bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of T Cell Signaling by Polyunsaturated Fatty Acids: Selectivity in Inhibition of Mitogen-Activated Protein Kinase and Nuclear Factor Activation
Authors: Zeyda Maximilian , Szekeres Andreas B. , Säemann Marcus D. , Geyeregger René , Stockinger Hannes , Zlabinger Gerhard J. , Waldhäusl Werner , Stulnig Thomas M.
Published: April 2014

Polyunsaturated fatty acids (PUFAs) are known to suppress inflammatory and autoimmune responses and, therefore, clinical applications of PUFAs as immunomodulatory substances are extensively studied. PUFAs are known to inhibit T cell responses, but with respect to TCR/CD3-mediated signal transduction only a block in CD3-induced phospholipase Cγ1/calcium signaling has been shown so far. In this study, we investigated PUFA-mediated changes in downstream T cell signal transduction. We show that among the mitogen-activated protein kinase families activation of c-Jun NH2-terminal kinase, but not phosphorylation of extracellular signal-regulated kinase-1/-2 or p38 is inhibited. CD3/CD28-induced activity of NF-AT was markedly reduced by PUFA treatment, while activation of other nuclear receptors (AP-1 and NF-κB) remained unaltered. Furthermore, IL-2 promoter activity, IL-2 and IL-13 mRNA levels, IL-2 secretion, and IL-2R α-chain expression were significantly diminished by PUFA treatment, whereas the expression of IFN-γ, IL-4, IL-10, and CD69 remained essentially unaffected by PUFAs. In conclusion, PUFA treatment of T cells inhibits selectively c-Jun NH2-terminal kinase and NF-AT activation, resulting in diminished production of IL-2 and IL-13.
Immunologically Induced, Complement-Dependent Up-Regulation of the Prion Protein in the Mouse Spleen: Follicular Dendritic Cells Versus Capsule and Trabeculae
Authors: Lötscher Marius , Recher Mike , Hunziker Lukas , Klein Michael A.
Published: April 2014

The expression of the prion protein (PrP) in the follicular dendritic cell network of germinal centers in the spleen is critical for the splenic propagation of the causative agent of prion diseases. However, a physiological role of the prion protein in the periphery remains elusive. To investigate the role and function of PrP expression in the lymphoid system we treated naive mice i.v. with preformed immune complexes or vesicular stomatitis virus. Immunohistochemistry and Western blot analysis of the spleen revealed that 8 days after immunization, immune complexes and vesicular stomatitis virus had both induced a strong increase of PrP expression in the follicular dendritic cell network. Remarkably, this up-regulation did not occur in mice that lack an early factor of the complement cascade, C1q, a component which has been shown previously to facilitate early prion pathogenesis. In addition to the variable PrP level in the germinal centers, we detected steady and abundant PrP expression in the splenic capsule and trabeculae, which are structural elements that have not been associated before with PrP localization. The abundant trabeculo-capsular PrP expression was also evident in spleens of Rag-1-deficient mice, which have been shown before to be incapable of prion expansion. We conclude that trabeculocapsular PrP is not sufficient for splenic prion propagation. Furthermore, our observations may provide important clues for a physiological function of the prion protein and allow a new view on the role of complement and PrP in peripheral prion pathogenesis.
Functional Epitope on Human Neutrophil Flavocytochrome b558
Authors: Burritt James B. , Foubert Thomas R. , Baniulis Danas , Lord Connie I. , Taylor Ross M. , Mills John S. , Baughan Travis D. , Roos Dirk , Parkos Charles A. , Jesaitis Algirdas J.
Published: April 2014

mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the gp91phox flavocytochrome b558 subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the 498EKDVITGLK506 region of gp91phox. In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47phox, p67phox, or Rac to the plasma membrane, and bound its epitope on gp91phox independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91phox on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47phox binding sites. We conclude that the 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.
Biochemical Features of the MHC-Related Protein 1 Consistent with an Immunological Function
Authors: Miley Michael J. , Truscott Steven M. , Yu Yik Yeung Lawrence , Gilfillan Susan , Fremont Daved H. , Hansen Ted H. , Lybarger Lonnie
Published: April 2014

MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with β2-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.
Structure-Function Relationships of Human C5a and C5aR
Authors: Huber-Lang Markus S. , Sarma J. Vidya , McGuire Stephanie R. , Lu Kristina T. , Padgaonkar Vaishalee A. , Younkin Ellen M. , Guo Ren Feng , Weber Christian H. , Zuiderweg Erik R. , Zetoune Firas S. , Ward Peter A.
Published: April 2014

Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four α helices of C5a, we have defined the ability of these peptides to reduce binding of 125I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H2O2 production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a.
A Role for Macrophage Inflammatory Protein-3α/CC Chemokine Ligand 20 in Immune Priming During T Cell-Mediated Inflammation of the Central Nervous System
Authors: Kohler Rachel E. , Caon Adriana C. , Willenborg David O. , Clark-Lewis Ian , McColl Shaun R.
Published: April 2014

Chemokines are a family of cytokines that exhibit selective chemoattractant properties for target leukocytes and play a significant role in leukocyte migration. In this study, we have investigated the role of the C-C chemokine, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20, in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of T cell-dependent inflammation. Expression in the CNS of MIP-3α, as determined by RT-PCR, increased in a time-dependent manner such that peak expression correlated with peak clinical disease. Similarly, levels of immunoreactive MIP-3α in the draining lymph nodes increased up to 10-fold 9 days postimmunization and remained elevated for up to 21 days postimmunization. The increased production of MIP-3α coincided with onset of clinical disease. Treatment of mice with specific neutralizing anti-MIP-3α Abs significantly reduced the severity of both clinical EAE and neuroinflammation by inhibiting the sensitization of lymphocytes to the specific Ag and release of lymphocytes from the draining lymph nodes. In contrast, adoptive transfer experiments indicated that MIP-3α was not essential for the effector phase of EAE. Together, these data demonstrate that MIP-3α plays a critical role in the sensitization phase of EAE.
Coordinate Regulation of IFN Consensus Sequence-Binding Protein and Caspase-1 in the Sensitization of Human Colon Carcinoma Cells to Fas-Mediated Apoptosis by IFN-γ
Authors: Liu Kebin , Abrams Scott I.
Published: April 2014

Interferon-γ is thought to be essential for the regulation of antitumor reactions. However, the degree of responsiveness of malignant cells to IFN-γ may have a profound influence on the overall efficacy of an antitumor response. In this study, we examined the molecular basis by which IFN-γ differentially sensitized human primary and metastatic colon carcinoma cells to Fas-mediated apoptosis. To that end, we analyzed IFN-γ-induced gene expression at the genome scale, followed by an analysis of the expression and function of specific genes associated with IFN-γ- and Fas-mediated signaling. We found that although both cell populations exhibited a similar gene expression profile at the genome scale in response to IFN-γ, the expression intensities of the IFN-γ-regulated genes were much greater in the primary tumor. Noteworthily, two genes, one involved in IFN-γ-mediated signaling, IFN consensus sequence-binding protein (ICSBP), and one involved in Fas-mediated signaling, caspase-1, were clearly shown to be differentially induced between the two cell lines. In the primary tumor cells, the expression of ICSBP and caspase-1 was strongly induced in response to IFN-γ, whereas they were weakly to nondetectable in the metastatic tumor cells. Functional studies demonstrated that both caspase-1 and ICSBP were involved in Fas-mediated apoptosis following IFN-γ sensitization, but proceeded via two distinct pathways. This study also reports for the first time the expression of ICSBP in a nonhemopoietic tumor exhibiting proapoptotic properties. Overall, in a human colon carcinoma cell model, we identified important functional contributions of two IFN-γ-regulated genes, ICSBP and caspase-1, in the mechanism of Fas-mediated death.
Intrinsic Differences in the Proliferation of Naive and Memory Human B Cells as a Mechanism for Enhanced Secondary Immune Responses
Authors: Tangye Stuart G. , Avery Danielle T. , Deenick Elissa K. , Hodgkin Philip D.
Published: April 2014

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with IL-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division 20–30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38+ blasts in a manner that increased with consecutive division number. These differentiated CD38+ B cells divided faster than did CD38− memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38+ and CD38− cells present in cultures of memory B cells was increased by IL-10. In contrast, IL-2 enhanced proliferation of CD38− and CD38+ memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.
The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor 9, Is Up-Regulated During Accessory Cell Maturation and Effector Cell Degranulation, and Its Overexpression Enhances CTL Potency
Authors: Hirst Claire E. , Buzza Marguerite S. , Bird Catherina H. , Warren Hilary S. , Cameron Paul U. , Zhang Manling , Ashton-Rickardt Philip G. , Bird Phillip I.
Published: April 2014

