bluuebunny/crossref_metadata_2025 huggingface dataset:
Foxp3 Expressing CD4+CD25high Regulatory T Cells Are Overrepresented in Human Metastatic Melanoma Lymph Nodes and Inhibit the Function of Infiltrating T Cells
Authors: Viguier Manuelle , Lemaître Fabrice , Verola Olivier , Cho Min-Sun , Gorochov Guy , Dubertret Louis , Bachelez Hervé , Kourilsky Philippe , Ferradini Laurent
Published: April 2014

Dominant tolerance is mediated by regulatory T cells (Treg) that control harmful autoimmune T cells in the periphery. In this study, we investigate the implication of Treg in modulating infiltrating T lymphocytes in human metastatic melanoma. We found that CD4+CD25high T cells are overrepresented in metastatic lymph nodes (LNs) with a 2-fold increased frequency compared with both tumor-free LNs and autologous PBMCs. These cells express the Foxp3 transcription factor, display an activated phenotype, and display a polyclonal TCR Vβ chain repertoire. They inhibit in vitro the proliferation and cytokine production of infiltrating CD4+CD25− and CD8+ T cells (IL-2, IFN-γ) through a cell-contact-dependent mechanism, thus behaving as Treg. In some cases, the presence of Treg type 1/Th3-like lymphocytes could also be demonstrated. Thus, Treg are a major component of the immunosuppressive microenvironment of metastatic melanoma LNs. This could explain the poor clinical response of cancer patients under immunotherapeutic protocols, and provides a new basis for future immunotherapeutic strategies counteracting in vivo Treg to reinforce local antitumor immune responses.
Peritoneal Exudate Cells Treated with Calcitonin Gene-Related Peptide Suppress Murine Experimental Autoimmune Uveoretinitis via IL-10
Authors: Kezuka Takeshi , Takeuchi Masaru , Keino Hiroshi , Usui Yoshihiko , Takeuchi Aya , Yamakawa Noriyuki , Usui Masahiko
Published: April 2014

Immunization with retinal Ag induces experimental autoimmune uveoretinitis (EAU) in mice. We investigated the suppression of murine EAU by peritoneal exudate cells (PEC) cultured with calcitonin gene-related peptide (CGRP). PEC derived from mice were treated with CGRP and residues 1–20 of human interphotoreceptor retinoid-binding protein (hIRBP 1–20). The hIRBP 1–20-immunized mice were injected i.v. with PEC treated with CGRP and hIRBP 1–20. After immunization, Ag-specific delayed hypersensitivity (DH) was measured and EAU was assessed histopathologically. Both EAU- and Ag-specific DH were suppressed by injection of PEC treated with CGRP (100 ng/ml) and hIRBP 1–20. However, hIRBP 1–20-mediated EAU was not suppressed by injection of PEC treated with CGRP and BSA. Both EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1–20 into splenectomized mice. In mice adoptively transferred spleen cells from hIRBP 1–20-immunized mice, EAU was also suppressed by injection of CGRP-treated PEC. EAU was markedly inhibited in hIRBP 1–20-immunized mice adoptively transferred T cells obtained from mice injected with hIRBP 1–20-pulsed, CGRP-treated PEC. Furthermore, EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1–20 when the recipient mice were given anti-IL-10 Ab i.p., or when the PEC were derived from IL-10 knockout mice. The present results indicate that PEC treated with CGRP suppress murine EAU in an Ag-specific manner, even in the efferent phase, and IL-10 secreted from PEC might play an important role in the CGRP-mediated suppression of murine EAU.
IL-15 and the Initiation of Cell Contact-Dependent Synovial Fibroblast-T Lymphocyte Cross-Talk in Rheumatoid Arthritis: Effect of Methotrexate
Authors: Miranda-Carús María-Eugenia , Balsa Alejandro , Benito-Miguel Marta , Pérez de Ayala Carlos , Martín-Mola Emilio
Published: April 2014

To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = 20) induced an up-regulation of ICAM-1, intracellular IL-8, IL-6, IL-15, and surface IL-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-α, IFN-γ, IL-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = 20) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = 20) were maximal. Blocking Abs to IL-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, IL-17, TNF-α, and IFN-γ significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4-μm inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of IL-15 induces the proinflammatory cytokines TNF-α, IFN-γ, and IL-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of IL-15, IL-8, and IL-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.
Chlamydia pneumoniae Stimulates IFN-γ Synthesis through MyD88-Dependent, TLR2- and TLR4-Independent Induction of IL-18 Release
Authors: Netea Mihai G. , Kullberg Bart Jan , Jacobs Liesbeth E. H. , Verver-Jansen Trees J. G. , van der Ven-Jongekrijg Johanna , Galama Jochem M. D. , Stalenhoef Anton F. H. , Dinarello Charles A. , Van der Meer Jos W. M.
Published: April 2014

