bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemokine-Guided CD4+ T Cell Help Enhances Generation of IL-6RαhighIL-7Rαhigh Prememory CD8+ T Cells
Authors: Castellino Flora , Germain Ronald N.
Published: April 2014

CD4+ T cells promote effective CD8+ T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8+ T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4+ T cell help early after priming is required for generating an optimal pool of functional memory CD8+ T cells. CD4+ T cell help increased the size of a previously unreported population of IL-6RαhighIL-7Rαhigh prememory CD8+ T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8+ T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interactions, the generation of IL-6RαhighIL-7Rαhigh prememory as well as functional memory CD8+ T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8+ T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8+ T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4+ T cells. They also reveal that elevated IL-6Rα expression by a subset of CD8+ T cells represents an early imprint of CD4+ T cell helper function that actively contributes to the survival of activated CD8+ T cells.
The IREM-1 (CD300f) Inhibitory Receptor Associates with the p85α Subunit of Phosphoinositide 3-Kinase
Authors: Álvarez-Errico Damiana , Sayós Joan , López-Botet Miguel
Published: April 2014

The immune receptor expressed by myeloid cell 1 (IREM-1) (CD300f) inhibitory receptor displays five cytoplasmic tyrosine residues, two of them (Y205 and Y249) fit with ITIMs, whereas Y236 and Y263 constitute putative binding sites for PI3K. In the present study, immunoprecipitation analysis revealed that both the p85α subunit of PI3K and Src homology region 2 domain-containing phosphatase-1 could be recruited by IREM-1 in transfected cells as well as in the U937 monocytic leukemia cells, which constitutively express the receptor. By assaying the ability of different IREM-1 mutants to regulate the secretion of β-hexosaminidase induced via FcRεI in rat basophilic leukemia cells, both Y205 and Y249 appeared crucial for IREM-1-mediated inhibition. Remarkably, engagement of an IREM-1 mutant (Y205,249,284F), which did not recruit Src homology region 2 domain-containing phosphatase-1 and lost its inhibitory function, induced rat basophilic leukemia cell degranulation. This effect was dependent on the recruitment of PI3K, requiring the integrity of Y236 and Y263, and was blocked by PI3K inhibitors (i.e., wortmannin and LY-294002). Altogether, these data reveal a putative functional duality of the IREM-1 myeloid cell receptor.
FcγRIIB Regulates Autoreactive Primary Antibody-Forming Cell, but Not Germinal Center B Cell, Activity
Authors: Rahman Ziaur S. M. , Alabyev Boris , Manser Tim
Published: April 2014

The low-affinity FcR for IgG FcγRIIB suppresses the development of IgG autoantibodies and autoimmune disease in normal individuals, but how this effect is mediated is incompletely understood. To investigate this issue, we created FcγRIIB-deficient versions of two previously described targeted BCR-transgenic lines of mice that contain follicular B cells with specificity for the hapten arsonate, but with different levels of antinuclear autoantigen reactivity. The primary development and tolerance of both types of B cells were unaltered by the absence of FcγRIIB. Moreover, the reduced p-azophenylarsonate-driven germinal center and memory responses characteristic of the highly autoreactive clonotype were not reversed by an intrinsic FcγRIIB deficiency. In contrast, the p-azophenylarsonate-driven primary Ab-forming cell responses of both clonotypes were equivalently increased by such a deficiency. In total, our data do not support the idea that FcγRIIB directly participates in the action of primary or germinal center tolerance checkpoints. In contrast, this receptor apparently contributes to the prevention of autoimmunity by suppressing the production of autoreactive IgGs from B cells that have breached tolerance checkpoints and entered the Ab-forming cell pathway due to spontaneous, or cross-reactive, Ag-mediated activation.
Disruption of Innate-Mediated Proinflammatory Cytokine and Reactive Oxygen Species Third Signal Leads to Antigen-Specific Hyporesponsiveness
Authors: Tse Hubert M. , Milton Martha J. , Schreiner Sheila , Profozich Jennifer L. , Trucco Massimo , Piganelli Jon D.
Published: April 2014