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4+ cells, CD8+ T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8+ T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3−, CD4+, CD8−, CD45+), tonsillar DCs, and DC subsets purified from peripheral blood (CD16+ monocytes and CD123+ plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-α-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.
IFN-γ Production Is Specifically Regulated by IL-10 in Mice Made Tolerant with Anti-CD40 Ligand Antibody and Intact Active Bone
Authors: Yin Dengping , Dujovny Nadav , Ma Lianli , Varghese Anncy , Shen JiKun , Bishop D. Keith , Chong Anita S.
Published: April 2014

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-γ production that is donor specific, and a reduction in the frequency of IFN-γ-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-γ responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-β, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-γ response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-γ production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-γ levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-γ. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-γ production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.
A Thyroid Tumor-Specific Antigen Formed by the Fusion of Two Self Proteins
Authors: Powell Daniel J. , Eisenlohr Laurence C. , Rothstein Jay L.
Published: April 2014

Thyroid epithelial cells frequently express one or more members of the rearranged during transfection/papillary thyroid carcinoma (RET/PTC) fusion oncogene family during early stages of cancer, and fusion gene transcripts have been found in inflammatory conditions of the thyroid such as the autoimmune disease, Hashimoto’s thyroiditis. Because these oncogenes encode chimeric proteins, novel RET/PTC epitopes may be targets of antitumor immune responses. We have been interested in the RET/PTC3 (RP3) fusion protein because this family member is more frequently expressed in radiation-induced and childhood papillary carcinomas than other members of the fusion oncogene family. We hypothesized that the activated kinase of c-RET, in the form of RP3, when expressed in patients with thyroid disease, presents an unusual altered self target for T cell recognition. Interestingly, we find that immunization with mouse RP3 protein can induce a strongly immunogenic response to RP3, although this response is not directed against the peptide comprising the unique fusion region. Rather, the responses are specific for the carboxyl-terminal portion of RP3 that is derived from the self protein c-RET. Furthermore, transplantation of RP3-expressing thyroid tumors into naive mice resulted in leukocytic infiltration, tumor rejection, and induction of RP3-specific T cells. Thus, the somatic fusion of two unrelated self proteins results in the development of a uniquely immunogenic response directed against self epitopes within RP3. These studies may better define the mechanisms controlling the initiation of thyroid-specific immune responses and provide insight into the design of novel molecules for invoking tumor-specific immunity.
Detection of Autoreactive Myelin Proteolipid Protein 139–151-Specific T Cells by Using MHC II (IAs) Tetramers
Authors: Reddy Jayagopala , Bettelli Estelle , Nicholson Lindsay , Waldner Hanspeter , Jang Mei-Huei , Wucherpfennig Kai W. , Kuchroo Vijay K.
Published: April 2014

Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IAs class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP139–151) and used it to analyze myelin PLP139–151-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IAs tetramers stimulated and stained the PLP139–151-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP139–151, but not a control T cell line specific for PLP178–191. We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH (∼8.0) and neuraminidase treatment enhances the staining capacity of PLP139–151 tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP139–151-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP139–151-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.
T Cell Chemokine Receptor Expression in Aging
Authors: Mo Ruran , Chen Jun , Han Yin , Bueno-Cannizares Cecelia , Misek David E. , Lescure Pascal A. , Hanash Samir , Yung Raymond L.
Published: April 2014

Changes in chemokine receptor expression are important in determining T cell migration and the subsequent immune response. To better understand the contribution of the chemokine system in immune senescence we determined the effect of aging on CD4+ T cell chemokine receptor function using microarray, RNase protection assays, Western blot, and in vitro chemokine transmigration assays. Freshly isolated CD4+ cells from aged (20–22 mo) mice were found to express a higher level of CCR1, 2, 4, 5, 6, and 8 and CXCR2–5, and a lower level of CCR7 and 9 than those from young (3–4 mo) animals. Caloric restriction partially or completely restored the aging effects on CCR1, 7, and 8 and CXCR2, 4, and 5. The aging-associated differences in chemokine receptor expression cannot be adequately explained by the age-associated shift in the naive/memory or Th1/Th2 profile. CD4+ cells from aged animals have increased chemotactic response to stromal cell-derived factor-1 and macrophage-inflammatory protein-1α, suggesting that the observed chemokine receptor changes have important functional consequences. We propose that the aging-associated changes in T cell chemokine receptor expression may contribute to the different clinical outcome in T cell chemokine receptor-dependent diseases in the elderly.
A Unique Population of Extrathymically Derived αβTCR+CD4−CD8− T Cells with Regulatory Functions Dominates the Mouse Female Genital Tract
Authors: Johansson Martina , Lycke Nils
Published: April 2014