Recent studies suggest that inflammation plays a central role in the pathogenesis of atherosclerosis, and IFN-γ is a prominent proinflammatory mediator in this context. However, it is unclear what stimuli are responsible for initial stimulation of IFN-γ synthesis in the vessel wall. In the present study, we demonstrate that Chlamydia pneumoniae is an important stimulus for IFN-γ synthesis, and this production depends on release of endogenous IL-18, IL-12, and IL-1, but not of TNF. The production of the proinflammatory cytokines TNF and IL-1β from PBMC by sonicated C. pneumoniae was mediated through TLR2-dependent pathways. In contrast, C. pneumoniae stimulated the production of IL-18 through MyD88-dependent, TLR2-, TLR4-, and CD14-independent pathways, mediated by posttranscriptional mechanisms not involving de novo protein synthesis. In conclusion, C. pneumoniae is a potent stimulus of IFN-γ production, in addition to the proinflammatory cytokines TNF and IL-1β, which may contribute to its proatherogenic effects. Most interestingly, C. pneumoniae is also a potent inducer of IL-18 production through pathways independent of TLR2 and TLR4.
Understanding the Pro- and Anti-Inflammatory Properties of IL-27
Authors: Villarino Alejandro V. , Huang Elaine , Hunter Christopher A.
Published: April 2014

The recent identification of IL-27 (IL-27p28/EBV-induced gene 3) and IL-27R (WSX-1/gp130) has provided new insights for the biology of IL-6/IL-12 family cytokines. Initial studies indicated that IL-27 can directly regulate T cell functions and suggested an important role for it in promoting Th1 type responses. However, subsequent studies have revealed that IL-27R signaling influences a variety of immune cell types and can inhibit either Th1 or Th2 type responses. Though elucidation of the Jak/STAT signaling pathways activated by IL-27R ligation has unveiled some of the molecular mechanisms used by IL-27 to promote inflammation, little is known about the anti-inflammatory activities of this cytokine. Thus, the aim of this review is to discuss the pleotropic nature of the IL-27/IL-27R interaction and attempt to reconcile the pro- and anti-inflammatory properties of this immunomodulator.
Cutting Edge: Pulmonary Immunopathology Mediated by Antigen-Specific Expression of TNF-α by Antiviral CD8+ T Cells
Authors: Xu Lumei , Yoon Heesik , Zhao Min Q. , Liu Jun , Ramana Chilakamarti V. , Enelow Richard I.
Published: April 2014

Respiratory virus infection results in considerable pulmonary immunopathology, a component of which results from the host immune responses. We have developed a murine model to specifically examine the lung injury due to CD8+ T cell recognition of an influenza hemagglutinin (HA) transgene on lung epithelium in the absence of replicating virus, after adoptive transfer. Lung injury is largely mediated by chemokines expressed by the epithelial cells upon T cell recognition mediated by TNF-α. To determine the critical source of TNF-α, HA-specific TNF−/− CD8+ T cells were transferred into HA transgenic animals, and lung injury was not observed, though these T cells exhibited no defect in antiviral activity in vivo. This indicates that the initiating event in the injury process is Ag-specific expression of TNF-α by antiviral CD8+ T cells upon recognition of alveolar epithelial Ag, and that the effector activities responsible for viral clearance may be dissociable from those resulting in immunopathology.
Cutting Edge: Selective Requirement for the Wiskott-Aldrich Syndrome Protein in Cytokine, but Not Chemokine, Secretion by CD4+ T Cells
Authors: Morales-Tirado Vanessa , Johannson Sara , Hanson Elaine , Howell Alan , Zhang Jinyi , Siminovitch Katherine A. , Fowell Deborah J.
Published: April 2014

The mechanism of cytokine secretion is not well understood, but cytokines appear to be synthesized and released in a polarized fashion toward an Ag-specific target cell. In this study, we demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an essential component of the cytokine secretory pathway in CD4+ T cells. Murine WASp-deficient CD4+ T cells fail to polarize cytokines toward a target and show an unexpected and striking block in cytokine secretion. In contrast, chemokine secretion and trafficking of plasma membrane proteins, transported via the constitutive secretory pathway, are unaffected by the lack of WASp. These results suggest that CD4+ T cell cytokines require a specialized, WASp-dependent pathway for cellular traffic and/or vesicle release that is distinct from that required for chemokine release. We propose that the use of different secretory pathways for cytokines and chemokines enables CD4+ T cell activity to be further fine-tuned to serve specialized effector functions.
Cutting Edge: Induction of the Antigen-Processing Enzyme IFN-γ-Inducible Lysosomal Thiol Reductase in Melanoma Cells Is STAT1-Dependent but CIITA-Independent
Authors: O’Donnell Patrick W. , Haque Azizul , Klemsz Michael J. , Kaplan Mark H. , Blum Janice S.
Published: April 2014