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-α, IL-1β, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN-γ synthesis by diminishing ROS production in the APC, which affected TNF-α levels produced by CD4+ T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4+ T cells to transition from expansion to effector function.
Involvement of Regulatory T Cells in the Experimental Autoimmune Encephalomyelitis-Preventive Effect of Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein plus TRAIL
Authors: Hirata Shinya , Matsuyoshi Hidetake , Fukuma Daiki , Kurisaki Akari , Uemura Yasushi , Nishimura Yasuharu , Senju Satoru
Published: April 2014

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4+CD25+ regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4+CD25+ T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3+ cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4+CD25+ T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4+CD25+ Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.
Redundancy in B Cell Developmental Pathways: c-Cbl Inactivation Rescues Early B Cell Development through a B Cell Linker Protein-Independent Pathway
Authors: Song Haifeng , Zhang Juan , Chiang Y. Jeffrey , Siraganian Reuben P. , Hodes Richard J.
Published: April 2014

Deficiency in the adaptor protein B cell linker protein (BLNK) results in a substantial but incomplete block in B cell development, suggesting that alternative pathways exist for B lineage differentiation. Another adaptor protein, c-Cbl, plays a negative regulatory role in several BCR-signaling pathways. We therefore investigated the role of c-Cbl during B cell development and addressed the possibility that redundancies in pathways for B cell differentiation could be further revealed by eliminating negative effects mediated by c-Cbl. Strikingly, c-Cbl inactivation reversed a number of the critical defects in early B cell differentiation that are seen in BLNK-deficient mice. c-Cbl−/−BLNK−/− mice exhibited normalized down-regulation of pre-BCR and CD43, up-regulation of MHC class II, and augmented L chain rearrangement, resulting in a successful transition from pre-B cells to immature B cells. c-Cbl inactivation also reversed the potentially tumor-predisposing hyperproliferative response of BLNK−/− pre-B cells to IL-7. Pre-BCR cross-linking induced enhanced and prolonged tyrosine phosphorylation in c-Cbl−/−BLNK−/− pre-BCR+ pre-B cells compared with c-Cbl+/−BLNK−/− cells, including elevated phosphorylation of Lyn, Syk, Btk, and phospholipase C-γ2. Our studies suggest that some, but not all, pre-BCR-triggered developmental events can be mediated by BLNK-independent pathways that are negatively regulated by c-Cbl, and further suggest that different events during early B cell development require different strength or duration of pre-BCR signaling.
First Glimpse of the Peptide Presentation by Rhesus Macaque MHC Class I: Crystal Structures of Mamu-A*01 Complexed with Two Immunogenic SIV Epitopes and Insights into CTL Escape
Authors: Chu Fuliang , Lou Zhiyong , Chen Yu Wai , Liu Yiwei , Gao Bin , Zong Lili , Khan Abdul Hamid , Bell John I. , Rao Zihe , Gao George F.
Published: April 2014

The infection of rhesus macaques (Macaca mulatta) by the SIV is the best animal model for studying HIV infection and for AIDS vaccine development. A prevalent MHC class I allele, Mamu-A*01, is known to correlate with containment of SIV, which has been extensively explored in studies of CTL-based vaccination concepts. We determined the crystal structures of Mamu-A*01 complexed with two immunodominant SIV epitopes: the nonamer CM9 of group-specific Ag (Gag, 181–189; CTPYDINQM) and the octamer TL8 of transcription activator (Tat, 28–35; TTPESANL). The overall structures of the two Mamu-A*01 complexes are similar to other MHC class I molecules. Both structures confirm the presence of an absolutely conserved proline anchor residue in the P3 position of the Ag, bound to a D pocket of the Mamu-A*01 H chain with optimal surface complementarity. Like other MHC/peptide complex structures, the P2 and C-terminal residues of the epitopes are also important for anchoring to the MHC molecule, whereas the middle residues form an arch and their side chains are directed into solvent. These two structures reveal details of how Mamu-A*01 interacts with two well-studied epitopes at the atomic level. We discuss the structural basis of CTL escape, based on molecular models made possible by these two structures. The results we present in this study are most relevant for the rational design of Mamu-A*01-restricted CTL epitopes with improved binding, as a step toward development of AIDS vaccines.
Promoter Region Architecture and Transcriptional Regulation of the Genes for the MHC Class I-Related Chain A and B Ligands of NKG2D
Authors: Venkataraman Gopalakrishnan M. , Suciu Dominic , Groh Veronika , Boss Jeremy M. , Spies Thomas
Published: April 2014

