bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of IFN-γ Signaling by Glucocorticoids
Authors: Hu Xiaoyu , Li Wai-Ping , Meng Charis , Ivashkiv Lionel B.
Published: April 2014

Recent reports suggest that a novel mechanism of glucocorticoid (GC) immunosuppressive action is inhibition of signaling by IL-2 and IL-12, cytokines that use the Janus kinase-STAT signaling pathway. We investigated whether GCs could also block activation of Janus kinase-STAT signaling by IFN-γ, a potent proinflammatory cytokine. Addition of dexamethasone to PBMC cultures resulted in a dramatic inhibition of IFN-γ activation of STAT1. Several days of exposure to GCs were required for inhibition of IFN-γ signaling to become apparent, and the underlying mechanism was down-regulation of STAT1 expression. GCs suppressed the expression of STAT1 mRNA, but did not affect STAT1 protein stability. STAT1 expression and IFN-γ signaling were preferentially suppressed in macrophages. GCs did not act directly on macrophages, but worked indirectly by regulating macrophage-lymphocyte interactions that control STAT1 expression. GCs inhibited IFN-γ-inducible gene expression, thus demonstrating the physiological significance of inhibition of signal transduction. Our results identify a novel level of regulation of IFN-γ signaling, whereby GCs control the amplitude of IFN-γ signaling by regulating STAT1 expression. These results suggest that inhibition of IFN-γ signaling contributes to the immunosuppressive action of GCs.
Phosphatidylserine on HIV Envelope Is a Cofactor for Infection of Monocytic Cells
Authors: Callahan Melissa K. , Popernack Paul M. , Tsutsui Shigeki , Truong Linh , Schlegel Robert A. , Henderson Andrew J.
Published: April 2014

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.
Complementarity-Determining Region 3 Spectratyping Analysis of the TCR Repertoire in Multiple Sclerosis
Authors: Matsumoto Yoh , Yoon Wong Kee , Jee Youngheun , Fujihara Kazuo , Misu Tatsuro , Sato Shigeru , Nakashima Ichiro , Itoyama Yasuto
Published: April 2014

Multiple sclerosis (MS) is considered to be an autoimmune disease mediated by T cells reactive with Ags in the CNS. Therefore, it has been postulated that neuroantigen-reactive T cells bearing particular types of TCRs are expanded clonally during the course of the disease. However, there is a controversy with regard to the TCR usage by T cells associated with the development of MS. By the use of complementarity-determining region 3 spectratyping analysis that is shown to be a useful tool for identification of pathogenic TCR in autoimmune disease models, we tried to demonstrate that spectratype was T cells bearing particular types of TCR are activated in MS patients. Consequently, it was found that Vβ5.2 were often oligoclonally expanded in peripheral blood of MS patients, but not of healthy subjects. Sequence analysis of the complementarity-determining region 3 region of spectratype-derived TCR clones revealed that the predominant TCR clone was different from patient to patient, but that similar results were obtained in a patient examined at different time points. More importantly, examination of cerebrospinal fluid T cells and longitudinal studies of PBLs from selected patients revealed that Vβ5.2 expansion was detectable in the majority of patients examined. These findings suggest that Vβ5.2 spectratype expansion is associated with the development of MS and that TCR-based immunotherapy can be applicable to MS patients if the TCR activation pattern of each patient is determined at different stages of the disease.
Design, Construction, and In Vitro Analyses of Multivalent Antibodies
Authors: Miller Kathy , Meng Gloria , Liu Jun , Hurst Amy , Hsei Vanessa , Wong Wai-Lee , Ekert Rene , Lawrence David , Sherwood Steven , DeForge Laura , Gaudreault Jacques , Keller Gilbert , Sliwkowski Mark , Ashkenazi Avi , Presta Leonard
Published: April 2014

Some Abs are more efficacious after being cross-linked to form dimers or multimers, presumably as a result of binding to and clustering more surface target to either amplify or diversify cellular signaling. To improve the therapeutic potency of these types of Abs, we designed and generated Abs that express tandem Fab repeats with the aim of mimicking cross-linked Abs. The versatile design of the system enables the creation of a series of multivalent human IgG Ab forms including tetravalent IgG1, tetravalent F(ab′)2, and linear Fab multimers with either three or four consecutively linked Fabs. The multimerized Abs target the cell surface receptors HER2, death receptor 5, and CD20, and are more efficacious than their parent mAbs in triggering antitumor cellular responses, indicating they could be useful both as reagents for study as well as novel therapeutics.
Cryptic CTL Epitope on a Murine Sarcoma Meth A Generated by Exon Extension as a Novel Mechanism
Authors: Uenaka Akiko , Hirano Yoshiki , Hata Hidenori , Win Sanda , Aji Toshiki , Tanaka Motoyuki , Ono Toshiro , Skipper Jonathan C. A. , Shimizu Kenji , Nakayama Eiichi
Published: April 2014