Presentation and CD4+ T cell responses to Ag in the context of MHC class II molecules require processing of native proteins into short peptide fragments. Within this pathway, IFN-γ-inducible lysosomal thiol reductase (GILT) functions to catalyze thiol bond reduction, thus unfolding native protein Ag and facilitating further processing via cellular proteases. In contrast with professional APCs such as B cells, class II-positive human melanomas expressed relatively little to no GILT protein or mRNA. Tumor cell GILT expression was partially restored with IFN-γ treatment but unlike other genes required for class II Ag presentation, GILT was not regulated by CIITA. Rather, studies revealed STAT1 plays a direct role in IFN-γ-inducible GILT expression. These results define a molecular mechanism for the uncoupled regulation of MHC class II genes and the processing enzyme GILT in human melanomas.
Cell Proliferation and STAT6 Pathways Are Negatively Regulated in T Cells by STAT1 and Suppressors of Cytokine Signaling
Authors: Yu Cheng-Rong , Mahdi Rashid M. , Ebong Samuel , Vistica Barbara P. , Chen Jun , Guo Yonghong , Gery Igal , Egwuagu Charles E.
Published: April 2014

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-γ is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1−/− Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.
C3a and C3b Activation Products of the Third Component of Complement (C3) Are Critical for Normal Liver Recovery after Toxic Injury
Authors: Markiewski Maciej M. , Mastellos Dimitrios , Tudoran Ruxandra , DeAngelis Robert A. , Strey Christoph W. , Franchini Silvia , Wetsel Rick A. , Erdei Anna , Lambris John D.
Published: April 2014

Although the complement system has been implicated in liver regeneration after toxic injury and partial hepatectomy, the mechanism or mechanisms through which it participates in these processes remains ill-defined. In this study, we demonstrate that complement activation products (C3a, C3b/iC3b) are generated in the serum of experimental mice after CCl4 injection and that complement activation is required for normal liver regeneration. Decomplementation by cobra venom factor resulted in impaired entry of hepatocytes into S phase of the cell cycle. In addition, livers from C3-deficient (C3−/−) mice showed similarly impaired proliferation of hepatocytes, along with delayed kinetics of both hepatocyte hyperplasia and removal of injured liver parenchyma. Restoration of hepatocyte proliferative capabilities of C3−/− mice through C3a reconstitution, as well as the impaired regeneration of C3a receptor-deficient mice, demonstrated that C3a promotes liver cell proliferation via the C3a receptor. These findings, together with data showing two waves of complement activation, indicate that C3 activation is a pivotal mechanism for liver regeneration after CCl4 injury, which fulfills multiple roles; C3a generated early after toxin injection is relevant during the priming of hepatocytes, whereas C3 activation at later times after CCl4 treatment contributes to the clearance of injured tissue.
Cholera Toxin Prevents Th1-Mediated Autoimmune Disease by Inducing Immune Deviation
Authors: Su Shao-Bo , Silver Phyllis B. , Wang Peng , Chan Chi-Chao , Caspi Rachel R.
Published: April 2014

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, is known for its properties as a mucosal adjuvant that promotes Th2 or mixed Th1 + Th2 responses. In this study, we explore the ability of CT to act as a systemic adjuvant to counteract the Th1 response leading to experimental autoimmune uveitis. We report that susceptible B10.RIII mice immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein could be protected from disease by a single systemic injection of as little as 2 μg of CT at the time of immunization. The protected mice were not immunosuppressed, but rather displayed evidence of immune deviation. Subsequent adaptive responses to interphotoreceptor retinoid-binding protein showed evidence of Th2 enhancement, as indicated by reduced delayed-type hypersensitivity in the context of enhanced Ag-specific lymphocyte proliferation and IL-4 production. Ag-specific production of several other cytokines, including IFN-γ, was not appreciably altered. The inhibitory effect of CT was dependent on the enzymatic A subunit of CT, because the cell-binding B subunit alone could not block disease development. Mice given CT displayed detectable IL-4 levels in their serum within hours of CT administration. This innate IL-4 production was critical for protection, as infusion of neutralizing Ab against IL-4 to mice, given a uveitogenic immunization and treated with CT, counteracted immune deviation and abrogated protection. Our data indicate that systemic administration of CT inhibits experimental autoimmune uveitis by skewing the response to the uveitogenic autoantigen to a nonpathogenic phenotype.
Role of Gut-Associated Lymphoreticular Tissues in Antigen-Specific Intestinal IgA Immunity
Authors: Yamamoto Masafumi , Kweon Mi-Na , Rennert Paul D. , Hiroi Takachika , Fujihashi Kohtaro , McGhee Jerry R. , Kiyono Hiroshi
Published: April 2014

