bluuebunny/crossref_metadata_2025 huggingface dataset:
An IFN-γ-Dependent Pathway Controls Stimulation of Memory Phenotype CD8+ T Cell Turnover In Vivo by IL-12, IL-18, and IFN-γ
Authors: Tough David F. , Zhang Xiaohong , Sprent Jonathan
Published: April 2014

Unlike naive T cells, memory phenotype (CD44high) T cells exhibit a high background rate of turnover in vivo. Previous studies showed that the turnover of memory phenotype CD8+ (but not CD4+) cells in vivo can be considerably enhanced by products of infectious agents such as LPS. Such stimulation is TCR independent and hinges on the release of type I IFNs (IFN-I) which leads to the production of an effector cytokine, probably IL-15. In this study, we describe a second pathway of CD44high CD8+ stimulation in vivo. This pathway is IFN-γ rather than IFN-I dependent and is mediated by at least three cytokines, IL-12, IL-18, and IFN-γ. As for IFN-I, these three cytokines are nonstimulatory for purified T cells and under in vivo conditions probably act via production of IL-15.
Involvement of Bik, a Proapoptotic Member of the Bcl-2 Family, in Surface IgM-Mediated B Cell Apoptosis
Authors: Jiang Aimin , Clark Edward A.
Published: April 2014

Apoptosis plays a central role in shaping the repertoire of circulating mature B lymphocytes, but the underlying molecular mechanisms regulating B cell fate are not well understood. Human B104 B lymphoma cells undergo apoptosis after surface Ig (sIg)M, but not sIgD, ligation; sIgM-mediated apoptosis of B104 cells apparently requires new gene transcription because actinomycin D can inhibit the apoptotic response. Here we report that expression of Bik, a proapoptotic member of the Bcl-2 family, is greatly increased after sIgM ligation. Bik expression was tightly controlled at both transcriptional and post-transcriptional levels. Whereas a calcineurin-dependent pathway was essential for Bik mRNA induction, both the phosphatidylinositol 3-kinase (PI3K)- and the calcineurin-dependent pathways were required for the sustained production of Bik protein. Consistent with these findings, sIgD ligation, which leads to the similar calcium mobilization and increases in Bik mRNA, induced only a transient activation of PI3K and did not lead to sustained Bik protein expression. Furthermore, sustained Bik protein expression correlated with B cell apoptosis, as treatment with either a calcineurin inhibitor or PI3K inhibitors blocked both sIgM-mediated sustained Bik protein induction and apoptosis. In addition, sIgM ligation strongly increased the amount of Bik associated with endogenous Bcl-x, but sIgD ligation did not. Studies with caspase inhibitors also revealed that Bik and Bcl-x interacted upstream of caspases in the B cell apoptosis cascade. Thus, Bik protein induction and, subsequently, sequestering of antiapoptotic Bcl-x by Bik may play an important role in regulating B cell apoptosis.
CD45 Tyrosine Phosphatase Controls Common γ-Chain Cytokine-Mediated STAT and Extracellular Signal-Related Kinase Phosphorylation in Activated Human Lymphoblasts: Inhibition of Proliferation Without Induction of Apoptosis
Authors: Blank Norbert , Kriegel Martin , Hieronymus Thomas , Geiler Thomas , Winkler Silke , Kalden Joachim R. , Lorenz Hanns-Martin
Published: April 2014

