bluuebunny/crossref_metadata_2025 huggingface dataset:
A Subset of NKT Cells That Lacks the NK1.1 Marker, Expresses CD1d Molecules, and Autopresents the α-Galactosylceramide Antigen
Authors: Hameg Agathe , Apostolou Irina , Leite-de-Moraes Maria , Gombert Jean-Marc , Garcia Corinne , Koezuka Yasuhiko , Bach Jean-François , Herbelin André
Published: April 2014

In the present report, we characterize a novel T cell subset that shares with the NKT cell lineage both CD1d-restriction and high reactivity in vivo and in vitro to the α-galactosylceramide (α-GalCer) glycolipid. These cells preferentially use the canonical Vα14-Jα281 TCR-α-chain and Vβ8 TCR-β segments, and are stimulated by α-GalCer in a CD1d-dependent fashion. However, in contrast to classical NKT cells, they lack the NK1.1 marker and express high surface levels of CD1d molecules. In addition, this NK1.1− CD1dhigh T subset, further referred to as CD1dhigh NKT cells, can be distinguished by its unique functional features. Although NK1.1+ NKT cells require exogenous CD1d-presenting cells to make them responsive to α-GalCer, CD1dhigh NKT cells can engage their own surface CD1d in an autocrine and/or paracrine manner. Furthermore, in response to α-GalCer, CD1dhigh NKT cells produce high amounts of IL-4 and moderate amounts of IFN-γ, a cytokine profile more consistent with a Th2-like phenotype rather than the Th0-like phenotype typical of NK1.1+ NKT cells. Our work reveals a far greater level of complexity within the NKT cell population than previously recognized and provides the first evidence for T cells that can be activated upon TCR ligation by CD1d-restricted recognition of their ligand in the absence of conventional APCs.
Potent Costimulation of Effector T Lymphocytes by Human Collagen Type I
Authors: Rao Wei Hong , Hales Jonathan M. , Camp Richard D. R.
Published: April 2014

Purified, resting peripheral blood T lymphocytes were previously reported to undergo β1 integrin-dependent activation when cultured with anti-CD3 mAb coimmobilized with fibronectin, but not type I collagen. However, the extravascular T cells that encounter immobilized extracellular matrix proteins and are involved in disease pathogenesis have different properties from resting peripheral blood cells. In this study, we confirm that resting CD4+ and CD8+ T cells from peripheral blood are costimulated by immobilized fibronectin, but not type I collagen. In contrast, Ag- or mitogen-stimulated CD4+ and CD8+ T cell lines, used as models of the effector cells involved in disease, are more potently costimulated by type I collagen than fibronectin. The collagen-induced effects are similar in assays with serum-free medium and in more physiological assays in which anti-CD3 mAb is replaced by a threshold concentration of Ag and irradiated autologous PBMC as APC. The responses are β1 integrin dependent and mediated largely by very late Ag (VLA) 1 and 2, as shown by their up-regulation on the T cell lines as compared with freshly purified resting PBL, and by the effects of blocking mAb. Reversed phase HPLC located the major costimulatory sequence(s) in the α1 chain of type I collagen, the structure of which was confirmed by amino acid sequencing. The results demonstrate the potential importance of type I collagen, an abundant extracellular matrix protein, in enhancing the activation of extravascular effector T cells in inflammatory disease, and point to a new immunotherapeutic target.
Regulation of β1 Integrin-Mediated Adhesion by T Cell Receptor Signaling Involves ZAP-70 but Differs from Signaling Events That Regulate Transcriptional Activity
Authors: Epler Jennifer A. , Liu Rugao , Chung Heekyoung , Ottoson Nadine C. , Shimizu Yoji
Published: April 2014