Using the recently developed ELISPOT cloning methodology, we obtained cDNA clone S35 coding for the Ag epitope recognized by a murine sarcoma Meth A-specific CTL clone AT-1. Analysis of truncated S35 constructs and overlapping peptides revealed that the peptide epitope was LGAEAIFRL. AT-1 CTL lysed peptide-pulsed CMS8 cells at a nanomolar concentration, and the peptide strongly stimulated IFN-γ production in AT-1 CTL. Sequence homology indicated that the S35 was derived from a mouse homologue of human retinoic acid-regulated nuclear matrix-associated protein (ramp). The ramp gene consisted of 15 exons. The majority of the ramp mRNA was the transcript normally spliced between exons 14 and 15, but a minor population of mRNA with an extended exon 14 was also present in Meth A cells. The epitope was derived from the newly created open reading frame, which resulted from extension of exon 14 after splicing of the adjacent intronic sequence.
Ephrin B2 Induces T Cell Costimulation
Authors: Yu Guang , Luo Hongyu , Wu Yulian , Wu Jiangping
Published: April 2014

Eph kinases form the largest family of receptor tyrosine kinases, and their ligands are ephrins (EFNs), which are cell surface proteins. Some Eph kinases and EFNs are expressed on T cells, B cells, and dendritic cells, but their functions in the immune system are largely unknown. In this study, we investigated the effect of EFNB2 on murine T cells. EFNB2 mRNA was expressed in the cortex of the thymus and white pulp of the spleen. At the protein level, it was expressed on T cells and monocytes/macrophages, but not on B cells. EFNB2Rs were expressed mainly on T cells. Solid-phase EFNB2 along with suboptimal anti-CD3 strongly stimulated T cell proliferation, with concomitant augmentation of IFN-γ but not IL-2 or IL-4 secretion. The activity of cytotoxic T cells was also significantly enhanced in the presence of solid-phase EFNB2. These results indicate that EFNB2R cross-linking results in costimulation of T cells. EFNB2Rs were normally scattered on the T cell surface; after TCR cross-linking, they rapidly congregated to capped TCR complexes and then to patched rafts. This provides a morphological base for EFNB2Rs to participate in T cell costimulation. We also demonstrated that EFNB2R signaling led to augmented p38 and p44/42 mitogen-activated protein kinase activation. Our study shows that EFNB2 plays important roles in immune regulation.
A Role for the Lymphotoxin/LIGHT Axis in the Pathogenesis of Murine Collagen-Induced Arthritis
Authors: Fava Roy A. , Notidis Evangelia , Hunt Jane , Szanya Veronika , Ratcliffe Nora , Ngam-ek Apinya , de Fougerolles Antonin R. , Sprague Andrew , Browning Jeffrey L.
Published: April 2014

A lymphotoxin-β (LTβ) receptor-Ig fusion protein (LTβR-Ig) was used to evaluate the importance of the lymphotoxin/LIGHT axis in the development and perpetuation of arthritis. Prophylactic treatment with the inhibitor protein LTβR-Ig blocked the induction of collagen-induced arthritis in mice and adjuvant arthritis in Lewis rats. Treatment of mice with established collagen-induced arthritis reduced the severity of arthritic symptoms and joint tissue damage. However, in a passive model of anti-collagen Ab-triggered arthritis, joint inflammation was not affected by LTβR-Ig treatment precluding LT/LIGHT involvement in the very terminal immune complex/complement/FcR-mediated effector phase. Collagen-II and Mycobacterium-specific T cell responses were not impaired, yet there was evidence that the overall response to the mycobacterium was blunted. Serum titers of anti-collagen-II Abs were reduced especially during the late phase of disease. Treatment with LTβR-Ig ablated follicular dendritic cell networks in the draining lymph nodes, suggesting that impaired class switching and affinity maturation may have led to a decreased level of pathological autoantibodies. These data are consistent with a model in which the LT/LIGHT axis controls microenvironments in the draining lymph nodes. These environments are critical in shaping the adjuvant-driven initiating events that impact the subsequent quality of the anti-collagen response in the later phases. Consequently, blockade of the LT/LIGHT axis may represent a novel approach to the treatment of autoimmune diseases such as rheumatoid arthritis that involve both T cell and Ab components.
Recombinant TCR Ligand Induces Tolerance to Myelin Oligodendrocyte Glycoprotein 35-55 Peptide and Reverses Clinical and Histological Signs of Chronic Experimental Autoimmune Encephalomyelitis in HLA-DR2 Transgenic Mice
Authors: Vandenbark Arthur A. , Rich Cathleen , Mooney Jeff , Zamora Alex , Wang Chunhe , Huan Jianya , Fugger Lars , Offner Halina , Jones Richard , Burrows Gregory G.
Published: April 2014