This study assessed the roles of the postnatal lymphotoxin-β receptor (LTβR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTβR-dependent events by postnatal administration of the fusion protein of LTβR and IgG Fc (LTβR-Ig) reduced both the size and numbers of Peyer’s patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTβR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTβR-Ig eliminated the formation of ILF. LTβR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTβR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.
Tec Kinase Migrates to the T Cell-APC Interface Independently of Its Pleckstrin Homology Domain
Authors: Garçon Fabien , Bismuth Georges , Isnardon Daniel , Olive Daniel , Nunès Jacques A.
Published: April 2014

Tec is the prototypical member of the Tec tyrosine kinases family, which plays an important role in T cell signaling. We show in this study that Tec translocates to the immunological synapse when a T cell contacts a dendritic cell. Surprisingly, the presence of the pleckstrin homology (PH) domain of Tec is not required for this accumulation, and despite a strong activation of 3′-phosphorylated phosphoinositide lipids synthesis during the synapse formation, the Tec PH domain is not redistributed to the T cell plasma membrane. In contrast, we demonstrate that an active Src homology 3 domain is absolutely required, underlining the essential role played by this part of the molecule in the recruitment and/or stabilization of Tec at the immunological synapse. Our results nevertheless suggest that the PH domain controls the kinase activity of the molecule in vivo. We finally demonstrate that the two domains are necessary to trigger transcriptional events following Ag presentation. These data support a model in which the plasma membrane recruitment of the PH-containing protein Tec is not dependent on the production of 3′-phosphorylated phosphoinositide lipids by the PI3K, but rather on an intact Src homology 3 domain.
K+ Channel Expression during B Cell Differentiation: Implications for Immunomodulation and Autoimmunity
Authors: Wulff Heike , Knaus Hans-Günther , Pennington Michael , Chandy K. George
Published: April 2014

Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD+CD27+ memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca2+-activated intermediate-conductance IKCa1 channel when quiescent, and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast, quiescent class-switched memory B cells express high levels of Kv1.3 (∼2000 channels/cell) and maintain their Kv1.3high expression after activation. Consistent with their channel phenotypes, proliferation of naive and IgD+CD27+ memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore, specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders.
Expression of the B7.1 Costimulatory Molecule on Pancreatic β Cells Abrogates the Requirement for CD4 T Cells in the Development of Type 1 Diabetes
Authors: Havari Evis , Lennon-Dumenil Ana Maria , Klein Ludger , Neely Devon , Taylor Jacqueline A. , McInerney Marcia F. , Wucherpfennig Kai W. , Lipes Myra A.
Published: April 2014

Although HLA-DQ8 has been implicated as a key determinant of genetic susceptibility to human type 1 diabetes, spontaneous diabetes has been observed in HLA-DQ8 transgenic mice that lack expression of murine MHC class II molecules (mII−/−) only when the potent costimulatory molecule, B7.1, is transgenically expressed on pancreatic β cells. To study the contribution of HLA-DQ8 to the development of diabetes in this model, we crossed RIP-B7.1mII−/− mice with a set of transgenic mouse lines that differed in their HLA-DQ8 expression patterns on APC subpopulations, in particular dendritic cells and cortical thymic epithelial cells. Surprisingly, we found that even in the absence of HLA-DQ8 and CD4 T cells, a substantial fraction of the RIP-B7.1mII−/− mice developed diabetes. This disease process was remarkable for not only showing insulitis, but also inflammatory destruction of the exocrine pancreas with diffusely up-regulated expression of MHC class I and ICAM-1 molecules. Expression of HLA-DQ8 markedly increased the kinetics and frequency of diabetes, with the most severe disease in the lines with the highest levels of HLA-DQ8 on cortical thymic epithelial cells and the largest numbers of CD4 T cells. However, the adoptive transfer of diabetes was not HLA-DQ8-dependent and disease could be rapidly induced with purified CD8 T cells alone. Expression of B7.1 in the target tissue can thus dramatically alter the cellular and molecular requirements for the development of autoimmunity.
Mechanism by Which Orally Administered β-1,3-Glucans Enhance the Tumoricidal Activity of Antitumor Monoclonal Antibodies in Murine Tumor Models
Authors: Hong Feng , Yan Jun , Baran Jarek T. , Allendorf Daniel J. , Hansen Richard D. , Ostroff Gary R. , Xing Pei Xiang , Cheung Nai-Kong V. , Ross Gordon D.
Published: April 2014