Stimulation of the CD3/TCR results within minutes in an increase in T cell adhesion mediated by β1 integrins. The biochemical pathways that control CD3-mediated increases in β1 integrin-mediated adhesion remain poorly characterized. In this study, the role of the tyrosine kinase ZAP-70 in the regulation of β1 integrin activity by the CD3/TCR was investigated. CD3 stimulation did not increase β1 integrin-mediated adhesion of the ZAP-70-deficient Jurkat T cell line, P116, to the β1 integrin ligand fibronectin. Reintroduction of wild-type ZAP-70, but not a kinase-inactive variant, K369R, corrected the adhesive defect observed in P116 T cells. In addition, the kinase-inactive ZAP-70 mutant inhibited CD3-induced adhesion of primary human T cell blasts. Interestingly, a ZAP-70 mutant with a tyrosine to phenylalanine substitution at position 319 (Y319F) restored the adhesive defect in P116 T cells, even though Y319F ZAP-70 failed to fully reconstitute CD3-initiated NF-AT-dependent transcription and tyrosine phosphorylation of the LAT adapter protein. Finally, expression of mutants of LAT and the SLP-76 adapter protein that modulate CD3-mediated activation of an NF-AT reporter gene failed to block CD3-induced increases in β1 integrin-mediated adhesion. These observations support a model in which the tyrosine kinase activity of ZAP-70 kinase is critical for regulation of β1 integrin activity by CD3/TCR. However, the signaling events downstream of ZAP-70 that regulate CD3/TCR-mediated activation of β1 integrin function exhibit key differences when compared with the signaling pathways that regulate transcriptional events initiated by CD3/TCR stimulation.
TGF-β1 and IFN-γ Direct Macrophage Activation by TNF-α to Osteoclastic or Cytocidal Phenotype
Authors: Fox Simon W. , Fuller Karen , Bayley Kathryn E. , Lean Jenny M. , Chambers Timothy J.
Published: April 2014

TNF-related activation-induced cytokine (TRANCE; also called receptor activator of NF-κB ligand (RANKL), osteoclast differentiation factor (ODF), osteoprotegerin ligand (OPGL), and TNFSF11) induces the differentiation of progenitors of the mononuclear phagocyte lineage into osteoclasts in the presence of M-CSF. Surprisingly, in view of its potent ability to induce inflammation and activate macrophage cytocidal function, TNF-α has also been found to induce osteoclast-like cells in vitro under similar conditions. This raises questions concerning both the nature of osteoclasts and the mechanism of lineage choice in mononuclear phagocytes. We found that, as with TRANCE, the macrophage deactivator TGF-β1 strongly promoted TNF-α-induced osteoclast-like cell formation from immature bone marrow macrophages. This was abolished by IFN-γ. However, TRANCE did not share the ability of TNF-α to activate NO production or heighten respiratory burst potential by macrophages, or induce inflammation on s.c. injection into mice. This suggests that TGF-β1 promotes osteoclast formation not only by inhibiting cytocidal behavior, but also by actively directing TNF-α activation of precursors toward osteoclasts. The osteoclast appears to be an equivalent, alternative destiny for precursors to that of cytocidal macrophage, and may represent an activated variant of scavenger macrophage.
Stimulatory Function of gp49A, a Murine Ig-Like Receptor, in Rat Basophilic Leukemia Cells
Authors: Lee Kwang Ho , Ono Masao , Inui Masanori , Yuasa Takae , Takai Toshiyuki
Published: April 2014

Murine gp49, a 49-kDa type I transmembrane glycoprotein, is a member of the Ig-like receptors expressed on the surface of cells involved in natural immunity such as mast cells, NK cells, and macrophages. The two major subtypes, gp49A and gp49B, are encoded by two different genes adjacent to each other. gp49B contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic region and is known to function as an inhibitory molecule. In contrast, gp49A does not harbor any specific motif for signal transduction, nor has its physiological role been determined. Here we report on the stimulatory nature of gp49A by analyzing biochemical characteristics of chimeric molecules consisting of an ectodomain of Fc receptor and a C-terminal half of gp49A, namely the pretransmembrane, transmembrane, and cytoplasmic portions, expressed on the rat basophilic leukemia mast cell line. Cross-linking of the chimeric receptors evoked cytoplasmic calcium mobilization, PGD2 release, and transcription of IL-3 and IL-4 genes, but did not elicit degranulation of the cells. The chimeric molecule could be expressed as a singlet and a homodimeric form on the cell surface. A pretransmembrane cysteine residue of gp49A was necessary for dimer formation. Dimerization was be necessary for their incorporation into glycolipid-enriched membrane fraction (GEM) upon cross-linking stimuli. The calcium mobilization response was inhibited by treatment of cells with methyl-β-cyclodextrin, an inhibitor of GEM formation. Together with these results, it was strongly suggested that gp49A could be expressed as a homodimer and elicit activation signals that lead to calcium mobilization, eicosanoid production, and cytokine gene transcription through its incorporation into GEM.
Dendritic Cells Prime In Vivo Alloreactive CD4 T Lymphocytes Toward Type 2 Cytokine- and TGF-β-Producing Cells in the Absence of CD8 T Cell Activation
Authors: Foucras Gilles , Coudert Jérôme D. , Coureau Christiane , Guéry Jean-Charles
Published: April 2014