In a previous study, we demonstrated that myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide could induce severe chronic experimental autoimmune encephalomyelitis (EAE) in HLA-DR2+ transgenic mice lacking all mouse MHC class II genes. We used this model to evaluate clinical efficacy and mechanism of action of a novel recombinant TCR ligand (RTL) comprised of the α1 and β1 domains of DR2 (DRB1*1501) covalently linked to the encephalitogenic MOG-35-55 peptide (VG312). We found that the MOG/DR2 VG312 RTL could induce long-term tolerance to MOG-35-55 peptide and reverse clinical and histological signs of EAE in a dose- and peptide-dependent manner. Some mice treated with lower doses of VG312 relapsed after cessation of daily treatment, but the mice could be successfully re-treated with a higher dose of VG312. Treatment with VG312 strongly reduced secretion of Th1 cytokines (TNF-α and IFN-γ) produced in response to MOG-35-55 peptide, and to a lesser degree purified protein derivative and Con A, but had no inhibitory effect on serum Ab levels to MOG-35-55 peptide. Abs specific for both the peptide and MHC moieties of the RTLs were also present after treatment with EAE, but these Abs had only a minor enhancing effect on T cell activation in vitro. These data demonstrate the powerful tolerance-inducing therapeutic effects of VG312 on MOG peptide-induced EAE in transgenic DR2 mice and support the potential of this approach to inhibit myelin Ag-specific responses in multiple sclerosis patients.
Inhibition of Graft-Versus-Host Disease by Double-Negative Regulatory T Cells
Authors: Young Kevin J. , DuTemple Barb , Phillips M. James , Zhang Li
Published: April 2014

Pretransplant infusion of lymphocytes that express a single allogeneic MHC class I Ag has been shown to induce tolerance to skin and heart allografts that express the same alloantigens. In this study, we demonstrate that reconstitution of immunoincompetent mice with spleen cells from MHC class I Ld-mismatched donors does not cause graft-vs-host disease (GVHD). Recipient mice become tolerant to skin allografts of lymphocyte donor origin while retaining immunity to third-party alloantigens. The mechanism involves donor-derived CD3+CD4−CD8− double-negative T regulatory (DN Treg) cells, which greatly increase and form the majority of T lymphocytes in the spleen of recipient mice. DN Treg cells isolated from tolerant recipient mice can suppress the proliferation of syngeneic antihost CD8+ T cells in vitro. Furthermore, we demonstrate that DN Treg cells can be generated in vitro by stimulating them with MHC class I Ld-mismatched lymphocytes. These in vitro generated Ld-specific DN Treg cells are able to down-regulate the activity of antihost CD8+ T cells in vitro by directly killing activated CD8+ T cells. Moreover, infusing in vitro generated Ld-mismatched DN Treg cells prevented the development of GVHD caused by allogeneic CD8+ T cells. Together these data demonstrate that infusion of single MHC class I locus-mismatched lymphocytes may induce donor-specific transplantation tolerance through activation of DN Treg cells, which can suppress antihost CD8+ T cells and prevent the development of GVHD. This finding indicates that using single class I locus-mismatched grafts may be a viable alternative to using fully matched grafts in bone marrow transplantation.
IL-3 Acts Directly on Osteoclast Precursors and Irreversibly Inhibits Receptor Activator of NF-κB Ligand-Induced Osteoclast Differentiation by Diverting the Cells to Macrophage Lineage
Authors: Khapli Shruti M. , Mangashetti Latha S. , Yogesha S. D. , Wani Mohan R.
Published: April 2014

Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of the monocyte/macrophage lineage in the presence of M-CSF and receptor activator of NF-κB ligand (RANKL). In this study we investigated the role of IL-3 in osteoclast differentiation. We show here that IL-3, a cytokine secreted by activated T lymphocytes, inhibits RANKL-induced osteoclast differentiation by a direct action on early osteoclast precursors. Anti-IL-3 Ab neutralized the inhibitory effect of IL-3 on osteoclast differentiation. In addition, IL-3 inhibits TNF-α-induced osteoclast differentiation in bone marrow-derived macrophages. However, IL-3 has no inhibitory effect on mature osteoclasts. In osteoclast precursors, IL-3 prevents RANKL-induced nuclear translocation of NF-κB by inhibiting the phosphorylation and degradation of IκB. RT-PCR analysis revealed that IL-3 down-regulated c-Fos transcription. Interestingly, the osteoclast precursors in the presence of IL-3 showed strong expression of macrophage markers such as Mac-1, MOMA-2, and F4/80. Furthermore, the inhibitory effect of IL-3 on osteoclast differentiation was irreversible, and the osteoclast precursors preincubated in IL-3 were resistant to RANKL action. Thus, our results reveal for the first time that IL-3 acts directly on early osteoclast precursors and irreversibly blocks RANKL-induced osteoclast differentiation by diverting the cells to macrophage lineage.
Dendritic Cells Support Sequential Reprogramming of Chemoattractant Receptor Profiles During Naive to Effector T Cell Differentiation
Authors: Kim Chang H. , Nagata Kinya , Butcher Eugene C.
Published: April 2014