The objective of this study was to test whether CD45 signals can influence signaling processes in activated human lymphoblasts. To this end, we generated lymphoblasts which proliferate in response to common γ-chain cytokines, but readily undergo apoptosis after cytokine withdrawal. In experiments with the CD45R0 mAb UCHL-1, but not control CD45 mAbs, we found significant inhibition of proliferation. Interestingly, the pan-CD45 mAb GAP8.3, which is most effective in inhibition of OKT-3-mediated proliferation in quiescent lymphocytes, was ineffective in lymphoblasts. Addition of CD3 mAb OKT-3 had no influence on IL-2-mediated proliferation (with or without UCHL-1). In contrast, after addition of OKT-3 to IL-4- and IL-7-stimulated proliferation assays, UCHL-1 signals could not significantly alter cellular proliferation. We did not find induction of apoptosis following CD45R0 signaling. In Western blots using mAbs detecting phosphorylated STAT-3, STAT-5, STAT-6, or extracellular signal-related kinase 1/2, we found that CD45R0 signaling could effectively diminish phosphorylation of these intracellular signaling components. Using RT-PCR, we found that CD45R0 signaling inhibited IL-2 mRNA production without major influence on IL-13, IL-5, or IFN-γ mRNA levels. Costimulation with OKT-3 and IL-2 optimally induced secretion of IFN-γ, TNF-α, and IL-5, which was not decreased by CD45 signals. In conclusion, we illustrate that CD45R0 signals control early cytokine receptor-associated signaling processes and mRNA and DNA synthesis in activated human lymphoblasts. Furthermore, we show the existence of CD45 epitopes (GAP8.3), which are active and critical for signaling in quiescent lymphocytes, but are nonfunctional in activated human lymphoblasts.
Recombinant Allergens with Reduced Allergenicity but Retaining Immunogenicity of the Natural Allergens: Hybrids of Yellow Jacket and Paper Wasp Venom Allergen Antigen 5s
Authors: King Te Piao , Jim Sui Y. , Monsalve Rafael I. , Kagey-Sobotka Anne , Lichtenstein Lawrence M. , Spangfort Michael D.
Published: April 2014

The homologous venom allergen Ag 5s from the yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis) have 59% sequence identity of their respective 204 and 205 amino acid residues, and they have low degrees of antigenic cross-reactivity in insect allergic patients and in animal models. Hybrids containing different segments of these two vespid Ag 5s were expressed in yeast. Circular dichroism spectroscopy suggests the hybrids to have the secondary structure of natural Ag 5. Inhibition ELISA with human and murine Abs suggests the hybrids to have the discontinuous B cell epitopes of the natural Ag 5 but with an altered epitope density. The hybrids were immunogenic in mice for B and T cell responses to both Ag 5s. The N-terminal region of Ag 5 was found to contain its dominant B cell epitope(s). Hybrids containing 10–49 residues of yellow jacket Ag 5 showed 100- to 3000-fold reduction in allergenicity when tested by histamine release assay with basophils of yellow jacket-sensitive patients. Our findings suggest that hybrids represent a useful approach to map the discontinuous B cell epitope-containing regions of proteins. They also suggest that Ag 5 hybrids may be useful immunotherapeutic reagents in man.
Development of Intestinal Intraepithelial Lymphocytes, NK Cells, and NK 1.1+ T Cells in CD45-Deficient Mice
Authors: Martin Steven M. , Mehta Indira K. , Yokoyama Wayne M. , Thomas Matthew L. , Lorenz Robin G.
Published: April 2014

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6−/− mouse. As NK1.1+ CD3+ (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9−/−) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9−/− mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8+) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-γ. In the absence of CD45, a significant population of extrathymically derived CD8αα+ iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8+ NK-T cells, NK cells, or CD8αα+ iIELs, but is essential for TCR-mediated function and cytokine production.
Transcriptional Regulation of Intracellular IL-1 Receptor Antagonist Gene by IL-1α in Primary Mouse Keratinocytes
Authors: La Eunhye , Fischer Susan M.
Published: April 2014

The inflammatory cytokine IL-1α mediates inflammatory reactions in skin and up-regulates the expression of other proinflammatory genes. We previously found that IL-1α also increases steady state mRNA levels for intracellular IL-1 receptor antagonist (icIL-1Ra) in primary mouse keratinocytes; however, the mechanism for this was unknown. Here we show that increased expression in primary keratinocytes is due to increased rates of transcription. To study the transcriptional regulation of icIL-1Ra expression induced by IL-1α, we functionally characterized 4.5 kb of the 5′-flanking region of the human icIL-1Ra gene. Deletion analysis showed that regulatory elements were contained in the −598- and −288-bp region upstream of the transcription start site. Then we investigated cis- and trans-acting factors required for icIL-1Ra expression and found that a NF-IL-6 site and a NF-κB site in the icIL-1Ra promoter were responsible for IL-1α-induced icIL-1Ra expression. Moreover, gel shift assays and cotransfection experiments showed that CCAAT/enhancer-binding proteins α, β, and p65 bind to the NF-IL-6 site and NF-κB site, respectively, and functionally trans-activate the icIL-1Ra promoter. Finally, mutational analysis confirmed that these elements were both essential for maximal transcription induced by IL-1α.
Multiple Transcription Factors Regulate the Inducible Expression of the Human Complement Receptor 2 Promoter
Authors: Vereshchagina Lyudmila A. , Tolnay Mate , Tsokos George C.
Published: April 2014