Ligands of the NKG2D receptor, which activates NK cells and costimulates effector T cells, are inducibly expressed under harmful conditions, such as malignancies and microbial infections. Moreover, aberrant expression in autoimmune disease lesions may contribute to disease progression. Among these ligands are the closely related human MHC class I-related chains (MIC) A and B, which appear to be regulated by cellular stress. Analyses of MIC gene 5′-end flanking regions in epithelial tumor cells defined minimal core promoters that directed near maximum heat shock- or oxidative stress-induced transcriptional activation. Considerably larger fully functional promoters were required for maximum proliferation-associated activation. These activities were dependent on core promoter sequences that included heat shock elements, which inducibly bound heat shock factor 1, TATA-like elements, and constitutively occupied Sp1 and inverted CCAAT box factor sites. By contrast, MIC gene activation by CMV infection was largely independent of these and upstream promoter sequences, and expression of viral immediate early gene (IE1 or IE2) products was sufficient for induction of transcription and surface protein expression. Altogether, these results reveal distinct modes of activation of the genes for the MIC ligands of NKG2D and provide a molecular framework for analyses of gene regulation under different cellular insult conditions.
Age-Associated Defect in Human TLR-1/2 Function
Authors: van Duin David , Mohanty Subhasis , Thomas Venetta , Ginter Sandra , Montgomery Ruth R. , Fikrig Erol , Allore Heather G. , Medzhitov Ruslan , Shaw Albert C.
Published: April 2014

The effects of aging on human TLR function remain incompletely understood. We assessed TLR function and expression in peripheral blood monocytes from 159 subjects in 2 age categories, 21–30 and >65 years of age, using a multivariable mixed effect model. Using flow cytometry to assess TLR-induced cytokine production, we observed a substantial, highly significant defect in TLR1/2-induced TNF-α (p = 0.0003) and IL-6 (p < 0.0001) production, in older adults compared with young controls. In contrast to findings in aged mice, other TLR (including TLR2/6)-induced cytokine production appeared largely intact. These differences were highly significant even after correcting for covariates including gender, race, medications, and comorbidities. This defect in TLR1/2 signaling may result from alterations in baseline TLR1 surface expression, which was decreased by 36% in older adults (p < 0.0001), whereas TLR2 surface expression was unaffected by aging. Production of IL-6 (p < 0.0001) and TNF-α (p = 0.003) after stimulation by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-Cys-[S]-Ser1-[S]-Lys(4) trihydrochloride was strongly associated with TLR1 surface expression. Diminished TLR1/2 signaling may contribute to the increased infection-related morbidity and mortality and the impaired vaccine responses observed in aging humans.
TLR4, Ethanol, and Lipid Rafts: A New Mechanism of Ethanol Action with Implications for other Receptor-Mediated Effects
Authors: Szabo Gyongyi , Dolganiuc Angela , Dai Qun , Pruett Stephen B.
Published: April 2014

Ethanol (EtOH) is the most widely abused substance in the United States, and it contributes to well-documented harmful (at high dosages) and beneficial (at low dosages) changes in inflammatory and immune responses. Lipid rafts have been implicated in the regulation and activation of several important receptor complexes in the immune system, including the TLR4 complex. Many questions remain about the precise mechanisms by which rafts regulate the assembly of these receptor complexes. Results summarized in this review indicate that EtOH acts by altering the LPS-induced redistribution of components of the TLR4 complex within the lipid raft and that this is related to changes in actin cytoskeleton rearrangement, receptor clustering, and subsequent signaling. EtOH provides an example of an immunomodulatory drug that acts at least in part by modifying lipid rafts, and it could represent a model to probe the relationships between rafts, receptor complexes, and signaling.
Cutting Edge: A Regulatory T Cell-Dependent Novel Function of CD25 (IL-2Rα) Controlling Memory CD8+ T Cell Homeostasis
Authors: Sharma Rahul , Zheng Lingjie , Deshmukh Umesh S. , Jarjour Wael N. , Sung Sun-sang J. , Fu Shu Man , Ju Shyr-Te
Published: April 2014