T cells undergo chemokine receptor switches during activation and differentiation in secondary lymphoid tissues. Here we present evidence that dendritic cells can induce changes in T cell expression of chemokine receptors in two continuous steps. In the first switch over a 4–5 day period, dendritic cells up-regulate T cell expression of CXCR3 and CXCR5. Additional stimulation leads to the second switch: down-regulation of lymphoid tissue homing related CCR7 and CXCR5, and up-regulation of Th1/2 effector tissue-targeting chemoattractant receptors such as CCR4, CCR5, CXCR6, and CRTH2. We show that IL-4 and IL-12 can determine the fate of the secondary chemokine receptor switch. IL-4 enhances the generation of CCR4+ and CRTH2+ T cells, and suppresses the generation of CXCR3+ T cells and CCR7− T cells, while IL-12 suppresses the level of CCR4 in responding T cells. Furthermore, IL-4 has positive effects on generation of CXCR5+ and CCR7+ T cells during the second switch. Our study suggests that the sequential switches in chemokine receptor expression occur during naive T cell interaction with dendritic cells. The first switch of T cell chemokine receptor expression is consistent with the fact that activated T cells migrate within lymphoid tissues for interaction with B and dendritic cells, while the second switch predicts the trafficking behavior of effector T cells away from lymphoid tissues to effector tissue sites.
CC Chemokine Receptor 9 Expression Defines a Subset of Peripheral Blood Lymphocytes with Mucosal T Cell Phenotype and Th1 or T-Regulatory 1 Cytokine Profile
Authors: Papadakis Konstantinos A. , Landers Carol , Prehn John , Kouroumalis Elias A. , Moreno Sofia T. , Gutierrez-Ramos Jose-Carlos , Hodge Martin R. , Targan Stephan R.
Published: April 2014

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9+ lymphocytes in blood from normal donors. A subset of CCR9+ T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9−, CCR9+CD4+ peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-γ and IL-10. IL-10-producing cells were exclusively detected within the CCR9+ subset of CD4+ T cells by intracellular staining and were distinct from IL-2- and IFN-γ-producing cells. Moreover, memory CCR9+CD4+ lymphocytes respond to CD2 stimulation with proliferation and IFN-γ/IL-10 production, whereas memory CCR9−CD4+ cells were unresponsive. In addition, memory CCR9+CD4+ T cells support Ig production by cocultured CD19+ B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9+CD4+ T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.
CD28 and Inducible Costimulatory Protein Src Homology 2 Binding Domains Show Distinct Regulation of Phosphatidylinositol 3-Kinase, Bcl-xL, and IL-2 Expression in Primary Human CD4 T Lymphocytes
Authors: Parry Richard V. , Rumbley Catherine A. , Vandenberghe Luk H. , June Carl H. , Riley James L.
Published: April 2014

Ligation of either CD28 or inducible costimulatory protein (ICOS) produces a second signal required for optimal T cell activation and proliferation. One prominent difference between ICOS- and CD28-costimulated T cells is the quantity of IL-2 produced. To understand why CD28 but not ICOS elicits major increases in IL-2 expression, we compared the abilities of these molecules to activate the signal transduction cascades implicated in the regulation of IL-2. Major differences were found in the regulation of phosphatidylinositol 3-kinase activity (PI3K) and c-jun N-terminal kinase. ICOS costimulation led to greatly augmented levels of PI3K activity compared with CD28 costimulation, whereas only CD28 costimulation activated c-jun N-terminal kinase. To examine how these differences in signal transduction affected IL-2 production, we transduced primary human CD4 T cells with a lentiviral vector that expressed the murine CD28 extracellular domain with a variety of human CD28 and ICOS cytoplasmic domain swap constructs. These domains were able to operate as discrete signaling units, suggesting that they can function independently. Our results show that even though the ICOS Src homology (SH) 2 binding domain strongly activated PI3K, it was unable to substitute for the CD28 SH2 binding domain to induce high levels of IL-2 and Bcl-xL. Moreover, the CD28 SH2 binding domain alone was sufficient to mediate optimal levels of Bcl-xL induction, whereas the entire CD28 cytoplasmic tail was required for high levels of IL-2 expression. Thus, differences within their respective SH2 binding domains explain, at least in part, the distinct regulation of IL-2 and Bcl-xL expression following ICOS- or CD28-mediated costimulation.
Cutting Edge: Rapid In Vivo CTL Activity by Polyoma Virus-Specific Effector and Memory CD8+ T Cells
Authors: Byers Anthony M. , Kemball Christopher C. , Moser Janice M. , Lukacher Aron E.
Published: April 2014

For viruses that establish persistent infection, continuous immunosurveillance by effector-competent antiviral CD8+ T cells is likely essential for limiting viral replication. Although it is well documented that virus-specific memory CD8+ T cells synthesize cytokines after short term in vitro stimulation, there is limited evidence that these T cells exhibit cytotoxicity, the dominant antiviral effector function. Here, we show that antiviral CD8+ T cells in mice acutely infected by polyoma virus, a persistent mouse pathogen, specifically eliminate viral peptide-pulsed donor spleen cells within minutes after adoptive transfer and do so via a perforin-dependent mechanism. Antiviral memory CD8+ T cells were similarly capable of rapidly mobilizing potent Ag-specific cytotoxic activity in vivo. These findings strongly support the concept that a cytotoxic effector-memory CD8+ T cell population operates in vivo to control this persistent viral infection.
Inhibition of IFN-γ-Induced Class II Transactivator Expression by a 19-kDa Lipoprotein fromMycobacterium tuberculosis: A Potential Mechanism for Immune Evasion
Authors: Pai Rish K. , Convery Marilyn , Hamilton Thomas A. , Boom W. Henry , Harding Clifford V.
Published: April 2014

Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-γ induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-γ-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-γ-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1−/− macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-γ-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-γ signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-γ-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4+ T cells.
Genetic Disassociation of Autoimmunity and Resistance to Costimulation Blockade-Induced Transplantation Tolerance in Nonobese Diabetic Mice
Authors: Pearson Todd , Markees Thomas G. , Serreze David V. , Pierce Melissa A. , Marron Michele P. , Wicker Linda S. , Peterson Laurence B. , Shultz Leonard D. , Mordes John P. , Rossini Aldo A. , Greiner Dale L.
Published: April 2014

Curing type 1 diabetes by islet transplantation requires overcoming both allorejection and recurrent autoimmunity. This has been achieved with systemic immunosuppression, but tolerance induction would be preferable. Most islet allotransplant tolerance induction protocols have been tested in nonobese diabetic (NOD) mice, and most have failed. Failure has been attributed to the underlying autoimmunity, assuming that autoimmunity and resistance to transplantation tolerance have a common basis. Out of concern that NOD biology could be misleading in this regard, we tested the hypothesis that autoimmunity and resistance to transplantation tolerance in NOD mice are distinct phenotypes. Unexpectedly, we observed that (NOD × C57BL/6)F1 mice, which have no diabetes, nonetheless resist prolongation of skin allografts by costimulation blockade. Further analyses revealed that the F1 mice shared the dendritic cell maturation defects and abnormal CD4+ T cell responses of the NOD but had lost its defects in macrophage maturation and NK cell activity. We conclude that resistance to allograft tolerance induction in the NOD mouse is not a direct consequence of overt autoimmunity and that autoimmunity and resistance to costimulation blockade-induced transplantation tolerance phenotypes in NOD mice can be dissociated genetically. The outcomes of tolerance induction protocols tested in NOD mice may not accurately predict outcomes in human subjects.
Peroxisome Proliferator-Activated Receptor α Negatively Regulates T-bet Transcription Through Suppression of p38 Mitogen-Activated Protein Kinase Activation
Authors: Jones Dallas C. , Ding Xiaohong , Zhang Tian Y. , Daynes Raymond A.
Published: April 2014

Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα) in resting lymphocytes was recently established, although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study, we used CD4+ T cells isolated from PPARα−/− and wild-type mice, as well as cell lines that constitutively express PPARα, in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4+ T cells lacking PPARα produce increased levels of IFN-γ, but significantly lower levels of IL-2 when compared with activated wild-type CD4+ T cells. Furthermore, we demonstrate that PPARα regulates the expression of these cytokines by CD4+ T cells in part, through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4+ T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation, and the presence of unliganded PPARα effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARα with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARα.
Development of Infectious Tolerance After Donor-Specific Transfusion and Rat Heart Transplantation
Authors: Kataoka Masaaki , Margenthaler Julie A. , Ku Grace , Flye M. Wayne
Published: April 2014

Regulatory cells developed after donor-specific transfusion (DST)-induced acceptance of a LEW heart transplanted into a DA rat. Both DST and the cardiac transplant were necessary to generate the regulatory cells. This donor-specific tolerance can then be transferred into a new DA recipient by adoptive transfer of lymphocytes from the DST-treated long term survivor (LTS) in a dose-dependent manner. The effectiveness of tolerance did not diminish over five generations of adoptive transfer, thus supporting its infectious nature. Although both spleen and lymph node cells were equally effective, graft-infiltrating lymphocytes were more potent. A high level of indirect CTL activity and MLC proliferation were observed in lymphocytes from LTS. In vivo tracking of adoptively transferred CFSE-labeled splenocytes from LTS showed equivalent FACS proliferation and a higher percentage of graft-infiltrating lymphocytes 7 days after heart transplantation, compared with adoptively transferred naive splenocytes. Adoptive transfer of CD8+-depleted LTS splenocytes resulted in 100% subsequent LEW allograft acceptance; whereas CD4+ depletion decreased acceptance to 40%, and depletion of both CD4 and CD8 resulted in 0% acceptance. When positively selected CD4+ or CD8+ cells were adoptively transferred, 100% or 62.5% of LEW cardiac allografts survived, respectively. In conclusion, DST alone promotes a donor-specific infectious tolerance of a heart graft that can be adoptively transferred to subsequent naive allograft recipients despite the undiminished in vitro immunological response to donor Ag. Although both CD4+ and CD8+ populations are responsible for the regulatory mechanism in DST-induced tolerance, the CD4+ population appears to dominate.
Plasmacytoid Dendritic Cell-Derived IFN-α Induces TNF-Related Apoptosis-Inducing Ligand/Apo-2L-Mediated Antitumor Activity by Human Monocytes Following CpG Oligodeoxynucleotide Stimulation
Authors: Kemp Troy J. , Elzey Bennett D. , Griffith Thomas S.
Published: April 2014