The mechanisms that influence the polarization of CD4 T cells specific for allogeneic MHC class II molecules in vivo are still poorly understood. We have examined the pathway of alloreactive CD4 T cell differentiation in a situation in which only CD4 T cells could be activated in vivo. In this report we show that priming of adult mice with allogeneic APC, in the absence of MHC class I-T cell interactions, induces a strong expansion of type 2 cytokine-producing allohelper T cells. These alloantigen-specific CD4 T cells directly recognize native allogeneic MHC class II molecules on APC and secrete, in addition to the prototypic Th2 cytokines IL-4, IL-5, and IL-10, large amounts of TGF-β. The default Th2-phenotype acquisition is not genetically controlled and occurred both in BALB/c and C57BL/6 mice. CD8 T cells are the principal cell type that controls CD4 T cell differentiation in vivo. Furthermore, we demonstrate that strong Th2 priming can be induced not only with allogeneic splenocytes but also with a low number of bone marrow-derived dendritic cells. Finally, using a passive transfer system, we provide direct evidence that CD8 T cell expansion in situ promotes alloreactive Th1 cell development principally by preventing their default development to the Th2 pathway in a mechanism that is largely IFN-γ independent. Therefore, this work demonstrates that type 2 cytokine production represents a dominant pathway of alloreactive CD4 T cell differentiation in adult mice, a phenomenon that was initially thought to occur only during the neonatal period.
Mouse Inducible Costimulatory Molecule (ICOS) Expression Is Enhanced by CD28 Costimulation and Regulates Differentiation of CD4+ T Cells
Authors: McAdam Alexander J. , Chang Tammy T. , Lumelsky Anna E. , Greenfield Edward A. , Boussiotis Vassiliki A. , Duke-Cohan Jonathan S. , Chernova Tatyana , Malenkovich Nelly , Jabs Claudia , Kuchroo Vijay K. , Ling Vincent , Collins Mary , Sharpe Arlene H. , Freeman Gordon J.
Published: April 2014

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4+ and CD8+ T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4+ T cells. First, CD4+ cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4+ cells and production of IFN-γ, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4+ T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4+ T cells produced more IFN-γ and less IL-4 and IL-10 than CD4+ cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.
Blockade of Costimulation Through B7/CD28 Inhibits Experimental Autoimmune Uveoretinitis, But Does Not Induce Long-Term Tolerance
Authors: Silver Phyllis B. , Hathcock Karen S. , Chan Chi-Chao , Wiggert Barbara , Caspi Rachel R.
Published: April 2014

It has been reported that costimulation blockade can result in T cell anergy. We investigated the effects of blocking costimulatory molecules in vivo on the development of experimental autoimmune uveoretinitis (EAU), a model for autoimmune uveitis in humans that is induced in mice by immunization with the retinal Ag interphotoreceptor retinoid binding protein. B10.A mice immunized with a uveitogenic regimen of interphotoreceptor retinoid-binding protein were treated with Abs to B7.1 and B7.2 for 2 wk. Evaluation of EAU and immunological responses 1 wk later showed that disease had been abrogated, and cellular responses were suppressed. To determine whether the costimulation blockade resulted in tolerance, adult-thymectomized mice immunized for uveitis and treated with anti-B7 or anti-CD28 were rechallenged for uveitis induction 5 wk after the initial immunization. Although confirmed to be disease free after the initial immunization, both anti-B7- and anti-CD28-treated mice developed severe EAU and elevated cellular responses after the rechallenge, equivalent to those of control mice. We conclude that in this model costimulatory blockade in vivo prevents the development of autoimmune disease, but does not result in long-term tolerance. The data are compatible with the interpretation that B7/CD28 blockade prevents generation of effector, but not of memory, T cells.
Allergen-Induced Airway Hyperreactivity Is Diminished in CD81-Deficient Mice
Authors: Deng Jun , Yeung V. Pete , Tsitoura Daphne , DeKruyff Rosemarie H. , Umetsu Dale T. , Levy Shoshana
Published: April 2014

We demonstrated previously that CD81−/− mice have an impaired Th2 response. To determine whether this impairment affected allergen-induced airway hyperreactivity (AHR), CD81−/− BALB/c mice and CD81+/+ littermates were sensitized i.p. and challenged intranasally with OVA. Although wild type developed severe AHR, CD81−/− mice showed normal airway reactivity and reduced airway inflammation. Nevertheless, OVA-specific T cell proliferation was similar in both groups of mice. Analysis of cytokines secreted by the responding CD81−/− T cells, particularly those derived from peribronchial draining lymph nodes, revealed a dramatic reduction in IL-4, IL-5, and IL-13 synthesis. The decrease in cytokine production was not due to an intrinsic T cell deficiency because naive CD81−/− T cells responded to polyclonal Th1 and Th2 stimulation with normal proliferation and cytokine production. Moreover, there was an increase in T cells and a decrease in B cells in peribronchial lymph nodes and in spleens of immunized CD81−/− mice compared with wild-type animals. Interestingly, OVA-specific Ig levels, including IgE, were similar in CD81−/− and CD81+/+ mice. Thus, CD81 plays a role in the development of AHR not by influencing Ag-specific IgE production but by regulating local cytokine production.
Vaginal Mucosa Serves as an Inductive Site for Tolerance
Authors: Black C. Allen , Rohan Lisa C. , Cost Marilyn , Watkins Simon C. , Draviam Romesh , Alber Sean , Edwards Robert P.
Published: April 2014

