregnancy Suppresses Relapses in Experimental Autoimmune Encephalomyelitis: Evidence for a Suppressive Pregancy-Related Serum Factor
Authors: Langer-Gould Annette , Garren Hideki , Slansky Amy , Ruiz Pedro J. , Steinman Lawrence
Published: April 2014

Women with multiple sclerosis have significantly diminished disease activity during pregnancy. The purpose of our study was to identify the underlying mechanism for the diminished disease activity. We found that during the period of late pregnancy there is protection against paralysis, during both the induction and effector phases of relapsing experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. We did not find any changes in the cytokine secretion profiles or the proliferative activity of autoreactive T cells from mice induced during late pregnancy compared with virgin controls. In mice mated after disease onset, the inflammatory histologic lesions did not clear, despite marked clinical improvement during pregnancy. We found evidence for a serum factor present in late pregnancy that suppresses T cell activation. In the presence of sera taken from mice late in pregnancy, the proliferative response and IL-2 production of proteolipid protein p139–151-specific T cells were significantly diminished as compared with stimulation in the presence of normal mouse sera. In conclusion, serum from late pregnancy has the capacity to down-regulate T cell responses and might be associated with the amelioration of disease activity in experimental autoimmune encephalomyelitis.
Cutting Edge: IL-17D, a Novel Member of the IL-17 Family, Stimulates Cytokine Production and Inhibits Hemopoiesis
Authors: Starnes Trevor , Broxmeyer Hal E. , Robertson Michael J. , Hromas Robert
Published: April 2014

A novel cytokine termed IL-17D was cloned using nested RACE PCR. It is a secreted cytokine with homology to the IL-17 family of proteins. IL-17D is preferentially expressed in skeletal muscle, brain, adipose tissue, heart, lung, and pancreas. Treatment of endothelial cells with purified rIL-17D protein stimulated the production of IL-6, IL-8, and GM-CSF. The increased expression of IL-8 was found to be NF-κB-dependent. rIL-17D also demonstrated an inhibitory effect on hemopoiesis of myeloid progenitor cells in colony formation assays.
Suppressive Effect of Glutamic Acid Decarboxylase 65-Specific Autoimmune B Lymphocytes on Processing of T Cell Determinants Located Within the Antibody Epitope
Authors: Jaume Juan Carlos , Parry Sarah Louise , Madec Anne-Marie , Sønderstrup Grete , Baekkeskov Steinunn
Published: April 2014

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.
Thymic and Extrathymic T Cell Development Pathways Follow Different Rules
Authors: Terra Rafik , Labrecque Nathalie , Perreault Claude
Published: April 2014

Separation between primary and secondary lymphoid organs is a universal feature in jawed vertebrates. Strikingly, oncostatin M (OM)-transgenic mice present massive extrathymic T cell development, localized exclusively in the lymph nodes (LN). According to the prevailing paradigm, the thymus is the main source of T lymphocytes in gnathostomes mainly because thymic epithelial cells have a unique ability to support early steps in T cell development. It is therefore remarkable that productive T cell development occurs in the OM+ LN, despite the absence of epithelial cells. The present study shows that in the OM+ LN: 1) MHC class I expression strictly on hemopoietic cells is sufficient to support the development of a diversified repertoire of CD8 T cells; 2) the efficiency of positive selection of specific TCR-transgenic T cells is not the same as in the thymus; 3) negative selection is very effective, despite the lack of an organized thymic-like medulla. Furthermore, our data suggest that extrathymic T lymphocytes developing in the OM+ LN undergo extensive postselection expansion because they live in the microenvironment in which they were positively selected. This work illustrates how the division of labor between primary and secondary lymphoid organs influences the repertoire and homeostasis of T lymphocytes.
Induction of CD4 T Cell Changes in Murine AIDS Is Dependent on Costimulation and Involves a Dysregulation of Homeostasis
Authors: Yen Michael H. , Lepak Nancy , Swain Susan L.
Published: April 2014