Complement receptor 2 (CR2) is regulated at the transcriptional level, but the promoter elements and the transcription factors that bind to them and contribute to its regulation are unknown. After documenting that PMA and cAMP induced the activity of the CR2 promoter by 10-fold, we conducted promoter truncation and mutagenesis experiments, in conjunction with shift assays, to determine the functionally important regions of the promoter and the proteins that bind to them. We identified two regions, separated by ∼900 nucleotides, which together were responsible for inducible promoter activity. Mutagenesis of single promoter elements demonstrated a functional upstream stimulatory factor/E box in the TATA box-proximal region and three equally important, closely spaced, CREB/AP-1 half-sites in the upstream promoter region. The cAMP response element-binding protein (CREB)/AP-1 half-sites bound in vitro Jun and CREB that are induced by protein kinases A and/or C. The 900-nucleotide segment stretching between the above two regions had no functional impact on the induced transcription, and its deletion increased the promoter activity. Finally, a region upstream of the distal site had a repressor activity on CR2 transcription. Moreover, IL-4 induced binding of CREB and AP-1 to the upstream promoter elements and resulted in increased CR2 surface protein expression. These studies have characterized regions of the CR2 promoter and the transcription factors that bind to them and are crucial to induced CR2 expression. Our studies may provide insights to novel approaches to modulate B cell function by regulating CR2 gene transcription.
Characteristics of HIV-1 Nef Regions Containing Multiple CD8+ T Cell Epitopes: Wealth of HLA-Binding Motifs and Sensitivity to Proteasome Degradation
Authors: Choppin Jeannine , Cohen William , Bianco Alberto , Briand Jean-Paul , Connan Francine , Dalod Marc , Guillet Jean-Gérard
Published: April 2014

First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90–97, 135–143, 71–81, 77–85, 90–100, 73–82, and 128–137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef66–100. This fragment was efficiently cleaved, and NH2-terminally extended precursors of epitope 71–81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.
RNA-Dependent Protein Kinase PKR Is Required for Activation of NF-κB by IFN-γ in a STAT1-Independent Pathway
Authors: Deb Amitabha , Haque S. Jaharul , Mogensen Trine , Silverman Robert H. , Williams Bryan R. G.
Published: April 2014

The IFN-inducible dsRNA-activated protein kinase PKR regulates protein synthesis through phosphorylation of eukaryotic initiation factor-2α. It also acts as a signal transducer for transcription factors NF-κB, IFN regulatory factor-1, and activating transcription factor-2. IFN-γ, a pleiotropic cytokine, elicits gene expression by activating the Janus kinase-STAT signaling pathway. IFN-γ can synergize with TNF-α to activate NF-κB in a number of cell lines. Here we show that IFN-γ alone can activate NF-κB, by a Janus kinase-1-mediated, but Stat1-independent, mechanism. NF-κB activation by IFN-γ is associated with degradation of IκB β. The IFN-γ response can be blocked by 2′,5′-oligoadenylate-linked antisense chimeras against PKR mRNA. There was no activation of NF-κB by IFN in PKR-null cells, indicating that PKR is required for IFN-γ signaling to NF-κB.
Positive and Negative Roles of theTrans-Acting T Cell Factor-1 for the Acquisition of Distinct Ly-49 MHC Class I Receptors by NK Cells
Authors: Kunz Béatrice , Held Werner
Published: April 2014