These data demonstrate that tolerance can be induced by vaginal Ag exposure. In these experiments, mice were given vaginal agarose gel suppositories containing either 5 mg OVA or saline for 6 h. Mice were given suppositories either during the estrous (estrogen dominant) or diestrous (progesterone dominant) stage of the estrous cycle. Mice were restrained during the inoculation period to prevent orovaginal transmission of the Ag. After 1 wk, mice were immunized s.c. with OVA in CFA. After 3 wk, mice were tested for delayed-type hypersensitivity responses by measuring footpad swelling and measuring in vitro proliferation of lymphocytes to Ag. Using ELISA, the magnitude of the serum Ab response was also measured. In some mice, FITC conjugated to OVA was used to track the dissemination of the protein into the systemic tissues. The magnitude of footpad swelling was significantly reduced in mice receiving OVA-containing suppositories during estrus compared with mice receiving saline suppositories. Concomitant decreases in the Ag-specific proliferative response were also observed in lymph node lymphocytes and splenocytes. Conversely, mice inoculated during diestrus did not show a decreased response to Ag by either footpad response or in vitro proliferation. Serum Ab titers in the estrus-inoculated mice did not decrease significantly. These data demonstrate that the reproductive tract can be an inductive site for mucosally induced tolerance. However, unlike other mucosal sites such as the lung and gastrointestinal tract, reproductive tract tolerance induction is hormonally regulated.
TNFR-Associated Factor Family Protein Expression in Normal Tissues and Lymphoid Malignancies
Authors: Zapata Juan M. , Krajewska Maryla , Krajewski Stanislaw , Kitada Shinichi , Welsh Kate , Monks Anne , McCloskey Natalie , Gordon John , Kipps Thomas J. , Gascoyne Randy D. , Shabaik Ahmed , Reed John C.
Published: April 2014

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.
Functional Uncoupling of the Janus Kinase 3-Stat5 Pathway in Malignant Growth of Human T Cell Leukemia Virus Type 1-Transformed Human T Cells
Authors: Kirken Robert A. , Erwin Rebecca A. , Wang Lihua , Wang Yuling , Rui Hallgeir , Farrar William L.
Published: April 2014

Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors Stat5a and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 (50 μM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, Stat5a (Tyr694), and Stat5b (Tyr699); 2) serine phosphorylation of Stat5a (Ser726) as determined by a novel phosphospecific Ab; and 3) Stat5a/b DNA binding to the Stat5-responsive β-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-κB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.
The Inference of Antigen Selection on Ig Genes
Authors: Lossos Izidore S. , Tibshirani Robert , Narasimhan Balasubramanian , Levy Ronald
Published: April 2014

Analysis of somatic mutations in V regions of Ig genes is important for understanding various biological processes. It is customary to estimate Ag selection on Ig genes by assessment of replacement (R) as opposed to silent (S) mutations in the complementary-determining regions and S as opposed to R mutations in the framework regions. In the past such an evaluation was performed using a binomial distribution model equation, which is inappropriate for Ig genes in which mutations have four different distribution possibilities (R and S mutations in the complementary-determining region and/or framework regions of the gene). In the present work, we propose a multinomial distribution model for assessment of Ag selection. Side-by-side application of multinomial and binomial models on 86 previously established Ig sequences disclosed 8 discrepancies, leading to opposite statistical conclusions about Ag selection. We suggest the use of the multinomial model for all future analysis of Ag selection.
Enhanced Murine Macrophage TNF Receptor Shedding by Cytosine-Guanine Sequences in Oligodeoxynucleotides
Authors: Jin Long , Raymond Daniel P. , Crabtree Traves D. , Pelletier Shawn J. , Houlgrave C. Webster , Pruett Timothy L. , Sawyer Robert G.
Published: April 2014