Antitumor mAb bind to tumors and activate complement, coating tumors with iC3b. Intravenously administered yeast β-1,3;1,6-glucan functions as an adjuvant for antitumor mAb by priming the inactivated C3b (iC3b) receptors (CR3; CD11b/CD18) of circulating granulocytes, enabling CR3 to trigger cytotoxicity of iC3b-coated tumors. Recent data indicated that barley β-1,3;1,4-glucan given orally similarly potentiated the activity of antitumor mAb, leading to enhanced tumor regression and survival. This investigation showed that orally administered yeast β-1,3;1,6-glucan functioned similarly to barley β-1,3;1,4-glucan with antitumor mAb. With both oral β-1,3-glucans, a requirement for iC3b on tumors and CR3 on granulocytes was confirmed by demonstrating therapeutic failures in mice deficient in C3 or CR3. Barley and yeast β-1,3-glucan were labeled with fluorescein to track their oral uptake and processing in vivo. Orally administered β-1,3-glucans were taken up by macrophages that transported them to spleen, lymph nodes, and bone marrow. Within the bone marrow, the macrophages degraded the large β-1,3-glucans into smaller soluble β-1,3-glucan fragments that were taken up by the CR3 of marginated granulocytes. These granulocytes with CR3-bound β-1,3-glucan-fluorescein were shown to kill iC3b-opsonized tumor cells following their recruitment to a site of complement activation resembling a tumor coated with mAb.
B Cell-Activating Factor Belonging to the TNF Family (BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells
Authors: Ng Lai Guan , Sutherland Andrew P. R. , Newton Rebecca , Qian Fang , Cachero Teresa G. , Scott Martin L. , Thompson Jeffrey S. , Wheway Julie , Chtanova Tatyana , Groom Joanna , Sutton Ian J. , Xin Cynthia , Tangye Stuart G. , Kalled Susan L. , Mackay Fabienne , Mackay Charles R.
Published: April 2014

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using specific mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti-μ responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells.
Decreased E47 in Senescent B Cell Precursors Is Stage Specific and Regulated Posttranslationally by Protein Turnover
Authors: Van der Put Elaine , Frasca Daniela , King Anne M. , Blomberg Bonnie B. , Riley Richard L.
Published: April 2014

The E2A-encoded transcription factor E47 is crucial to B lymphopoiesis. Senescent BALB/c mice (∼2 years old) had reduced pre-B cells ex vivo. Pro-B/early pre-B cells from these aged mice, both ex vivo and in vitro, were deficient in E47 protein. In vitro, IL-7 expanded pro-B/early pre-B cells from young BALB/c mice expressed E47 protein that was relatively stable over a 5-h period. Cultured senescent pro-B/early pre-B cells exhibited reduced E47 protein stability with ∼50–90% loss of E47 over the same time period. Degradation of E47 was effectively blocked by the proteasome inhibitor lactacystin as well as calpain I and II inhibitors; E2A proteins were also shown to undergo ubiquitination. Although senescent B cell precursors expressed less E47 protein, E47 mRNA levels and turnover were normal. Therefore, E47 protein levels are reduced relatively early in B lineage differentiation in senescence and the decline in E47 protein occurs via increased protein degradation by proteasome and, possibly, calpain pathways. In contrast, normal E47 protein levels were observed within the highly reduced pre-B cell pool in aged mice. This suggests that pre-B cells in senescence undergo selection based on E47 expression. Increased degradation rates and lower steady-state levels were also observed for the transcription factors Pax-5/BSAP, Bob-1, and Ikaros, but this was not a general property of all proteins in aged B cell precursors. Therefore, altered turnover of multiple, select proteins crucial to B cell development may contribute to diminished B lymphopoiesis in old age.
IL-10 Plays an Important Role in the Homeostatic Regulation of the Autoreactive Repertoire in Naive Mice
Authors: Anderson Ana C. , Reddy Jayagopala , Nazareno Remedios , Sobel Raymond A. , Nicholson Lindsay B. , Kuchroo Vijay K.
Published: April 2014