Immunostimulatory oligodeoxynucleotides (ODN) containing the CpG motif are being tested as immune adjuvants in many disease settings. Of the human PBMC examined, plasmacytoid dendritic cells (pDC) are a major source of type I IFN upon stimulation with CpG ODN. IFNs have numerous immunostimulatory effects, including the induction of TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L on monocytes, NK cells, and T cells. Importantly, IFN has also been linked to antitumor responses. Thus, we tested whether CpG ODN stimulation of PBMC led to TRAIL/Apo-2L-induced tumor cell death. When PBMC were stimulated with CpG ODN, TRAIL/Apo-2L-dependent tumor cell death was observed. Further examination of CpG ODN-stimulated PBMC revealed that TRAIL/Apo-2L expression was limited to CD14+ cells, which, when depleted, led to a loss of the TRAIL/Apo-2L-mediated tumor cell killing. Moreover, pDC depletion also abolished the TRAIL/Apo-2L-mediated killing of tumor cell targets. Analysis of the pDC showed IFN-α production after CpG ODN stimulation. Finally, inclusion of neutralizing IFN-α antiserum with the PBMC during CpG ODN stimulation abrogated TRAIL/Apo-2L-mediated tumor cell killing. These results define a mechanism by which CpG ODN induces TRAIL/Apo-2L-dependent killing of tumor cells by CD14+ PBMC, in which CpG ODN-activated pDC produce IFN-α that stimulates CD14+ PBMC to express functional TRAIL/Apo-2L.
A MAGE-3 Peptide Presented by HLA-DR1 to CD4+ T Cells That Were Isolated from a Melanoma Patient Vaccinated with a MAGE-3 Protein
Authors: Zhang Yi , Chaux Pascal , Stroobant Vincent , Eggermont Alexander M. M. , Corthals Jurgen , Maillère Bernard , Thielemans Kris , Marchand Marie , Boon Thierry , van der Bruggen Pierre
Published: April 2014

“Cancer-germline” genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE-3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE-3 peptide, which is recognized by three different CD4+ T cell clones isolated from a melanoma patient vaccinated with a MAGE-3 protein. These clones, which express different TCRs, recognize on HLA-DR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE-3267–282 and the MAGE-12267–282 protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE-3. Another of these DR1-restricted CD4+ clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.
Cutting Edge: Class I Presentation of Host Peptides Following HIV Infection
Authors: Hickman Heather D. , Luis Angela D. , Bardet Wilfried , Buchli Rico , Battson Casey L. , Shearer Michael H. , Jackson Kenneth W. , Kennedy Ronald C. , Hildebrand William H.
Published: April 2014

Class I MHC molecules bind intracellular peptides for presentation to cytotoxic T lymphocytes. Identification of peptides presented by class I molecules during infection is therefore a priority for detecting and targeting intracellular pathogens. To understand which host-encoded peptides distinguish HIV-infected cells, we have developed a mass spectrometric approach to characterize HLA-B*0702 peptides unique to or up-regulated on infected T cells. In this study, we identify 15 host proteins that are differentially presented on infected human T cells. Peptides with increased expression on HIV-infected cells were derived from multiple categories of cellular proteins including RNA binding proteins and cell cycle regulatory proteins. Therefore, comprehensive analysis of the B*0702 peptide repertoire demonstrates that marked differences in host protein presentation occur after HIV infection.
T Cell Receptor Revision Does Not Solely Target Recent Thymic Emigrants
Authors: Cooper Cristine J. , Orr Mark T. , McMahan Catherine J. , Fink Pamela J.
Published: April 2014

CD4+Vβ5+ T cells enter one of two tolerance pathways after recognizing a peripherally expressed superantigen encoded by an endogenous retrovirus. One pathway leads to deletion, while the other, termed TCR revision, results in cellular rescue upon expression of an alternate TCR that no longer recognizes the tolerogen. TCR revision requires the rearrangement of novel TCR β-chain genes and depends on recombinase-activating gene (RAG) expression in peripheral T cells. In line with recent findings that RAG+ splenic B cells are immature cells that have maintained RAG expression, it has been hypothesized that TCR revision is limited to recent thymic emigrants that have maintained RAG expression and TCR loci in a recombination-permissive configuration. Using mice in which the expression of green fluorescent protein is driven by the RAG2 promoter, we now show that in vitro stimulation can drive reporter expression in noncycling, mature, peripheral CD4+ T cells. In addition, thymectomized Vβ5 transgenic RAG reporter mice are used to demonstrate that TCR revision can target peripheral T cells up to 2 mo after thymectomy. Both sets of experiments strongly suggest that reinduction of RAG genes triggers TCR revision. Approximately 3% of CD4+Vβ5+ T cells in thymectomized Vβ5 transgenic reporter mice have undergone TCR revision within the previous 4–5 days. TCR revision can also occur in Vβ5+ T cells from nontransgenic mice, illustrating the relevance of this novel tolerance mechanism in unmanipulated animals.
NK Cells Regulate CD4 Responses Prior to Antigen Encounter
Authors: Dowdell Kennichi C. , Cua Daniel J. , Kirkman Erlinda , Stohlman Stephen A.
Published: April 2014