The immunomodulatory role of unmethylated cytosine-guanine sequences (CpG) in bacterial DNA has been well documented. We have previously demonstrated that murine macrophage-like RAW 264.7 cells respond to CpG DNA with an increase in the proinflammatory cytokine, TNF-α, in both a dose-dependent and time-dependent manner. In addition, CpG DNA stimulates a significant, though delayed, secretion of the anti-inflammatory cytokine IL-10. Because TNF-α and TNFR (TNFRI and II) expression are tightly regulated responses, we hypothesized that CpG containing oligodeoxynucleotide (CpG ODN) would also affect TNFRI and II shedding. Using both murine peritoneal macrophages and RAW 264.7 cells, we demonstrated a significant, time-dependent increase in soluble TNFRI and TNFRII production with CpG ODN stimulation. RAW 264.7 cells treated with CpG ODN had a transient increase in membrane TNFRII expression, but not TNFRI. Both types of TNFR mRNA were also up-regulated by CpG ODN, and addition of the transcriptional inhibitor actinomycin D abrogated the effect of CpG ODN on TNFR mRNA and protein expression. Addition of anti-IL-10 and anti-TNF-α Abs did not change these results. The addition of plate-bound anti-TNF receptor Abs to this system increased the amount of bioactive TNF, implying that these receptors are acting as inhibitors of TNF activity. These results suggest that the de novo, non-IL-10- and non-TNF-α-dependent transcription, translation, and shedding of TNFRs are additional potential counterinflammatory effects of CpG DNA.
The Serpin Secreted byBrugia malayiMicrofilariae, Bm-SPN-2, Elicits Strong, but Short-Lived, Immune Responses in Mice and Humans
Authors: Zang Xingxing , Atmadja Agnes Kurniawan , Gray Paul , Allen Judith E. , Gray Carolyn A. , Lawrence Rachel A. , Yazdanbakhsh Maria , Maizels Rick M.
Published: April 2014

Understanding the basic immunology of an infectious disease requires insight into the pattern of T cell reactivity and specificity. Although lymphatic filariasis is a major tropical disease, the predominant T cell Ags of filarial species such as Brugia malayi are still undefined. We have now identified a prominent T cell Ag from B. malayi microfilariae (Mf) as Bm-SPN-2, a serpin secreted exclusively by this stage. Mf-infected mice mounted strong, but short-lived, Bm-SPN-2-specific Th1 responses, measured by in vitro production of IFN-γ, but not IL-4 or IL-5, 14 days postinfection. By day 35, responsiveness to Bm-SPN-2 was lost despite enhanced reactivity to whole Mf extract. Single immunization with Mf extract also stimulated typical Th1 reactions to Bm-SPN-2, but IgG1 Ab responses dominated after repeated immunizations. Human patients displayed potent humoral responses to Bm-SPN-2 in both IgG1 and IgG4 subclasses. Thus, 100% (20 of 20) of the microfilaremic (MF+) patients bore IgG4 responses to Bm-SPN-2, while only 30% of endemic normal subjects were similarly positive. Following chemotherapy, Bm-SPN-2-specific Abs disappeared in 12 of 13 MF+ patients, although the majority remained seropositive for whole parasite extract. PBMC from most, but not all, endemic subjects were induced to secrete IFN-γ when stimulated with Bm-SPN-2. These findings demonstrate that Bm-SPN-2 is recognized by both murine and human T and B cells and indicate that their responses are under relatively stringent temporal control. This study also provides the first example of a stage-specific secreted molecule that acts as a major T cell Ag from filarial parasites and is a prime candidate for a serodiagnostic probe.
Inhibition of IL-6 and IL-10 Signaling and Stat Activation by Inflammatory and Stress Pathways
Authors: Ahmed Simi T. , Ivashkiv Lionel B.
Published: April 2014