A better understanding of the regulatory role of genital tract T cells is much needed. In this study, we have analyzed the phenotype, distribution, and function of T lymphocytes in the female genital tract of naive, pregnant, or Chlamydia trachomatis-infected C57BL/6 mice. Unexpectedly, we found that the dominant lymphocyte population (70–90%) in the genital tract was that of CD3+αβTCRintCD4−CD8− T cells. Moreover, these cells were CD90low but negative for the classical T cell markers CD2 and CD5. The CD3+B220low cells were NK1.1 negative and found in nude mice as well as in mice deficient for MHC class II, β2-microglobulin, and CD1, indicating extrathymic origin. They dominated the KJ126+Vβ8.2+ population in the genital tract of DO11.10 OVA TCR-transgenic mice, further supporting the idea that the CD3+B220low cells are truly T cells. The function of these T cells appeared not to be associated with immune protection, because only CD4+ and CD8+ T cells increased in the genital tract following chlamydial infection. Notwithstanding this, the infected, as well as the uninfected and the pregnant, uterus was dominated by a high level of the CD3+CD4−CD8−B220low cells. Following in vitro Ag or polyclonal stimulation of the CD3+CD4−CD8−B220low cells, poor proliferative responses were observed. However, these cells strongly impaired splenic T cell proliferation in a cell density-dependent manner. A large fraction of the cells expressed CD25 and produced IFN-γ upon anti-CD3 plus anti-CD28 stimulation, arguing for a strong regulatory role of this novel T cell population in the mouse female genital tract.
Pertussis Toxin Enhances Th1 Responses by Stimulation of Dendritic Cells
Authors: Hou Wanqiu , Wu Yadi , Sun Shuhui , Shi Mude , Sun Yue , Yang Cuihong , Pei Gang , Gu Yundi , Zhong Cuiping , Sun Bing
Published: April 2014

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-γ and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-γ. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-α production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-γ production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.
Protective Mucosal Immunity in Aging Is Associated with Functional CD4+ T Cells in Nasopharyngeal-Associated Lymphoreticular Tissue
Authors: Hagiwara Yukari , McGhee Jerry R. , Fujihashi Keiko , Kobayashi Ryoki , Yoshino Naoto , Kataoka Kosuke , Etani Yuri , Kweon Mi-Na , Tamura Shinichi , Kurata Takeshi , Takeda Yoshifumi , Kiyono Hiroshi , Fujihashi Kohtaro
Published: April 2014

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4+ T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4+, CD45RB+ T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer’s patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.
Suppressive DNA Vaccination in Myelin Oligodendrocyte Glycoprotein Peptide-Induced Experimental Autoimmune Encephalomyelitis Involves a T1-Biased Immune Response
Authors: Lobell Anna , Weissert Robert , Eltayeb Sana , de Graaf Katrien L. , Wefer Judit , Storch Maria K. , Lassmann Hans , Wigzell Hans , Olsson Tomas
Published: April 2014

Vaccination with DNA encoding a myelin basic protein peptide suppresses Lewis rat experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. Additional myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), may be important in multiple sclerosis. Here we demonstrate that DNA vaccination also suppresses MOG peptide-induced EAE. MOG91–108 is encephalitogenic in DA rats and MHC-congenic LEW.1AV1 (RT1av1) and LEW.1N (RT1n) rats. We examined the effects of DNA vaccines encoding MOG91–108 in tandem, with or without targeting of the hybrid gene product to IgG. In all investigated rat strains DNA vaccination suppressed clinical signs of EAE. There was no requirement for targeting the gene product to IgG, but T1-promoting CpG DNA motifs in the plasmid backbone of the construct were necessary for efficient DNA vaccination, similar to the case in DNA vaccination in myelin basic protein-induced EAE. We failed to detect any effects on ex vivo MOG-peptide-induced IFN-γ, TNF-α, IL-6, IL-4, IL-10, and brain-derived neurotropic factor expression in splenocytes or CNS-derived lymphocytes. In CNS-derived lymphocytes, Fas ligand expression was down-regulated in DNA-vaccinated rats compared with controls. However, MOG-specific IgG2b responses were enhanced after DNA vaccination. The enhanced IgG2b responses together with the requirement for CpG DNA motifs in the vaccine suggest a protective mechanism involving induction of a T1-biased immune response.
Stat Signals Release Activated Naive Th Cells from an Anergic Checkpoint
Authors: Mohrs Markus , Lacy Dee A. , Locksley Richard M.
Published: April 2014