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 β2-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.
Enhancement of Sindbis Virus Self-Replicating RNA Vaccine Potency by Linkage ofMycobacterium tuberculosisHeat Shock Protein 70 Gene to an Antigen Gene
Authors: Cheng Wen-Fang , Hung Chien-Fu , Chai Chee-Yin , Hsu Keng-Fu , He Liangmai , Rice Charles M. , Ling Morris , Wu T.-C.
Published: April 2014

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8+ T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.
Herpes Simplex Inhibits the Capacity of Lymphoblastoid B Cell Lines to Stimulate CD4+ T Cells
Authors: Barcy Serge , Corey Lawrence
Published: April 2014

HSV establish a lifelong persistent infection in their host even among immunocompetent persons. The viruses use a variety of immune evasion strategies, presumably to assist persistent replication in the human host. We have observed that infection of human B lymphoblastoid cells (B-LCL) by HSV resulted in a strong inhibition of their ability to induce CD4+ T cell clone proliferation and cytokine secretion. This inhibitory effect occurs in a variety of both HSV- and HIV-specific clones from three different patients. The inhibition is observed when the Ag is provided either as a soluble protein or as a synthetic peptide and is not associated with detectable down-modulation of the MHC class II molecules or costimulatory molecules. Expression of the HSV-1 unique sequence 1 gene (US1) is necessary and sufficient to induce this inhibition of APC function. US1 gene expression also made B-LCL less susceptible to CD4+ T cell-mediated lysis. These data indicate a novel immune evasion strategy by HSV-1 in which Ag-processing cells that become infected by HSV-1 are inhibited in their ability to induce subsequent CD4+ T cell activation.
Activity of Human IgG and IgA Subclasses in Immune Defense Against Neisseria meningitidis Serogroup B
Authors: Vidarsson Gestur , van der Pol W.-Ludo , van den Elsen Jean M. H. , Vilé Henriëtte , Jansen Marc , Duijs Jacques , Morton H. Craig , Boel Edwin , Daha Mohamed R. , Corthésy Blaise , van de Winkel Jan G. J.
Published: April 2014

Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1–3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.
Apoptosis: One of the Mechanisms That Maintains Unresponsiveness of the Intestinal Mucosal Immune System
Authors: Bu Ping , Keshavarzian Ali , Stone David D. , Liu Jianzhong , Le Phong T. , Fisher Susan , Qiao Liang
Published: April 2014

Intestinal mucosa is constantly exposed to environmental Ags. Activation of lamina propria (LP) T cells by luminal Ags may lead to the production of inflammatory cytokines and subsequent mucosal inflammation and tissue damage. However, in normal circumstances, LP T cells do not respond to antigenic stimulation. The mechanisms of this unresponsiveness in healthy subjects are not fully understood. In this study, we found by in vivo analysis that, except for T cells in lymph nodules of the mucosa, 15% of LP T cells underwent apoptosis in normal individuals. In contrast, there was a marked reduction in apoptosis of LP T cells in patients with inflammatory bowel disease (Crohn’s disease and ulcerative colitis) and those with specific colitis. Our findings suggest that apoptosis might be a mechanism that turns off mucosal T cell responses to environmental Ags in healthy subjects, and resistance to apoptosis could be an important cause of mucosal immune dysregulation and tissue inflammation in colitis.
Complement Component C3 Is Not Required for Full Expression of Immune Complex Glomerulonephritis in MRL/lprMice
Authors: Sekine Hideharu , Reilly Christopher M. , Molano Ivan D. , Garnier Gérard , Circolo Antonella , Ruiz Philip , Holers V. Michael , Boackle Susan A. , Gilkeson Gary S.
Published: April 2014

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3−/−), heterozygous (C3+/−), and C3 wild-type (C3+/+) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3−/− mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3−/− mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.
Impairment of CD8+ T Suppressor Cell Function in Patients with Active Systemic Lupus Erythematosus
Authors: Filaci Gilberto , Bacilieri Sabrina , Fravega Marco , Monetti Monia , Contini Paola , Ghio Massimo , Setti Maurizio , Puppo Francesco , Indiveri Francesco
Published: April 2014