A massive systemic expansion of CD8+ memory T (TM) cells and a remarkable increase in circulating IL-2 were observed only in IL-2Rα (CD25) knockout (KO) mice but not in IL-2 KO and scurfy mice, although all three mutants lack regulatory T (Treg) cells. However, both phenotypes were suppressed by the transfer of Treg cells. The data presented indicate that Treg cell deficiency drives naive T cells to TM cells. The lack of high-affinity IL-2R in IL-2Rα KO mice increases circulating IL-2 that is then preferentially used by CD8+ TM cells through its abundant low-affinity IL-2R, resulting in systemic CD8+ TM cell dominance. Our study demonstrates the critical control of CD8+ TM cell homeostasis by a Treg cell-dependent novel function of CD25 and resolves its mechanism of action.
Cutting Edge: Monarch-1 Suppresses Non-Canonical NF-κB Activation and p52-Dependent Chemokine Expression in Monocytes
Authors: Lich John D. , Williams Kristi L. , Moore Chris B. , Arthur Janelle C. , Davis Beckley K. , Taxman Debra J. , Ting Jenny P-Y.
Published: April 2014

CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-κB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-κB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.
Tolerization of Tumor-Specific T Cells Despite Efficient Initial Priming in a Primary Murine Model of Prostate Cancer
Authors: Anderson Michael J. , Shafer-Weaver Kimberly , Greenberg Norman M. , Hurwitz Arthur A.
Published: April 2014

In this report, we studied T cell responses to a prostate cancer Ag by adoptively transferring tumor Ag-specific T cells into prostate tumor-bearing mice. Our findings demonstrate that CD8+ T cells initially encountered tumor Ag in the lymph node and underwent an abortive proliferative response. Upon isolation from the tumor, the residual tumor-specific T cells were functionally tolerant of tumor Ag as measured by their inability to degranulate and secrete IFN-γ and granzyme B. We next sought to determine whether providing an ex vivo-matured, peptide-pulsed dendritic cell (DC) vaccine could overcome the tolerizing mechanisms of tumor-bearing transgenic adenocarcinoma of the mouse prostate model mice. We demonstrate that tumor Ag-specific T cells were protected from tolerance following provision of the DC vaccine. Concurrently, there was a reduction in prostate tumor size. However, even when activated DCs initially present tumor Ag, T cells persisting within the tolerogenic tumor environment gradually lost Ag reactivity. These results suggest that even though a productive antitumor response can be initiated by a DC vaccine, the tolerizing environment created by the tumor still exerts suppressive effects on the T cells. Furthermore, our results demonstrate that when trying to elicit an effective antitumor immune response, two obstacles must be considered: to maintain tumor Ag responsiveness, T cells must be efficiently primed to overcome tumor Ag presented in a tolerizing manner and protected from the suppressive mechanisms of the tumor microenvironment.
An In Vivo Cytotoxicity Threshold for Influenza A Virus-Specific Effector and Memory CD8+ T Cells
Authors: Stambas John , Doherty Peter C. , Turner Stephen J.
Published: April 2014

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8+ T cell responses to H2Db complexed with peptides from the viral nucleoprotein (NP366, ASNENMETM) and acid polymerase (PA224, SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of DbNP366- and DbPA224-specific acute and memory CD8+ T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8+ T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8+DbNP366+ T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.
An Alternative Pathway of NF-κB Activation Results in Maturation and T Cell Priming Activity of Dendritic Cells Overexpressing a Mutated IκBα
Authors: Moore Fabrice , Buonocore Sofia , Aksoy Ezra , Ouled-Haddou Najate , Goriely Stanislas , Lazarova Elena , Paulart Frédéric , Heirman Carlo , Vaeremans Elsy , Thielemans Kris , Goldman Michel , Flamand Véronique
Published: April 2014