Activation of naive Th lymphocytes by the TCR and the costimulatory molecule, CD28, is believed to provide competent signals for differentiation to effector cells. Such activated cells proliferated and expressed IL-2, but arrested in an immature state maintained by CTLA-4. Although unresponsive to restimulation by TCR/CD28 alone, restimulation with TCR/CD28 and either Stat4- or Stat6-mediated cytokine signals rescued cells to proliferate and differentiate to the appropriately matched canonical Th subsets. Addition of IL-4 at defined periods revealed that naive T cells were receptive to IL-4-mediated differentiation for up to 3 days after their initial priming. A Stat-dependent anergic checkpoint between clonal expansion and effector cell differentiation may defer the cytokine profile to be instructed at the site of infection, thus preventing the unregulated development of potentially damaging effector cells.
TNF Plays an Essential Role in Tumor Regression after Adoptive Transfer of Perforin/IFN-γ Double Knockout Effector T Cells
Authors: Poehlein Christian H. , Hu Hong-Ming , Yamada Jane , Assmann Ilka , Alvord W. Gregory , Urba Walter J. , Fox Bernard A.
Published: April 2014

We have recently shown that effector T cells (TE) lacking either perforin or IFN-γ are highly effective mediators of tumor regression. To rule out compensation by either mechanism, TE deficient in both perforin and IFN-γ (perforin knockout (PKO)/IFN-γ knockout (GKO)) were generated. The adoptive transfer of PKO/GKO TE mediated complete tumor regression and cured wild-type animals with established pulmonary metastases of the B16BL6-D5 (D5) melanoma cell line. PKO/GKO TE also mediated tumor regression in D5 tumor-bearing PKO, GKO, or PKO/GKO recipients, although in PKO/GKO recipients efficacy was reduced. PKO/GKO TE exhibited tumor-specific TNF-α production and cytotoxicity in a 24-h assay, which was blocked by the soluble TNFRII-human IgG fusion protein (TNFRII:Fc). Blocking TNF in vivo by administering soluble TNFR II fusion protein (TNFRII:Fc) significantly reduced the therapeutic efficacy of PKO/GKO, but not wild-type TE. This study identifies perforin, IFN-γ, and TNF as a critical triad of effector molecules that characterize therapeutic antitumor T cells. These insights could be used to monitor and potentially tune the immune response to cancer vaccines.
Cutting Edge: Dysfunctional CD8+ T Cells Reside in Nonlymphoid Tissues During Chronic Trypanosoma cruzi Infection
Authors: Leavey Jennifer K. , Tarleton Rick L.
Published: April 2014

Chagas disease is caused by persistent Trypanosoma cruzi infection in muscle tissue that ultimately results in chronic inflammation and tissue destruction. It is unclear why T. cruzi is cleared from some tissues but persists in others, despite an active inflammatory response. In this study, we show that the majority of CD8+ T cells present in muscle tissue express memory and effector cell surface markers but have sharply attenuated effector function compared with their splenic counterparts. The dysfunction of CD8+ T cells in the muscle tissue suggests a mechanism by which T. cruzi can persist in that location and cause inflammatory damage.
Cutting Edge: Histamine Inhibits IFN-α Release from Plasmacytoid Dendritic Cells
Authors: Mazzoni Alessandra , Leifer Cynthia A. , Mullen Gregory E. D. , Kennedy Margaret N. , Klinman Dennis M. , Segal David M.
Published: April 2014