We have previously shown that naive SJL (H-2s) mice, which are highly susceptible to myelin proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), have a very high frequency (1/20,000 CD4 T cells) of PLP139–151-reactive T cells in the naive repertoire. In this study, we examine the function of this endogenous PLP139–151-reactive repertoire in vivo and find that this repertoire encompasses the precursors of pathogenic T cells. Because SJL mice do not develop spontaneous EAE, we have explored the mechanisms that keep this autopathogenic repertoire in check and prevent the development of spontaneous autoimmunity. We crossed IL-4 and IL-10 deficiency onto the SJL background and analyzed the roles of these two immunoregulatory cytokines in regulating the size and effector function of the endogenous PLP139–151-reactive repertoire and development of autoimmune disease. We find that IL-10 is important in the homeostatic regulation of the endogenous PLP139–151-reactive repertoire in that it both limits the size of the repertoire and prevents development of effector autoaggressive T cells. SJL IL-10−/− mice with high numbers of PLP139–151-specific precursors in the repertoire did not develop spontaneous EAE, but when they were injected with pertussis toxin, they showed atypical clinical signs of EAE with small numbers of typical mononuclear cell infiltrates predominantly in the meninges. EAE could be inhibited by prior tolerization of the mice with soluble PLP139–151 peptide. These findings indicate that IL-10 may contribute to the regulation of the endogenous autoimmune repertoire.
No Intrinsic Deficiencies in CD8+ T Cell-Mediated Antitumor Immunity with Aging
Authors: Norian Lyse A. , Allen Paul M.
Published: April 2014

Aging is associated with a decline in immune function, particularly within the T cell compartment. Because CD8+ T cells are critical mediators of protective immunity against cancer, which arises more frequently with advancing age, it is important to understand how aging affects T cell-based antitumor responses. We used our DUC18 T cell/CMS5 tumor model system to examine the ability of both aged APCs and aged, tumor-specific CD8+ T cells to mount protective responses to tumors in vivo. An assessment of aged DUC18 T cells in vitro showed a naive phenotype, but impaired proliferation in response to anti-CD3 and anti-CD28 stimulation. We found that DCs from young and old recipient mice are comparable phenotypically, and endogenous APCs in these mice are equally able to prime adoptively transferred young DUC18 T cells. Even when aged DUC18 T cells are transferred into aged CMS5-challenged mice, Ag-specific proliferation and CD25 expression are similar to those found when young DUC18 T cells are transferred into young mice. Although trafficking to tumor sites appears unequal, old and young DUC18 T cells reject primary CMS5 challenges to the same degree and with similar kinetics. Overall, we found no loss of endogenous APC function or intrinsic defects in CD8+ DUC18 T cells with advanced age. Therefore, when young and old tumor-specific T cell populations are equivalently sized, CD8+ T cell-mediated antitumor immunity in our system is not impaired by age, a finding that has positive implications for T cell-based immunotherapies.
Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning
Authors: Wysocki Christian A. , Burkett Susan B. , Panoskaltsis-Mortari Angela , Kirby Suzanne L. , Luster Andrew D. , McKinnon Karen , Blazar Bruce R. , Serody Jonathan S.
Published: April 2014

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5−/−) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5+/+ cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5−/− or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5−/− T cells, which was associated with significant increases in the accumulation of alloreactive CD4+ and CD8+ T cells in liver and lung. Conversely, the transfer of CCR5−/− donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5−/− T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5−/− T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.
FTY720-Enhanced T Cell Homing Is Dependent on CCR2, CCR5, CCR7, and CXCR4: Evidence for Distinct Chemokine Compartments
Authors: Yopp Adam C. , Fu Shuang , Honig Shaun M. , Randolph Gwendalyn J. , Ding Yaozhong , Krieger Nancy R. , Bromberg Jonathan S.
Published: April 2014

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.
Antigen-Specific Lymphocyte Sequestration in Lymphoid Organs: Lack of Essential Roles for αL and α4 Integrin-Dependent Adhesion or Gαi Protein-Coupled Receptor Signaling
Authors: Arnold Carrie N. , Butcher Eugene C. , Campbell Daniel J.
Published: April 2014

Selective lymphocyte sequestration was described over 30 years ago as the transient withdrawal of Ag-specific lymphocytes from the circulation as a result of their activation in secondary lymphoid organs. We used a TCR-transgenic adoptive transfer system to further characterize the Ag and adjuvant dependence of this process in mice. In addition, we examined the contribution of the αL and α4 integrin chains as well as Gαi protein-coupled receptor signaling to the retention of Ag-specific T cells in peripheral lymph nodes. Our results demonstrate that selective lymphocyte sequestration is T cell autonomous and adjuvant independent, and that the duration of sequestration is not controlled by the continued presence of Ag in secondary lymphoid organs. This process is not critically dependent on the αL and α4 integrin chains or Gαi protein-coupled receptor signaling. Selective lymphocyte sequestration may be mediated by redundant mechanisms and/or controlled by novel or nonclassical adhesion or trafficking molecules.
Nitric Oxide Regulates Immune Cell Bioenergetic: A Mechanism to Understand Immunomodulatory Functions of Nitric Oxide-Releasing Anti-Inflammatory Drugs
Authors: Fiorucci Stefano , Mencarelli Andrea , Distrutti Eleonora , Baldoni Monia , del Soldato Piero , Morelli Antonio
Published: April 2014