The development and resolution of an inflammatory process are regulated by a complex interplay among cytokines that have pro- and anti-inflammatory effects. Effective and sustained action of a proinflammatory cytokine depends on synergy with other inflammatory cytokines and antagonism of opposing cytokines that are often highly expressed at inflammatory sites. We analyzed the effects of the inflammatory and stress agents, IL-1, TNF-α, LPS, sorbitol, and H2O2, on signaling by IL-6 and IL-10, pleiotropic cytokines that activate the Jak-Stat signaling pathway and have both pro- and anti-inflammatory actions. IL-1, TNF-α, and LPS blocked the activation of Stat DNA binding and tyrosine phosphorylation by IL-6 and IL-10, but not by IFN-γ, in primary macrophages. Inhibition of Stat activation correlated with inhibition of expression of IL-6-inducible genes. The inhibition was rapid and independent of de novo gene induction and occurred when the expression of suppressor of cytokine synthesis-3 was blocked. Inhibition of IL-6 signaling was mediated by the p38 subfamily of stress-activated protein kinases. Jak1 was inhibited at the level of tyrosine phosphorylation, indicating that inhibition occurred at least in part upstream of Stats in the Jak-Stat pathway. Experiments using Stat3 mutated at serine 727 and using truncated IL-6Rs suggested that the target of inhibition is contained within the membrane-proximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor and is different from the SH2 domain-containing protein-tyrosine phosphatase/suppressor of cytokine synthesis-3 docking site. These results identify a new level at which IL-1 and TNF-α modulate signaling by pleiotropic cytokines such as IL-6 and IL-10 and provide a molecular basis for the previously described antagonism of certain IL-6 actions by IL-1.
The Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 1/2 Pathway Is Involved in formyl-Methionyl-Leucyl-Phenylalanine-Induced p47phox Phosphorylation in Human Neutrophils
Authors: Dewas Cédric , Fay Michèle , Gougerot-Pocidalo Marie-Anne , El-Benna Jamel
Published: April 2014

Phosphorylation of p47 phagocyte oxidase, (p47phox), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47phox is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47phox phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47phox. However, PD98059 weakly affected PMA-induced p47phox phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47phox induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47phox phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47phox phosphorylation either directly or indirectly via other kinases. In PMA-induced p47phox phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47phox phosphorylation. However, in fMLP-induced p47phox phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47phox when tested alone, and exerted additive inhibitory effects on p47phox phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47phox. Furthermore, they strongly suggest that p47phox is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.
The CXC Chemokine Receptor 2, CXCR2, Is the Putative Receptor for ELR+ CXC Chemokine-Induced Angiogenic Activity
Authors: Addison Christina L. , Daniel Thomas O. , Burdick Marie D. , Liu Hua , Ehlert Jan E. , Xue Ying Ying , Buechi Linda , Walz Alfred , Richmond Ann , Strieter Robert M.
Published: April 2014

We have previously shown that members of the ELR+ CXC chemokine family, including IL-8; growth-related oncogenes α, β, and γ; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR+ CXC chemokines has not been determined. Because all ELR+ CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR+ CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR+ CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR+ CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR+ CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR+ CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2−/− mice. We thus conclude that CXCR2 is the receptor responsible for ELR+ CXC chemokine-mediated angiogenesis.
Human Peyer’s Patch T Cells Are Sensitized to Dietary Antigen and Display a Th Cell Type 1 Cytokine Profile
Authors: Nagata Satoru , McKenzie Catriona , Pender Sylvia L. F. , Bajaj-Elliott Mona , Fairclough Peter D. , Walker-Smith John A. , Monteleone Giovanni , MacDonald Thomas T.
Published: April 2014

Animal studies have demonstrated that feeding Ags induces regulatory (Th2, Th3) cells in Peyer’s patches (PP), which migrate to the periphery and produce immunomodulatory cytokines such as IL-4, IL-10, or TGF-β. In this work we have attempted to extend this paradigm to man by analyzing the response of human PP T cells to in vitro challenge with the common dietary Ag β-lactoglobulin (βlg) of cow’s milk. PP T cells stimulated with βlg showed enhanced proliferation compared with blood T cells from the same patient. Increased expression of CD25 and the Th1-associated chemokine receptor CCR5 was also seen on CD4+ and CD8+ PP T cells, but not blood T cells, stimulated with βlg. By enzyme-linked immunospot assay and RT-PCR, the PP T cell recall response to βlg and casein was dominated by IFN-γ, with negligible IL-4, IL-5, IL-10, or TGF-β. To help explain the PP T cell response to βlg, we examined IL-12 expression. Both IL-12p40 and -p35 transcripts were abundantly expressed in PP, but not in adjacent normal ileal mucosa. Immunoreactive IL-12p40-containing cells were present below the PP dome epithelium. Furthermore, in culture, PP, but not paired PBMC, spontaneously released IL-12p70. These results suggest that the human response to oral Ags in the gut may be different from that in rodents.
Anti-SSA/Ro and Anti-SSB/La Autoantibodies Bind the Surface of Apoptotic Fetal Cardiocytes and Promote Secretion of TNF-α by Macrophages
Authors: Miranda-Carús Maria-Eugenia , Askanase Anca Dinu , Clancy Robert M. , Di Donato Francis , Chou Tseng-Ming , Libera Matthew R. , Chan Edward K. L. , Buyon Jill P.
Published: April 2014