Strong CD4 T cell activation and proliferation are seen in susceptible mice infected with the murine retroviral inoculum, LP-BM5, which produces an immunodeficiency syndrome called murine AIDS (MAIDS). We developed a short term adoptive transfer model of MAIDS to examine the requirements for the CD4 T cell response. Naive CD4 T cells from uninfected donors responded quickly after adoptive transfer into MAIDS-infected hosts, becoming activated and proliferating within several days. Using blocking mAbs to costimulatory ligands and CD4 T cells deficient in expression of their receptors, we found that the CD4 T cell response requires CD28:B7.1/B7.2 interactions, but not CTLA4 or CD40-CD40 ligand interactions. Naive CD4 T cells did not respond in H-2M-deficient mice with MAIDS, suggesting that disease requires recognition of self peptide-MHC complexes. The self MHC-dependent division and accumulation of large numbers of CD4 T cells suggest that MAIDS involves a disruption of the balance of homeostatic signals. Supporting this hypothesis, CD4 T cells from mice with MAIDS failed to regulate the homeostatic division of naive CD4 T cells in a cotransfer model. Thus, a combination of up-regulation of costimulatory ligands and disruption of homeostatic control may be responsible for CD4 lymphoproliferation in MAIDS.
Regulation of Retinoid X Receptor Responsive Element-Dependent Transcription in T Lymphocytes by Ser/Thr Phosphatases: Functional Divergence of Protein Kinase C (PKC)θ and PKCα in Mediating Calcineurin-Induced Transactivation
Authors: Ishaq Mohammad , Fan Ming , Wigmore Kip , Gaddam Arunasri , Natarajan Ven
Published: April 2014

T lymphocyte activation signals regulate the expression and transactivation function of retinoid X receptor (RXR) α through an interplay of complex signaling cascades that are not yet fully understood. We show that cellular Ser/Thr protein phosphatases (PPs) play an important role in mediating these processes. Inhibitors specific for PP1 and PP2A decreased basal expression of RXRα RNA and protein in T lymphocyte leukemia Jurkat cells and prevented activation-induced RXRα accumulation in these cells. In addition, these inhibitors attenuated the RXR responsive element (RXRE)-dependent transcriptional activation in transient transfection assays. Inhibitors of calcineurin (CN), by contrast, did not have any effect on the basal RXRα expression and even augmented activation-induced RXRα expression. Expression of a dominant-active (DA) mutant of CN together with a DA mutant of protein kinase C (PKC)θ, a novel PKC isoform, significantly increased RXRE-dependent transcription. Expression of catalytically inactive PKCθ or a dominant-negative mutant of PKCθ failed to synergize with CN and did not increase RXRE-dependent transcription. Expression of a DA mutant of PKCα or treatment with PMA was found to attenuate PKCθ and CN synergism. We conclude that PP1, PP2A, and CN regulate levels and transcriptional activation function of RXRα in T cells. In addition, CN synergizes with PKCθ to induce RXRE-dependent activation, a cooperative function that is antagonized by the activation of the conventional PKCα isoform. Thus, PKCθ and PKCα may function as positive and negative modulators, respectively, of CN-regulated RXRE-dependent transcription during T cell activation.
The Majority of Immunogenic Epitopes Generate CD4+ T Cells That Are Dependent on MHC Class II-Bound Peptide-Flanking Residues
Authors: Arnold Paula Y. , La Gruta Nicole L. , Miller Tim , Vignali Kate M. , Adams P. Scott , Woodland David L. , Vignali Dario A. A.
Published: April 2014

Peptides bind to MHC class II molecules with a defined periodicity such that the peptide-flanking residues (PFRs) P-1 and P11, which lie outside the core binding sequence (P1–P9), are solvent exposed and accessible to the TCR. Using a novel MHC class II:peptide binding assay, we defined the binding register for nine immunogenic epitopes to formally identify the flanking residues. Seven of the nine epitopes, restricted by H-2Ak, H-2Ag7, or H-2Ek, were found to generate T cells that were completely dependent on either P-1 or P11, with dependency on P-1 favored over P11. Such PFR dependency appears to be influenced by the type of amino acid exposed, in that residues that can form salt bridges or hydrogen bonds are favored over small or hydrophobic residues. Peptides containing alanine substitutions at P-1 or P11 in place of PFRs that mediate dependency were considerably less immunogenic and mediated a substantially reduced in vitro recall response to the native protein, inferring that PFR recognition increases immunogenicity. Our data suggest that PFR recognition is a common event characteristic of all MHC class II-restricted T cell responses. This key feature, which is not shared by MHC class I-restricted responses, may underlie the broad functional diversity displayed by MHC class II-restricted T cells.