Maturation of dendritic cells (DC) is a critical step in the induction of T cell responses and depends on the activation of NF-κB transcription factors. Therefore, inhibition of NF-κB activation has been proposed as a strategy to maintain DC in an immature stage and to promote immune tolerance. Herein, we generated murine myeloid DC expressing a mutated IκBα acting as a superrepressor of the classical NF-κB pathway (s-rIκB DC) to investigate the consequences of NF-κB inhibition on the ability of DC to prime T cell responses. Upon in vitro LPS activation, maturation of s-rIκB DC was profoundly impaired as indicated by defective up-regulation of MHC class II and costimulatory molecules and reduced secretion of IL-12 p70 and TNF-α. In contrast, after injection, s-rIκB DC had the same capacity as control DC to migrate to draining lymph node and to induce Th1- and Th2-type cytokine production in a MHC class II-incompatible host mice. Likewise, s-rIκB DC pulsed with OVA were as efficient as control DC to induce Ag-specific T cell responses in vivo. Indeed, further in vitro experiments established that s-rIκB DC undergo efficient maturation upon prolonged contact with activated T cells via the alternative pathway of NF-κB activation triggered at least partly by lymphotoxin β receptor ligation and involving processing of p100/RelB complexes.
Environmental and Intrinsic Factors Lead to Antigen Unresponsiveness in CD4+ Recent Thymic Emigrants from Aged Mice
Authors: Clise-Dwyer Karen , Huston Gail E. , Buck Amanda L. , Duso Debra K. , Swain Susan L.
Published: April 2014

Naive CD4 cells from aged mice respond inefficiently to Ag, but the factors that underlie the age-associated defects remain unclear. We have used two approaches to isolate recent thymic emigrants (RTE) in young and aged mice and have compared their capacity to respond to antigenic stimulation ex vivo. An in situ intrathymic CFSE injection labeled developing thymocytes and allowed the identification of RTE in secondary lymphoid tissues. Analysis of CFSE-labeled RTE and control unlabeled naive CD4 cells indicated that cells from aged mice were defective in their ability to increase intracellular Ca2+ concentration following TCR cross-linking. Aged naive and RTE CD4 also secreted less IL-2 and proliferated less than that of comparable young CD4 populations. Defects in effector generation in aged RTE were overcome by the addition of IL-2 to cultures. RTE from both polyclonal and TCR transgenic mice were compromised, indicating that defects were independent of TCR specificity. In the second model, the cotransfer of congenic marker-labeled young and aged BM cells into young and aged syngeneic hosts revealed that hyporesponsiveness in aged RTE was caused by a combination of defects intrinsic to CD4 progenitors and defects induced by the aged environment. Depletion of peripheral CD4 cells in aged mice led to production of new RTE that were not defective. The results of this study suggest that defects induced by environmental and lineage intrinsic factors act together to reduce responses to Ag in aged naive CD4 cells and that these defects can be overcome in aged CD4 cells produced during recovery from lymphopenia.
T-bet Regulates the Fate of Th1 and Th17 Lymphocytes in Autoimmunity
Authors: Gocke Anne R. , Cravens Petra D. , Ben Li-Hong , Hussain Rehana Z. , Northrop Sara C. , Racke Michael K. , Lovett-Racke Amy E.
Published: April 2014