The 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX-4016) is a NO-releasing derivative of aspirin. In this study, we provide evidence that NCX-4016 delivered to PMBC-derived T lymphocytes and monocytes causes a transitory inhibition of cell respiration and ≈50% reduction of cellular ATP, which translates in a time-reversible inhibition of cell proliferation and IL-2, IL-4, IL-5, and IFN-γ secretion. Exposure of lymphocytes and monocytes to aspirin, 2-(acetyloxy)benzoic acid 3-(hydroxymethyl)phenyl ester (NCX-4017), a non-NO-releasing analog of NCX-4016, and cyclooxygenase inhibitors, reduced PG formation, but has no effect on cytokine/chemokine release. In contrast, delivering NO with (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) reproduced most of the metabolic and anti-cytokine activities of NCX-4016. Scavenging NO with hemoglobin or adding selective substrates of complex II, III, and IV of the mitochondrial respiratory chain reverses NCX-4016′ inhibitory activities. Exposure to DETA-NO and NCX-4016 enhances glucose uptake, glycolytic rate, and lactate generation in CD3/CD28-costimulated lymphocytes, while reduced citric acid cycle intermediates. These effects were not reproduced by selective and nonselective cyclooxygenase 2 inhibitors. In summary, we demonstrated that exposure of lymphocytes to NCX-4016 causes a metabolic hypoxia that inhibits lymphocyte reactivity to costimulatory molecules, providing a potential counteregulatory mechanism to control activated immune system.
Bcl6 Acts as an Amplifier for the Generation and Proliferative Capacity of Central Memory CD8+ T Cells
Authors: Ichii Hirohito , Sakamoto Akemi , Kuroda Yoshikazu , Tokuhisa Takeshi
Published: April 2014

Central memory CD8+ T cells (TCM) are considered to be more efficient than effector ones (TEM) for mediating protective immunity. The molecular mechanism involved in the generation of these cells remains elusive. Because Bcl6 plays a role in the generation and maintenance of memory CD8+ T cells, we further examined this role in the process in relation to TCM and TEM subsets. In this study, we show that TCM and TEM were functionally identified in CD62L+ and CD62L− memory (CD44+Ly6C+) CD8+ T cell subsets, respectively. Although TCM produced similar amounts of IFN-γ and IL-2 to TEM after anti-CD3 stimulation, the cell proliferation capacity after stimulation and tissue distribution profiles of TCM differed from those of TEM. Numbers of TCM were greatly reduced and elevated in spleens of Bcl6-deficient and lck-Bcl6 transgenic mice, respectively, and those of TEM were constant in nonlymphoid organs of these same mice. The majority of Ag-specific memory CD8+ T cells in spleens of these mice 10 wk after immunization were TCM, and the number correlated with Bcl6 expression in T cells. The proliferation of Ag-specific memory CD8+ T cells upon secondary stimulation was dramatically up-regulated in lck-Bcl6 transgenic mice, and the adoptive transfer experiments with Ag-specific naive CD8+ T cells demonstrated that some of the up-regulation was due to the intrinsic effect of Bcl6 in the T cells. Thus, Bcl6 is apparently a crucial factor for the generation and secondary expansion of TCM.
Human B Cells Express Functional TRAIL/Apo-2 Ligand after CpG-Containing Oligodeoxynucleotide Stimulation
Authors: Kemp Troy J. , Moore Jill M. , Griffith Thomas S.
Published: April 2014

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-α production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-α from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14+ PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-α production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3+, CD14+, CD19+, and CD56+ PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19+ PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14+ PBMC, CD19+ sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-α alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-α-induced TRAIL/Apo-2L expression on CD19+ B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.
IL-21 Enhances and Sustains CD8+ T Cell Responses to Achieve Durable Tumor Immunity: Comparative Evaluation of IL-2, IL-15, and IL-21
Authors: Moroz Adrianna , Eppolito Cheryl , Li Qingsheng , Tao Jianming , Clegg Christopher H. , Shrikant Protul A.
Published: April 2014