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16–24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-α from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.
Cell Division Is Not a “Clock” Measuring Acquisition of Competence to Produce IFN-γ or IL-4
Authors: Ben-Sasson Shlomo Z. , Gerstel Regina , Hu-Li Jane , Paul William E.
Published: April 2014

Naive CD4 T cells acquire the potential to produce IFN-γ and IL-4 by culture in the presence of their cognate Ag, APC, and appropriate cytokines. In this study, we show that commitment to IFN-γ production on the part of rigorously purified naive CD4 T cells can occur without cell division. Indeed, even entry into S phase is not essential. Moreover, both CD4 and CD4/CD8 thymocytes from TCR-transgenic mice (5CC7 mice) on a Rag2−/− background can acquire IFN-γ-producing capacity when stimulated by peptide, APC, and IL-12. These cells can do so without dividing and some acquire IFN-γ-producing activity without entry into S phase. Not only is cell division not required for acquisition of cytokine-producing potential, cell populations that have undergone the same numbers of divisions can have quite different proportions of IFN-γ- or IL-4-producing cells, depending on the duration of priming or, in the case of IL-4, on the concentration of peptide. Thus, cell division is not a clock for the expression of these cytokines. Factors associated with priming conditions including strength of stimulation, duration of priming, and number of divisions each play a role.
IL-6 Secretion by a Rat T9 Glioma Clone Induces a Neutrophil-Dependent Antitumor Response with Resultant Cellular, Antiglioma Immunity
Authors: Graf Martin R. , Prins Robert M. , Merchant Randall E.
Published: April 2014

Previously, we reported that IL-6 transduction attenuates tumor formation of a rat T9 glioma clone (termed T9.F). This study focuses on the mechanisms of the antitumor response elicited by IL-6 and the generation of glioma immunity. Ten days post s.c. inoculation of T9.F- or IL-6-secreting T9.F cells (T9.F/IL6/hi), tumor nodules were removed and their leukocytic infiltrate was analyzed by FACS with Ab markers for T cells, B cells, granulocytes, and monocytes. T9.F/IL6/hi tumors showed a marked increase in granulocytes as compared with parental T9.F tumors, and histological examination revealed that the granulocytes were neutrophils. Animals made neutropenic failed to reject T9.F/IL6/hi tumors. FACS analysis of 17-day T9.F/IL6/hi regressing tumors and T9.F progressing tumors did not reveal any significant differences in the leukocytic infiltrates. Tumor-specific effector cells were detected in the spleens harvested from animals bearing 17-day, regressing, T9.F/IL6/hi tumors. In vitro, these effector cells lysed T9.F cells, proliferated in response to T9.F stimulator cells, and produced Th1 cytokines (IL-2 and IFN-γ) but not the Th2 cytokine, IL-4, when cocultured with T9.F stimulator cells. Rats that had rejected s.c. T9.F/IL6/hi tumors displayed a delayed-type hypersensitivity response when injected with viable T9.F cells in the contralateral flank. Passive transfer of spleen cells from these animals transferred glioma immunity to naive recipients and depletion of CD3+ T cells, before transfer, completely abolished immunity, whereas depletion of CD8+ T cells had moderate inhibitory effects on the transfer of immunity.
VCAM-1 Activates Phosphatidylinositol 3-Kinase and Induces p120CblPhosphorylation in Human Airway Smooth Muscle Cells
Authors: Lazaar Aili L. , Krymskaya Vera P. , Das Susan K. P.
Published: April 2014

VCAM-1 is a member of the Ig superfamily of receptors the expression of which is up-regulated on human airway smooth muscle (ASM) cells following stimulation with inflammatory mediators. The function of these receptors in adhesion is well known, but there is growing recognition that they also possess “outside-in” signaling functions, such as cytoskeletal reorganization, calcium mobilization, and cytokine release. The present study examined the activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K) in ASM cells following VCAM-1 engagement. VCAM-1 ligation activated extracellular signal-regulated kinase 2 and resulted in increased expression of cyclin D1, yet there was neither p27kip1 degradation nor an increase in smooth muscle cell DNA synthesis. VCAM-1 ligation, however, augmented the proliferative response to submitogenic concentrations of epidermal growth factor. VCAM-1 engagement also stimulated a rapid increase in PI3K activity. This was associated with phosphorylation of the adapter protein p120Cbl and an increase in Cbl-associated PI3K activity. These studies suggest that VCAM-1 is linked to multiple signaling pathways in human ASM cells and may function to augment growth factor-induced responses.
IL-7 Enhances the Responsiveness of Human T Cells That Develop in the Bone Marrow of Athymic Mice
Authors: Tsark Eleanor C. , Dao Mo A. , Wang Xiuli , Weinberg Kenneth , Nolta Jan A.
Published: April 2014