IL-17-producing T cells (Th17) have recently been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. However, little is known about the transcription factors that regulate these cells. Although it is clear that the transcription factor T-bet plays an essential role in the differentiation of IFN-γ-producing CD4+ Th1 lymphocytes, the potential role of T-bet in the differentiation of Th17 cells is not completely understood. In this study, therapeutic administration of a small interfering RNA specific for T-bet significantly improved the clinical course of established EAE. The improved clinical course was associated with suppression of newly differentiated T cells that express IL-17 in the CNS as well as suppression of myelin basic protein-specific Th1 autoreactive T cells. Moreover, T-bet was found to directly regulate transcription of the IL-23R, and, in doing so, influenced the fate of Th17 cells, which depend on optimal IL-23 production for survival. We now show for the first time that suppression of T-bet ameliorates EAE by limiting the differentiation of autoreactive Th1 cells, as well as inhibiting pathogenic Th17 cells via regulation of IL-23R.
The p85α Regulatory Subunit of Class IA Phosphoinositide 3-Kinase Regulates β-Selection in Thymocyte Development
Authors: Shiroki Fumiko , Matsuda Satoshi , Doi Tomomitsu , Fujiwara Mari , Mochizuki Yoshito , Kadowaki Takashi , Suzuki Harumi , Koyasu Shigeo
Published: April 2014

We examined the role of class IA PI3K in pre-TCR controlled β-selection and TCR-controlled positive/negative selection in thymic development. Using mice deficient for p85α, a major regulatory subunit of the class IA PI3K family, the role of class IA PI3K in β-selection was examined by injection of anti-CD3ε mAb into p85α−/−Rag-2−/− mice, which mimics pre-TCR signals. Transition of CD4−CD8− double-negative (DN) to CD4+CD8+ double-positive (DP) thymocytes triggered by anti-CD3ε mAb was significantly impaired in p85α−/−Rag-2−/− compared with p85α+/−Rag-2−/− mice. Furthermore, DP cell numbers were lower in p85α−/−DO11.10/Rag-2−/− TCR-transgenic mice than in DO11.10/Rag-2−/− mice. In addition, inhibition by IC87114 of the major class IA PI3K catalytic subunit expressed in lymphocytes, p110δ, blocked transition of DN to DP cells in embryonic day 14.5 fetal thymic organ culture without affecting cell viability. In the absence of phosphatase and tensin homolog deleted on chromosome 10, where class IA PI3K signals would be amplified, the DN to DP transition was accelerated. In contrast, neither positive nor negative selection in Rag-2−/−TCR-transgenic mice was perturbed by the lack of p85α. These findings establish an important function of class IA PI3K in the pre-TCR-controlled developmental transition of DN to DP thymocytes.
Kinetics and Organ Distribution of IL-17-Producing CD4 Cells in Proteolipid Protein 139–151 Peptide-Induced Experimental Autoimmune Encephalomyelitis of SJL Mice
Authors: Hofstetter Harald H. , Toyka Klaus V. , Tary-Lehmann Magdalena , Lehmann Paul V.
Published: April 2014

In experimental autoimmune encephalomyelitis (EAE), the production of proinflammatory cytokines by neuroantigen-specific T cells is thought to initiate and maintain the inflammatory autoimmune pathology. Because gene knockout strategies have shown that IFN-γ and TNF are not essential for EAE development, there is increasing interest in establishing the role of other proinflammatory cytokines, primarily IL-17 in EAE. We used an IL-17 ELISPOT assay to track the neuroantigen-specific IL-17-producing T cells at single-cell resolution in various organs of SJL mice undergoing PLP 139–151-induced EAE. Overall, the migration patterns and population kinetics of the PLP 139–151-specific IL-17-producing CD4 cells were reminiscent of the IFN-γ-producing cells, with the exception of IL-17 producers far outnumbering the IFN-γ and IL-2 producers in the inflamed CNS. The selective enrichment of IL-17-producing CD4 cells in the CNS is suggestive of the pathogenic role of an independent (non-Th1) IL-17-producing proinflammatory effector T cell class in EAE.
Streptococcus agalactiae GAPDH Is a Virulence-Associated Immunomodulatory Protein
Authors: Madureira Pedro , Baptista Marina , Vieira Marta , Magalhães Vanessa , Camelo Ana , Oliveira Liliana , Ribeiro Adília , Tavares Delfina , Trieu-Cuot Patrick , Vilanova Manuel , Ferreira Paula
Published: April 2014