Alteration of T cell suppression function has been recognized in patients with systemic lupus erythematosus (SLE). Recently, CD8+ T suppressor lymphocytes (CD8+ Ts) have been generated in vitro by incubating purified CD8+ T cells with IL-2 and GM-CSF. Using this method, we generated CD8+ Ts from patients affected by SLE. No major differences were found in the CD8+ Ts phenotype between SLE patients and healthy subjects. CD8+ Ts from SLE patients with active disease did not inhibit the anti-CD3 mAb-induced proliferation of autologous PBMC, whereas CD8+ Ts from SLE patients in remission exerted an inhibitory activity comparable to normal subjects. The inhibitory effect of CD8+ Ts cells was neither mediated by cytotoxic activity nor by apoptosis induction. Two cytokines, IFN-γ and IL-6, were found to be responsible for the function of CD8+ Ts. In fact, counteraction of CD8+ Ts suppression activity was obtained by blocking IFN-γ with a specific Ab or by inhibiting CD8+ Ts-mediated IL-6 secretion by an antisense oligonucleotide. Interestingly, CD8+ Ts from SLE patients showed a peculiar cytokine pattern characterized by an impaired secretion of IL-6 and an increased secretion of IL-12. Thus, it appears that an altered balance between inhibitory (IL-6) and stimulatory (IL-12) cytokines might be responsible for the functional impairment of CD8+ Ts in SLE patients.
Cutting Edge: Cellular Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Inhibitory Protein Protects Germinal Center B Cells from Apoptosis During Germinal Center Reactions
Authors: van Eijk Marco , Medema Jan Paul , de Groot Cornelis
Published: April 2014

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIPL), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIPL decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIPL degradation and blocks all signs of apoptosis, even in the presence of anti-Fas Abs. cFLIPL expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.
Low-Level Monocyte Chemoattractant Protein-1 Stimulation of Monocytes Leads to Tumor Formation in Nontumorigenic Melanoma Cells
Authors: Nesbit Mark , Schaider Helmut , Miller Thomas H. , Herlyn Meenhard
Published: April 2014

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-α, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-α production.
Mechanism of Melphalan-Induced B7-1 Gene Expression in P815 Tumor Cells
Authors: Donepudi Manjula , Raychaudhuri Pradip , Bluestone Jeffrey A. , Mokyr Margalit B.
Published: April 2014

We have previously shown that exposure of P815 tumor cells to melphalan (l-phenylalanine mustard; l-PAM) leads to up-regulation of B7-1 surface expression, and this l-PAM-induced up-regulation requires de novo RNA synthesis and is associated with accumulation of B7-1 mRNA. Here we show that the effect of l-PAM on B7-1 surface expression can be mimicked by exposing P815 tumor cells to oxidative stress but not to heat shock. Moreover, the antioxidant N-acetyl-l-cysteine prevented the l-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells, suggesting that reactive oxygen species are involved in the transcriptional regulation of l-PAM-induced B7-1 gene expression. Although AP-1 and NF-κB are regarded as redox-sensitive transcription factors and the promoter/enhancer region of the B7-1 gene contains an AP-1 and an NF-κB binding site, exposure of P815 tumor cells to l-PAM led to rapid and transient activation only of NF-κB, but not AP-1, that bound specifically to a probe containing the respective binding site in the murine or human B7-1 gene. Moreover, exposure of P815 tumor cells to a cell-permeable peptide that selectively inhibits NF-κB activation by blocking the activation of the IκB-kinase complex was found to inhibit the l-PAM-induced B7-1 mRNA accumulation, indicating that NF-κB activation is essential for the l-PAM-induced B7-1 gene expression. Taken together, these results indicate that l-PAM leads to activation of B7-1 gene expression by activating NF-κB via a pathway that involves reactive oxygen species.
Identification of MHC Class II-Associated Peptides That Promote the Presentation of Toxic Shock Syndrome Toxin-1 to T Cells
Authors: Hogan Robert J. , VanBeek Josine , Broussard Dana R. , Surman Sherri L. , Woodland David L.
Published: April 2014