Cytokines that use the common receptor γ-chain for regulating CD8+ T cell responses to Ag include IL-2, IL-15, and the recently identified IL-21. The ability of these cytokines to regulate antitumor activity in mice has generated considerable interest in understanding their mode of action. In this study we compare the abilities of IL-2, IL-15, and IL-21 to stimulate immunity against tumors in a syngeneic thymoma model. Durable cures were only achieved in IL-21-treated mice. By monitoring both endogenous and adoptively transferred tumor Ag-specific CD8+ T cells, it was determined that IL-21 activities overlap with those of IL-2 and IL-15. Similar to IL-2, IL-21 enhanced Ag activation and clonal expansion. However, unlike IL-2 treatment, which induces activation-induced cell death, IL-21 sustained CD8+ T cell numbers long term as a result of increased survival, an effect often attributed to IL-15. These findings indicate that the mechanisms used by IL-21 to promote CD8+ T cell responses offer unique opportunities for its use in malignant diseases and infections.
Differential Requirement for IFN-γ in CTL Maturation in Acute Murine Graft-versus-Host Disease
Authors: Puliaev Roman , Nguyen Phuong , Finkelman Fred D. , Via Charles S.
Published: April 2014

Although IFN-γ is the archetypal Th1 cytokine, its role in CTL maturation is uncertain. We used an in vivo mouse model of CTL development, parent-into-F1 acute graft-vs-host disease (AGVHD), to evaluate this issue. In AGVHD, transfer of naive parental T cells into F1 hosts stimulates the development of allospecific CTL effectors that eliminate host lymphocytes, particularly B cells. Complete elimination of IFN-γ, using IFN-γ-deficient donors and administering anti-IFN-γ mAb, suppressed B cell elimination, down-regulated ΤNF-α production, and enhanced Th2 cytokine production, but did not allow the B cell expansion characteristic of chronic GVHD (CGVHD). Because complete CTL inhibition results in full-blown CGVHD that is IFN-γ independent, these observations indicate that IFN-γ elimination only partially blocks CTL development. IFN-γ elimination did not inhibit donor T cell engraftment or activation in the AGVHD model, but almost completely blocked Fas/Fas ligand (FasL) gene expression, protein up-regulation, and Fas/FasL-mediated CTL killing. In contrast, IFN-γ elimination only partially inhibited perforin gene expression and perforin-mediated CTL activity. The contributions of IFN-γ to CTL development were indirect, because IFN-γ receptor-deficient donor cells differentiated normally into allospecific CTLs. Consistent with the view that the Fas/FasL and perforin pathways each mediate CTL killing in AGVHD, the absence of both perforin and IFN-γ (perforin knockout donor cells and anti-IFN-γ mAb) converted AGVHD to CGVHD. Thus, both IFN-γ-dependent induction of Fas/FasL and IFN-γ-independent induction of perforin contribute to CTL-mediated elimination of host B cells in AGVHD. Suppression of both pathways is required for typical CGVHD development.
Rap1 Signaling Is Required for Suppression of Ras-Generated Reactive Oxygen Species and Protection Against Oxidative Stress in T Lymphocytes
Authors: Remans Philip H. J. , Gringhuis Sonja I. , van Laar Jacob M. , Sanders Marjolein E. , Papendrecht-van der Voort Ellen A. M. , Zwartkruis Fried J. T. , Levarht E. W. Nivine , Rosas Marcela , Coffer Paul J. , Breedveld Ferdinand C. , Bos Johannes L. , Tak Paul P. , Verweij Cornelis L. , Reedquist Kris A.
Published: April 2014

Transient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to TCR signaling in T lymphocytes. Conversely, chronic oxidative stress leads to decreased proliferative responses and enhanced transcription of inflammatory gene products, and is thought to underlie the altered pathogenic behavior of T lymphocytes in some human diseases, such as rheumatoid arthritis (RA). Although the signaling mechanisms regulating ROS production in T lymphocytes has not been identified, activation of the small GTPase Ras has been shown to couple agonist stimulation to ROS production in other cell types. We find that Ras signaling via Ral stimulates ROS production in human T lymphocytes, and is required for TCR and phorbol ester-induced ROS production. The related small GTPase Rap1 suppresses agonist, Ras and Ral–dependent ROS production through a PI3K–dependent pathway, identifying a novel mechanism by which Rap1 can distally antagonize Ras signaling pathways. In synovial fluid T lymphocytes from RA patients we observed a high rate of endogenous ROS production, correlating with constitutive Ras activation and inhibition of Rap1 activation. Introduction of dominant-negative Ras into synovial fluid T cells restored redox balance, providing evidence that deregulated Ras and Rap1 signaling underlies oxidative stress and consequent altered T cell function observed in RA.