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.
Identical β Cell-Specific CD8+ T Cell Clonotypes Typically Reside in Both Peripheral Blood Lymphocyte and Pancreatic Islets
Authors: Wong Carmen P. , Stevens Rosemary , Long Brian , Li Li , Wang Yaming , Wallet Mark A. , Goudy Kevin S. , Frelinger Jeffrey A. , Tisch Roland
Published: April 2014

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring β cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8+ T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3α and CDR3β sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. β cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.
Induction of IL-4 Expression in CD4+ T Cells by Thymic Stromal Lymphopoietin
Authors: Omori Miyuki , Ziegler Steven
Published: April 2014

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic inflammation in both humans and mice. Although the underlying mechanism is not known, TSLP-stimulated dendritic cells have been shown to prime human CD4+ T cells into Th2 cytokine-producing cells. However, its direct effect on CD4+ T cells has not been extensively investigated. In this study, we show that TSLP can drive Th2 differentiation in the absence of exogenous IL-4 and APCs. IL-4 blockade inhibited TSLP-mediated Th2 differentiation, demonstrating that IL-4 is involved in this process. Further analysis has shown that TSLP-induced Th2 differentiation is dependent on Stat6 and independent of IL-2 and that TSLP treatment leads to immediate, direct Il-4 gene transcription. Taken together, these data demonstrate that TSLP is directly involved in Th2-mediated responses via the induction of IL-4 production.
The B Cell Antigen Receptor Controls AP-1 and NFAT Activity through Ras-Mediated Activation of Ral
Authors: de Gorter David J. J. , Vos Johanna C. M. , Pals Steven T. , Spaargaren Marcel
Published: April 2014

Signaling by the BCR involves activation of several members of the Ras superfamily of small GTPases, among which is Ras itself. Ras can control the activity of multiple effectors, including Raf, PI3K, and guanine nucleotide exchange factors for the small GTPase Ral. Ras, Raf, and PI3K have been implicated in a variety of processes underlying B cell development, differentiation, and function; however, the role of Ral in B lymphocytes remains to be established. In this study, we show that Ral is activated upon BCR stimulation in human tonsillar and mouse splenic B lymphocytes and in B cell lines. Using signaling molecule-deficient B cells, we demonstrate that this activation is mediated by Lyn and Syk, Btk, phospholipase C-γ2, and inositol-1,4,5-trisphosphate receptor-mediated Ca2+ release. In addition, although Ral can be activated by Ras-independent mechanisms, we demonstrate that BCR-controlled activation of Ral is dependent on Ras. By means of expression of the dominant-negative mutants RasN17 and RalN28, or of RalBPΔGAP, a Ral effector mutant which sequesters active Ral, we show that Ras and Ral mediate BCR-controlled transcription of c-fos. Furthermore, while not involved in NF-κB activation, Ras and Ral mediate BCR-controlled activation of JUN/ATF2 and NFAT transcription factors. Taken together, our data show that Ral is activated upon BCR stimulation and mediates BCR-controlled activation of AP-1 and NFAT transcription factors. These findings suggest that Ral plays an important role in B cell development and function.
Improved Outcomes in NOD Mice Treated with a Novel Th2 Cytokine-Biasing NKT Cell Activator
Authors: Forestier Claire , Takaki Toshiyuki , Molano Alberto , Im Jin S. , Baine Ian , Jerud Elliot S. , Illarionov Petr , Ndonye Rachel , Howell Amy R. , Santamaria Pere , Besra Gurdyal S. , DiLorenzo Teresa P. , Porcelli Steven A.
Published: April 2014