Plasmacytoid dendritic cells (DC) are professional APC and a major source of type I IFN following viral infection. We previously showed that histamine alters the cytokine profiles of maturing monocyte-derived DC resulting in a change from Th1 to Th2 in their T cell polarizing function. In this study, we show that human plasmacytoid DC, activated by either CpG oligodeoxynucleotides or viral infection, also respond to histamine through H2 receptors, leading to a marked down-regulation of IFN-α and TNF-α and a moderate switch in their capacity to polarize naive T cells. Our findings provide an explanation for low levels of type I IFN frequently observed in atopic individuals.
Induction of Low Dose Oral Tolerance in Monocyte Chemoattractant Protein-1- and CCR2-Deficient Mice
Authors: Gonnella Patricia A. , Kodali Dhatri , Weiner Howard L.
Published: April 2014

The chemokine monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR2 have been shown to play an important role in the migration and trafficking of macrophages and Th1 effector cells in experimental autoimmune encephalomyelitis. Also, MCP-1 has been reported to regulate oral tolerance induction by inhibition of Th1 cell-related cytokines and by the ability of Abs to MCP-1 to inhibit oral tolerance. This study demonstrates that neither MCP-1 nor its receptor CCR2 is required for the induction of oral tolerance. Mice deletional for either MCP-1 or CCR2 had suppressed cell-proliferative and Th1 responses following oral administration and immunization with myelin oligodendrocyte glycoprotein (MOG35–55). TGF-β was up-regulated in fed and immunized deletional mice, while IL-4 was absent from deletional mice, but up-regulated in controls. Decreased experimental autoimmune encephalomyelitis severity was found in MOG35–55-fed MCP-1 deletional mice, indicating induction of oral tolerance. These results demonstrate that MCP-1 is not required for induction of oral tolerance and that MCP-1 and CCR2 are essential for up-regulation of IL-4 in tolerized mice.
BATF Transgenic Mice Reveal a Role for Activator Protein-1 in NKT Cell Development
Authors: Williams Kristi L. , Zullo Alfred J. , Kaplan Mark H. , Brutkiewicz Randy R. , Deppmann Christopher D. , Vinson Charles , Taparowsky Elizabeth J.
Published: April 2014

The importance of regulated AP-1 activity during T cell development was assessed using transgenic mice overexpressing BATF, a basic leucine zipper transcription factor and an AP-1 inhibitor. BATF transgenic animals possess normal thymic cellularity and all major T cell subsets, but show impaired thymocyte proliferation in vitro and no induction of IL-2, IL-4, IL-5, IL-10, and IL-13 expression. Since NKT cells are largely responsible for cytokine production in the thymus, this population was examined by detection of the Vα14-Jα281 TCR, flow cytometry of NK1.1+ TCRβ+ cells, and analysis of cytokine production by heat-stable Aglow thymocytes and peripheral NKT cells stimulated in vivo. Results show a severe under-representation of NKT cells in BATF transgenic animals, providing the first evidence that the precise control of AP-1-mediated transcription is critical for the proper emergence of thymus-derived NKT cells in the mouse.
Inhibition of Th2 Differentiation and GATA-3 Expression by BCL-6
Authors: Kusam Saritha , Toney Lisa M. , Sato Hiroshi , Dent Alexander L.
Published: April 2014

The B cell lymphoma (BCL)-6 transcriptional repressor protein is an important regulator of Th2 responses. Mice deficient in BCL-6 develop severe Th2-type inflammation that can develop even in the absence of IL-4 signaling. We have investigated the mechanism for how BCL-6 regulates Th2 cell differentiation and have found that IL-6 signaling can promote dramatically increased levels of Th2 differentiation in BCL-6−/− CD4 T cells compared with wild-type CD4 T cells. IL-6 can induce a low level of Th2 cytokine expression in BCL-6−/−STAT6−/− cells but not in STAT6−/− cells. Since the promoters for Th2 cytokines such as IL-4, IL-5, IL-10, and IL-13 do not contain consensus BCL-6 DNA binding sites, we investigated whether BCL-6 might regulate the GATA-3 transcription factor that activates the expression of multiple Th2 cytokines. Consistent with the idea that BCL-6 represses GATA-3 expression, we found that GATA-3 levels are up-regulated in BCL-6−/−STAT6−/− CD4 T cells compared with STAT6−/− CD4 T cells. Retrovirus-mediated expression of BCL-6 in BCL-6−/−STAT6−/− T cells as well as developing wild-type Th2 cells leads to a potent repression of IL-4 and IL-10 secretion. Retrovirus-mediated expression of BCL-6 in both BCL-6−/−STAT6−/− and wild-type T cells also leads to a significant decrease in GATA-3 protein levels. Surprisingly, BCL-6 does not appear to regulate GATA-3 mRNA levels and thus BCL-6 appears to regulate GATA-3 expression at a posttranscriptional level. Regulation of GATA-3 protein levels is likely a key mechanism for how BCL-6 regulates Th2 cytokine expression and Th2 differentiation independently of STAT6. These data also point to a novel regulatory mechanism for BCL-6 separate from transcriptional repression.
Immediate Early Effector Functions of Virus-Specific CD8+CCR7+ Memory Cells in Humans Defined by HLA and CC Chemokine Ligand 19 Tetramers
Authors: Ravkov Eugene V. , Myrick Christy M. , Altman John D.
Published: April 2014