The beige/nude/xid/human (bnx/hu) model of human hematopoiesis provides a unique opportunity to study extrathymic human T lymphocyte development in an in vivo system. Purified human hematopoietic stem cells develop into mature T lymphocytes and immature progenitors in the bone marrow of athymic bnx mice. The human T cells are all TCRαβ+ and display a restricted TCRVβ repertoire. In the current studies, we examined the effects of systemic human IL-7 (huIL-7) administration on the phenotype and the activation status of the bnx/hu T cells. In the majority of the mice that did not have huIL-7 administration, a higher frequency of human CD3+/CD8+ than CD3+/CD4+ T cells developed in the bone marrow. This phenomenon is also frequently observed in human bone marrow transplant recipients. Extremely low levels of IL-2 were expressed by human CD3+ cells isolated from these mice, in response to PMA plus ionomycin and to CD3 and CD28 cross-linking. IL-4 was not expressed by cells exposed to either stimulus, demonstrating a profound inability of the bnx/hu T cells to produce this cytokine. Systemic production of huIL-7 from engineered stromal cells transplanted into the mice increased the human CD4 to CD8 ratios, and increased the ratio of memory to naive CD4+ and CD8+ T cells. The human CD3+ cells recovered from mice that had systemic huIL-7 and equivalent numbers of CD3+/CD4+ and CD3+/CD8+ cells in the marrow were still unable to produce IL-4 in response to any condition tested, but were capable of normal levels of IL-2 production following stimulation.
Redirecting Mouse CTL Against Colon Carcinoma: Superior Signaling Efficacy of Single-Chain Variable Domain Chimeras Containing TCR-ζ vs FcεRI-γ
Authors: Haynes Nicole M. , Snook Marie B. , Trapani Joseph A. , Cerruti Loretta , Jane Stephen M. , Smyth Mark J. , Darcy Phillip K.
Published: April 2014

The structurally related TCR-ζ and Fc receptor for IgE (FcεRI)-γ are critical signaling components of the TCR and FcεRI, respectively. Although chimeric Ab receptors containing ζ and γ signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the ζ and γ subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-ζ and scFv-γ receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-ζ chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-γ receptors. T cells expressing scFv-ζ chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the ζ signaling pathway.
Activated T Lymphocytes Regulate Hyaluronan Binding to Monocyte CD44 Via Production of IL-2 and IFN-γ
Authors: Levesque Marc C. , Haynes Barton F.
Published: April 2014

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-α. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1α, IL-1β, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-γ, and TNF-α were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-α, anti-IL-2, and anti-IFN-γ Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-α induced high levels of monocyte HA binding. Likewise, IFN-γ augmented TNF-α-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4+ T cells in these assays. Fixed, PMA/ionomycin-activated CD4+ T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-γ and anti-TNF-α Abs blocked fixed PMA/ionomycin-activated CD4+ T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-γ, and a role for monocyte-derived TNF-α in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.
Negative-Feedback Regulation of CD28 Costimulation by a Novel Mitogen-Activated Protein Kinase Phosphatase, MKP6
Authors: Marti Francesc , Krause Anja , Post Nicholas H. , Lyddane Clay , Dupont Bo , Sadelain Michel , King Philip D.
Published: April 2014

TCR and CD28 costimulatory receptor-cooperative induction of T cell IL-2 secretion is dependent upon activation of mitogen-activated protein (MAP) kinases. Using yeast-hybrid technology, we cloned a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, MAP kinase phosphatase-6 (MKP6), which we demonstrate inactivates MAP kinases. Several lines of evidence indicate that MKP6 plays an important functional role in CD28 costimulatory signaling. First, in human peripheral blood T cells (PBT), expression of MKP6 is strongly up-regulated by CD28 costimulation. Second, transfer of dominant-negative MKP6 to PBT with the use of retroviruses primes PBT for the secretion of substantially larger quantities of IL-2, specifically in response to CD28 costimulation. A similar enhancement of IL-2 secretion is observed neither in response to TCR plus CD2 costimulatory receptor engagement nor in response to other mitogenic stimuli such as phorbol ester and ionomycin. Furthermore, this hypersensitivity to CD28 costimulation is associated with CD28-mediated hyperactivation of MAP kinases. Third, a retroviral transduced chimeric receptor with a CD28 CYT that is specifically unable to bind MKP6 costimulates considerably larger quantities of IL-2 from PBT than a similar transduced chimeric receptor that contains a wild-type CD28 CYT. Taken together, these results suggest that MKP6 functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls the activation of MAP kinases.