NK cells not only respond rapidly to infection, shaping subsequent adaptive immunity, but also play a role in regulating autoimmune disease. The ability of NK cells to influence adaptive immunity before Ag exposure was examined in a gender-dependent model of preferential Th1 and Th2 activation. The inability of young adult male SJL mice to activate Th1 cells was reversed via depletion of NK1.1+ cells, whereas the presence or the absence of NK1.1+ cells did not alter responses in age-matched females. Consistent with a gender-dependent role in regulating adaptive immunity, significantly more NK1.1+ cells were present in males compared with females, and this difference was reversed by castration. In contrast to NK1.1+ cells derived from C57BL/6 mice, no spontaneous cytokine secretion was detected in NK1.1+ cells derived from either male or female SJL mice, although an increased frequency of IL-10-secreting NK1.1+ cells was observed in males vs females following in vitro stimulation. Direct evidence that NK1.1+ cells in males influence CD4+ T cell activation before Ag exposure was demonstrated via the adoptive transfer of APC from control and NK1.1-depleted males. The absence of a functional NK T cell population in SJL mice suggests that NK cells influence adaptive immunity before Ag exposure via alterations in APC activity.
Local Cooperation Dominates Over Competition Between CD4+ T Cells of Different Antigen/MHC Specificity
Authors: Creusot Rémi J. , Thomsen Lindy L. , Tite John P. , Chain Benjamin M.
Published: April 2014

Interactions between CD4+ T cells in vivo are controlled by a balance between cooperation and competition. In this study the interaction between two populations of CD4+ T cells of different MHC/peptide specificity was probed at different precursor frequencies, delivering one or both Ags to APC using particle-mediated DNA delivery. Expansion of clonal populations of Ag (OVA and pigeon cytochrome c-specific) CD4+ T cells was limited at higher precursor frequencies, presumably reflecting intraclonal competition. In contrast, a strong enhancement of the number of cells expressing IFN-γ, IL-4, and IL-2 was observed in populations of cells at low precursor frequency in the presence of a high frequency of activated cells of a different Ag specificity. The helper effect was most potent when both Ags were delivered to the same dendritic cell (i.e., linked). This reflects the requirement of epicrine or paracrine help for optimal activation of T cell clones at low frequency. A measure of help was also delivered in an endocrine manner (unlinked), especially for Th1 responses, suggesting that there is also limited diffusion of cytokines between dendritic cell clusters. The dominant effects of cooperation over competition between CD4+ T cells responding to different Ags may have important implications in terms of the efficacy of multivalent vaccines.
The Requirements for Fas-Associated Death Domain Signaling in Mature T Cell Activation and Survival
Authors: Beisner Daniel R. , Chu Isaac H. , Arechiga Adrian F. , Hedrick Stephen M. , Walsh Craig M.
Published: April 2014

Fas-associated death domain (FADD) is a death domain containing cytoplasmic adapter molecule required for the induction of apoptosis by death receptors. Paradoxically, FADD also plays a crucial role in the development and proliferation of T cells. Using T cells from mice expressing a dominant negative form of FADD (FADDdd), activation with anti-TCR Ab and costimulation or exogenous cytokines is profoundly diminished. This is also seen in wild-type primary T cells transduced with the same transgene, demonstrating that FADD signaling is required in normally differentiated T cells. The defective proliferation does not appear to be related to the early events associated with TCR stimulation. Rather, with a block in FADD signaling, stimulated T cells exhibit a high rate of cell death corresponding to the initiation of cell division. Although CD4 T cells exhibit a moderate deficiency, this effect is most profound in CD8 T cells. In vivo, the extent of this defective accumulation is most apparent; lymphocytic choriomenigitis virus-infected FADDdd-expressing mice completely fail to mount an Ag-specific response. These results show that, in a highly regulated fashion, FADD, and most likely caspases, can transduce either a signal for survival or one that leads directly to apoptosis and that the balance between these opposing outcomes is crucial to adaptive immunity.
TNF-α Induces Tyrosine Phosphorylation and Recruitment of the Src Homology Protein-Tyrosine Phosphatase 2 to the gp130 Signal-Transducing Subunit of the IL-6 Receptor Complex
Authors: Bode Johannes G. , Schweigart Jens , Kehrmann Jan , Ehlting Christian , Schaper Fred , Heinrich Peter C. , Häussinger Dieter
Published: April 2014