Previous studies have shown that the DM-deficient cell line, T2-I-Ab, is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-Ab-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-Ab-binding peptide, staphylococcal enterotoxin B 121–136, onto T2-I-Ab cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-Ab. Here we show that peptides that do not promote TSST-1 presentation can be converted into “promoting” peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Eα proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.
Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 on Murine Dendritic Cells Is a Potent Regulator of T Cell Stimulation
Authors: Kammerer Robert , Stober Detlef , Singer Bernhard B. , Öbrink Björn , Reimann Jörg
Published: April 2014

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1α, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4+ T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.
Mechanisms of the Antimetastatic Effect in the Liver and of the Hepatocyte Injury Induced by α-Galactosylceramide in Mice
Authors: Nakagawa Ryusuke , Nagafune Ikuko , Tazunoki Yoshiko , Ehara Hiromi , Tomura Hitomi , Iijima Rieko , Motoki Kazuhiro , Kamishohara Masaru , Seki Shuhji
Published: April 2014

The role of mouse liver NK1.1 Ag+ T (NKT) cells in the antitumor effect of α-galactosylceramide (α-GalCer) has been unclear. We now show that, whereas α-GalCer increased the serum IFN-γ concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of α-GalCer. Injection of Abs to IFN-γ also inhibited the α-GalCer-induced increase in antitumor cytotoxicity of MNCs. α-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas α-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCs. The α-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-γ. Depletion of CD8+ T cells did not affect the α-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of α-GalCer on the survival of B6 mice. Thus, IFN-γ produced by α-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8+ T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate α-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.
The Fetal Liver Counterpart of Adult Common Lymphoid Progenitors Gives Rise to All Lymphoid Lineages, CD45+CD4+CD3− Cells, As Well As Macrophages
Authors: Mebius Reina E. , Miyamoto Toshihiro , Christensen Julie , Domen Jos , Cupedo Tom , Weissman Irving L. , Akashi Koichi
Published: April 2014

We identified an IL-7Rα+Sca-1lowc-Kitlow population in E14 fetal liver, which is the phenotypical analog of common lymphoid progenitors (CLP) in adult bone marrow. After transfer into newborn mice, the IL-7Rα+Sca-1lowc-Kitlow population rapidly differentiated into CD45+CD4+CD3− cells, which are candidate cells for initiating lymph node and Peyer’s patch formation. In addition, this population also gave rise to B, T, NK, and CD8α+ and CD8α− dendritic cells. The fetal liver precursors expressed a significantly lower level of the myeloid-suppressing transcription factor Pax-5, than adult CLP, and retained differentiation activity for macrophages in vitro. We propose that the transition from fetal liver IL-7Rα+Sca-1lowc-Kitlow cells to adult CLP involves a regulated restriction of their developmental potential, controlled, at least in part, by Pax-5 expression.
A Low Affinity TCR Ligand Restores Positive Selection of CD8+ T Cells In Vivo
Authors: Stefanski Heather E. , Mayerova Dita , Jameson Stephen C. , Hogquist Kristin A.
Published: April 2014

The T cell repertoire is shaped by the processes of positive and negative selection. During development, the TCR binds self peptide-MHC complexes in the thymus, and the kinetics of this interaction are thought to determine the thymocyte’s fate. For development of CD8+ T cells, the data supporting such a model have been obtained using fetal thymic organ culture. To confirm the fidelity of this model in vivo, we studied development of OT-I TCR-transgenic mice that expressed different individual Kb binding peptides in thymic epithelial cells under the control of the human keratin 14 promoter. We used a system that allowed TAP-independent expression of the peptide-MHC complex, such that the ability of given peptides to restore positive selection in TAPo mice could be assessed. We found that transgenic expression of a TCR antagonist peptide (E1) in vivo efficiently restored positive selection of OT-I T cells in TAPo mice. An unrelated transgenic peptide (SIY) did not restore selection of OT-I T cells, nor did the E1-transgenic peptide restore selection of an unrelated receptor (2C), showing that positive selection is peptide specific in vivo, as observed in organ cultures. Neither E1 nor SIY transgenes increased the polyclonal CD8 T cell repertoire size in non-TCR-transgenic animals, arguing that single class I binding peptides do not detectably affect the size of the CD8 T cell repertoire when expressed at low levels. We also observed that OT-I T cells selected in TAPo-E1 mice were functional in their response to Ag; however, there was a lag in this response, suggesting that the affinity of the TCR interaction with MHC-self peptide can result in fine-tuning of the T cell response.
Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic Reticulum Export Imaged by Fluorescence Resonance Energy Transfer
Authors: Pentcheva Tsvetelina , Edidin Michael
Published: April 2014

Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.
A Novel Pathway Regulating Lipopolysaccharide-Induced Shock by ST2/T1 Via Inhibition of Toll-Like Receptor 4 Expression
Authors: Sweet Matthew J. , Leung Bernard P. , Kang Daiwu , Sogaard Morten , Schulz Kerstin , Trajkovic Vladimir , Campbell Carol C. , Xu Damo , Liew Foo Y.
Published: April 2014

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-α but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-κB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-α. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.
An Alternative Form of IL-18 in Human Blood Plasma: Complex Formation with IgM Defined by Monoclonal Antibodies
Authors: Shida Kyoko , Shiratori Ikuo , Matsumoto Misak , Fukumori Yasuo , Matsuhisa Akio , Kikkawa Satomi , Tsuji Shoutaro , Okamura Haruki , Toyoshima Kumao , Seya Tsukasa
Published: April 2014

Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mAbs. Plasma IL-18, designated type 2, was detected by this ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in ∼30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25–100 ng/ml) compared with those of type 1 (0.05–0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its Mr was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-γ induction, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in ∼30% normal subjects.
Association of ERp57 with Mouse MHC Class I Molecules Is Tapasin Dependent and Mimics That of Calreticulin and not Calnexin
Authors: Harris Michael R. , Lybarger Lonnie , Yu Yik Y. L. , Myers Nancy B. , Hansen Ted H.
Published: April 2014

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-β2-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of Ld mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient .220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.
Flexibility in MHC and TCR Recognition: Degenerate Specificity at the T Cell Level in the Recognition of Promiscuous Th Epitopes Exhibiting No Primary Sequence Homology
Authors: Joshi Sunil K. , Suresh Padma R. , Chauhan Virander S.
Published: April 2014

Recognition of peptide Ags by T cells through the TCR can be highly specific. In this report we show the degeneracy of Ag recognition at both MHC and TCR levels. We present evidence that unrelated promiscuous Th cell epitopes from various protein sources exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive proliferative responses at the bulk T cell level. This epitopic mimicry was also observed when peptide (CS.T3378–395 and TT830–844)-specific CD4+ T cell lines and T cell hybridoma clones were used in proliferation and Ag presentation assays. A scrambled CS.T3378–395 peptide did not show any proliferation, indicating that the specificity of the cross-reactive responses may be linked with the primary structure of the peptides. Blocking of CS.T3378–395-specific CD4+ T cell proliferation by anti-MHC class II mAb showed that recognition of promiscuous T cell epitopes is largely in association with MHC class II molecules. These findings suggest that promiscuous Th epitopes may be useful in designing peptide-based vaccine constructs. At the same time these results show that at the T cell level there may be a great deal of immunological cross-reactivity between heterologous pathogens, and because of this the host’s response to a pathogen may be modified by its previous experience with other unrelated pathogens.
Immunological and Biochemical Characterization of Streptococcal Pyrogenic Exotoxins I and J (SPE-I and SPE-J) from Streptococcus pyogenes
Authors: Proft Thomas , Arcus Vickery L. , Handley Vanessa , Baker Edward N. , Fraser John D.
Published: April 2014

Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II β-chain in a zinc-dependent mode but not to the generic MHC class II α-chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DRβ–SPE-J–SPE-J–HLA-DRβ. SPE-I preferably stimulates T cells bearing the Vβ18.1 TCR, which is not targeted by any other known SAg. SPE-J almost exclusively stimulates Vβ2.1 T cells, a Vβ that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistinguishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C.