Memory T cells exhibit a high degree of heterogeneity in terms of their phenotype and functional characteristics. It has been proposed that the CCR7 chemokine receptor divides memory T cell populations into central memory T cells and effector memory T cells with distinct functions in secondary immune responses. We were interested whether this hypothesis holds true in experiments performed on Ag-specific CD8+ T cells. To identify CCR7+ cells, we engineered a fluorescent ligand for CCR7; results with the new CC chemokine ligand 19 chemotetramer were verified by staining with a CCR7 mAb. Staining with the CC chemokine ligand 19 chemotetramer reveals two subsets within CCR7+ cells: a CCR7int population containing memory cells and a CCR7high population containing naive T cells. Phenotypic analysis of MHC class I/peptide tetramer-positive cells revealed that HLA-A2-restricted CMV-specific CD8 T cells exhibit the lowest percentage of CCR7+ cells (0.5–5%), while HLA-A2-restricted flu- and HLA-B8-restricted EBV-specific CD8 T cells showed the highest (45–70%). Intracellular staining of unstimulated cells revealed that both CCR7int- and CCR7−-specific CD8 T cells exhibit a detectable level of perforin. Both CCR7int and CCR7− Ag-specific CD8+ T cells produced IFN-γ and TNF-α following short-term peptide stimulation. Therefore, our finding that CCR7+CD8+ T cells are able to exert immediate effector functions requires a substantial revision to the central and effector memory hypothesis.
C/EBPα and Ets Protein Family Members Regulate the Human Myeloid IgA Fc Receptor (FcαR, CD89) Promoter
Authors: Shimokawa Toshibumi , Ra Chisei
Published: April 2014

FcαR (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the FcαR 5′-flanking region from −102 to −64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at −74 and −92, respectively. EMSAs and cotransfection experiments show that C/EBPα acts as a major activator of the FcαR promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBPα at −139 and −127. On the other hand, the FcαR Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces FcαR promoter activity, suggesting some functional interaction between these factors. As the in vivo role of FcαR is still incompletely defined, these findings reveal the features controlling the FcαR promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of FcαR gene expression associated with its potential roles.
The T Cell Receptor β Locus of the Channel Catfish,Ictalurus punctatus, Reveals Unique Features
Authors: Zhou He , Bengtén Eva , Miller Norman W. , Clem L. William , Wilson Melanie
Published: April 2014

Previously, a series of clonal alloantigen-dependent T cell lines established from the channel catfish revealed distinctly different TCRβ rearrangements. Here, a follow-up study of the junctional diversity of these TCR gene rearrangements focuses on characterization of the genomic organization of the TCRB locus. Surprisingly, a total of 29 JB genes and two substantially different CB genes were identified downstream of a single DB gene. This is in contrast to the situation in mammals, where two clusters of a DB gene, six or seven JB genes, and a CB gene are found in tandem. The catfish CB genes are ∼36% identical at the amino acid level. All 29 catfish JB gene segments appear functional. Thirteen were used in the 19 cDNAs analyzed, of these eight were used by the 11 catfish clonal alloantigen-dependent T cell lines. As might be expected, CDR3 diversity is enhanced by N-nucleotide additions as well as nucleotide deletions at the V-D and D-J junctions. Taken together, compared with that in mammals, genomic sequencing of the catfish TCR DB-JB-CB region reveals a unique locus containing a greater number of JB genes and two distinct CB genes.
Up-Regulation of Proteinase-Activated Receptor 1 Expression in Astrocytes During HIV Encephalitis
Authors: Boven Leonie A. , Vergnolle Nathalie , Henry Scot D. , Silva Claudia , Imai Yoshinori , Holden Janet , Warren Kenneth , Hollenberg Morley D. , Power Christopher
Published: April 2014

Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide. Activated cells expressed significantly increased levels of IL-1β, inducible NO synthase, and PAR-1 mRNA. Moreover, supernatants of these same cells were neurotoxic, which was inhibited by an N-methyl-d-aspartate receptor antagonist. Striatal implantation of the PAR-1-activating peptide significantly induced brain inflammation and neurobehavioral deficits in mice compared with mice implanted with the control peptide or saline. Since HIV-related neurological disease is predicated on brain inflammation and neuronal injury, the expression of PAR-1 in HIV encephalitis (HIVE) was investigated. Immunohistochemical analysis revealed that PAR-1 and (pro)-thrombin protein expression was low in control brains, but intense immunoreactivity was observed on astrocytes in HIVE brains. Similarly, PAR-1 and thrombin mRNA levels were significantly increased in HIVE brains compared with control and multiple sclerosis brains. These data indicated that activation and up-regulation of PAR-1 probably contribute to brain inflammation and neuronal damage during HIV-1 infection, thus providing new therapeutic targets for the treatment of HIV-related neurodegeneration.
Paralytic Autoimmune Myositis Develops in Nonobese Diabetic Mice Made Th1 Cytokine-Deficient by Expression of an IFN-γ Receptor β-Chain Transgene
Authors: Serreze David V. , Pierce Melissa A. , Post Cristina M. , Chapman Harold D. , Savage Holly , Bronson Roderick T. , Rothman Paul B. , Cox Gregory A.
Published: April 2014

Nonobese diabetic (NOD) mice and some human type 1 diabetes (T1D) patients manifest low to high levels of other autoimmune pathologies. Skewing their cytokine production from a Th1 (primarily IFN-γ) to a Th2 (primarily IL-4 and IL-10) pattern is a widely proposed approach to dampen the pathogenicity of autoreactive diabetogenic T cells. However, it is important that altered cytokine balances not enhance any other autoimmune proclivities to dangerous levels. Murine CD4 T cells are characterized by a reciprocal relationship between the production of IFN-γ and expression of the β-chain component of its receptor (IFN-γRB). Thus, NOD mice constitutively expressing a CD2 promoter-driven IFN-γRB transgene in all T cells are Th1-deficient. Unexpectedly, NOD.IFN-γRB Tg mice were found to develop a lethal early paralytic syndrome induced by a CD8 T cell-dependent autoimmune-mediated myositis. Furthermore, pancreatic insulitis levels were not diminished in 9-wk-old NOD.IFN-γRB Tg females, and overt T1D developed in the few that survived to an older age. Autoimmune-mediated myositis is only occasionally detected in standard NOD mice. Hence, some manipulations diminishing Th1 responses can bring to the forefront what are normally secondary autoimmune pathologies in NOD mice, while also failing to dependably abrogate pancreatic β cell destruction. This should raise a cautionary note when considering the use of protocols that induce alterations in cytokine balances as a means of blocking progression to overt T1D in at-risk humans.
Endogenous Dendritic Cells Are Required for Amplification of T Cell Responses Induced by Dendritic Cell Vaccines In Vivo
Authors: Kleindienst Petra , Brocker Thomas
Published: April 2014

Dendritic cells (DCs) loaded in vitro with Ag are used as cellular vaccines to induce Ag-specific immunity. These cells are thought to be responsible for direct stimulation of Ag-specific T cells, which may subsequently mediate immunity. In this study, in transgenic mouse models with targeted MHC class II expression specifically on DCs, we show that the DC vaccine is responsible only for partial CD4+ T cell activation, but to obtain optimal expansion of T cells in vivo, participation of endogenous (resident) DCs, but not endogenous B cells, is crucial. Transfer of Ag to endogenous DCs seems not to be mediated by simple peptide diffusion, but rather by DC-DC interaction in lymph nodes as demonstrated by histological analysis. In contrast, injection of apoptotic or necrotic DC vaccines does not induce T cell responses, but rather represents an immunological null event, which argues that viability of DC vaccines can be crucial for initial triggering of T cells. We propose that viable DCs from the DC vaccine must migrate to the draining lymph nodes and initiate a T cell response, which thereafter requires endogenous DCs that present transferred Ag in order induce optimal T cell expansion. These results are of specific importance with regard to the applicability of DC vaccinations in tumor patients, where the function of endogenous DCs is suppressed by either tumors or chemotherapy.