Recently, it has been demonstrated that TNF-α and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-α also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-α causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-α, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-α, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-α. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-α on IL-6 signaling and TNF-α-dependent recruitment of SHP2 to gp130.
Activation of Protein Kinase Cδ by IFN-γ
Authors: Deb Dilip K. , Sassano Antonella , Lekmine Fatima , Majchrzak Beata , Verma Amit , Kambhampati Suman , Uddin Shahab , Rahman Arshad , Fish Eleanor N. , Platanias Leonidas C.
Published: April 2014

Engagement of the type II IFN (IFN-γ) receptor results in activation of the Janus kinase-Stat pathway and induction of gene transcription via IFN-γ-activated site (GAS) elements in the promoters of IFN-γ-inducible genes. An important event in IFN-γ-dependent gene transcription is phosphorylation of Stat1 on Ser727, which is regulated by a kinase activated downstream of the phosphatidylinositol 3′-kinase. Here we provide evidence that a member of the protein kinase C (PKC) family of proteins is activated downstream of the phosphatidylinositol 3′-kinase and is engaged in IFN-γ signaling. Our data demonstrate that PKCδ is rapidly phosphorylated during engagement of the type II IFNR and its kinase domain is induced. Subsequently, the activated PKCδ associates with a member of the Stat family of proteins, Stat1, which acts as a substrate for its kinase activity and undergoes phosphorylation on Ser727. Inhibition of PKCδ activity diminishes phosphorylation of Stat1 on Ser727 and IFN-γ-dependent transcriptional regulation via IFN-γ-activated site elements, without affecting the phosphorylation of the protein on Tyr701. Thus, PKCδ is activated during engagement of the IFN-γ receptor and plays an important role in IFN-γ signaling by mediating serine phosphorylation of Stat1 and facilitating transcription of IFN-γ-stimulated genes.
Cutting Edge: Rapid In Vivo Killing by Memory CD8 T Cells
Authors: Barber Daniel L. , Wherry E. John , Ahmed Rafi
Published: April 2014

In this study, we examined the cytotoxic activity of effector and memory CD8 T cells in vivo. At the peak of the CTL response following an acute lymphocytic choriomeningitis virus infection, effector CD8 T cells exhibited extremely rapid killing and started to eliminate adoptively transferred target cells within 15 min by a perforin-dependent mechanism. Although resting memory CD8 T cells are poorly cytolytic by in vitro 51Cr release assays, there was rapid elimination (within 1–4 h) of target cells after transfer into immune mice, and both CD62Lhigh and CD62Llow memory CD8 T cells were able to kill rapidly in vivo. Strikingly, when directly compared on a per cell basis, memory CD8 T cells were only slightly slower than effector cells in eliminating target cells. These data indicate that virus specific memory CD8 T cells can rapidly acquire cytotoxic function upon re-exposure to Ag and are much more efficient killers in vivo than previously appreciated.
A Novel Instance of Class I Modification (cim) Affecting Two of Three Rat Class I RT1-A Molecules Within One MHC Haplotype
Authors: González-Muñoz Andrea L. , Le Rolle Anne-France , Brun Hélène , Hedrich Hans J. , Wedekind Dirk , Powis Simon J. , Joly Etienne , Butcher Geoffrey W.
Published: April 2014

MHC class I expression by rats of the RT1o, RT1d, and RT1m MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1o and BDIX (RT1dv1) rats for three MHC class I molecules. RT1-A1o/d and -A2o/d are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3° is highly homologous to a class I gene identified by sequencing an RT1-An genomic contig and is named A3n. Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1° is the principal MHC class Ia (classical) restricting element of this haplotype. A2°, which is highly cross-reactive with A1°, and A3° probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1m haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1m contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1° and -A2° than it does on RT1-Aa in the established case of class I modification (cim). Historically, this led to the misclassification of RT1m class Ia molecules as separate and distinct.
IFN-α Induces the Human IL-10 Gene by Recruiting Both IFN Regulatory Factor 1 and Stat3
Authors: Ziegler-Heitbrock Löms , Lötzerich Mark , Schaefer Annette , Werner Thomas , Frankenberger Marion , Benkhart Elke
Published: April 2014

The anti-inflammatory cytokine IL-10 can be induced by type I IFNs, but the molecular mechanisms involved have remained elusive. With in silico analysis of the human IL-10 promoter we identified a module consisting of an IFN regulatory factor 1 (IRF-1) site and a Stat3 site. We demonstrate that IFN-α will induce the binding of IRF-1 and Stat3 to the respective motifs. Mutational analysis revealed that inactivation of the IRF-1 motif substantially reduces trans-activation from 5- to 2-fold and that inactivation of the Stat3 motif completely ablates trans-activation by IFN-α. The dominant role of Stat3 in this module was confirmed with the blockade of trans-activation by a dominant negative Stat3. By contrast, Stat1 contributes a minor proportion to the DNA binding to the Stat site, and overexpression will counteract Stat3-mediated trans-activation. The data show that IFN-α induces the IL-10 gene via a module consisting of interdependent IRF-1 and Stat3 motifs. Of note, LPS-induced trans-activation does not target this module, since it is independent of the IRF-1 motif but completely depends on Stat3.