Activation of CD1d-restricted invariant NKT (iNKT) cells by α-galactosylceramide (αGalCer) significantly suppresses development of diabetes in NOD mice. The mechanisms of this protective effect are complex, involving both Th1 and Th2 cytokines and a network of regulatory cells including tolerogenic dendritic cells. In the current study, we evaluated a newly described synthetic αGalCer analog (C20:2) that elicits a Th2-biased cytokine response for its impact on disease progression and immunopathology in NOD mice. Treatment of NOD mice with αGalCer C20:2 significantly delayed and reduced the incidence of diabetes. This was associated with significant suppression of the late progression of insulitis, reduced infiltration of islets by autoreactive CD8+ T cells, and prevention of progressive disease-related changes in relative proportions of different subsets of dendritic cells in the draining pancreatic lymph nodes. Multiple favorable effects observed with αGalCer C20:2 were significantly more pronounced than those seen in direct comparisons with a closely related analog of αGalCer that stimulated a more mixed pattern of Th1 and Th2 cytokine secretion. Unlike a previously reported Th2-skewing murine iNKT cell agonist, the αGalCer C20:2 analog was strongly stimulatory for human iNKT cells and thus warrants further examination as a potential immunomodulatory agent for human disease.
Influenza Virus Infection Causes Global Respiratory Tract B Cell Response Modulation via Innate Immune Signals
Authors: Chang W. L. William , Coro Elizabeth S. , Rau Friederike C. , Xiao Yuanyuan , Erle David J. , Baumgarth Nicole
Published: April 2014

Induction of primary B cell responses requires the presence of Ag and costimulatory signals by T cells. Innate signals further enhance B cell activation. The precise nature and kinetics of such innate immune signals and their functional effects are unknown. This study demonstrates that influenza virus-induced type I IFN is the main innate stimulus affecting local B cells within 48 h of infection. It alters the transcriptional profile of B cells and selectively traps them in the regional lymph nodes, presumably via up-regulation of CD69. Somewhat paradoxically, innate B cell stimulation inhibited the ability of regional lymph node B cells to clonally expand following BCR-mediated stimulation. This inhibition was due to IFNR-signaling independent B cell intrinsic, as well as IFNR-dependent B cell extrinsic, regulation induced following influenza infection. IFNR-mediated signals also reduced B cell migration to various chemotactic agents. Consistent with the lack of responsiveness to CCR7 ligands, unaltered or reduced expression of MHC class II and genes associated with MHC class II Ag processing/presentation and CD40, B cells were unable to induce proliferation of naive CD4 T cells. Instead, they showed increased expression of a subset of nonclassical MHC molecules that facilitate interaction with γδ T cells and NK T cells. We conclude that type I IFN is the main “third” B cell signal following influenza infection causing early trapping of B cells in regional lymph nodes and, at a time when cognate T cell help is rare, enhancing their propensity to interact with innate immune cells for noncognate stimulation.
Dendritic Cell Immunization Route Determines Integrin Expression and Lymphoid and Nonlymphoid Tissue Distribution of CD8 T Cells
Authors: Sheasley-O’Neill Stacey L. , Brinkman C. Colin , Ferguson Andrew R. , Dispenza Melanie C. , Engelhard Victor H.
Published: April 2014

Exogenous dendritic cells (bone marrow-derived dendritic cell (BMDC)) display restricted trafficking in vivo after injection into mice, but the route(s) by which they generate gut-homing effector cells is unclear. Mesenteric lymph nodes (LN) and spleen were differentially targeted by i.p. and i.v. administration of BMDC, respectively, whereas mediastinal LN were targeted by both routes. BMDC injected by either route activated CD8+ T cells to up-regulate both α4β1 and α4β7 integrins. However, the lymphoid compartment in which activation occurred determined their expression kinetics, magnitude, and population distribution. Only T cells activated in mesenteric LN after i.p. immunization expressed high levels of α4β7, which also correlated with localization to small intestine. These α4β7high cells also redistributed to mediastinal LN in a manner sensitive to treatment with α4β7 blocking Abs, but not to mucosal addressin cell adhesion molecule-1 blocking Abs. Our results demonstrate the importance of lymphoid compartment, as dictated by immunization route, in determining integrin expression on activated T cells and their distribution in lymphoid and nonlymphoid tissues.
Plasmacytoid Dendritic Cells Synergize with Myeloid Dendritic Cells in the Induction of Antigen-Specific Antitumor Immune Responses
Authors: Lou Yanyan , Liu Chengwen , Kim Grace J. , Liu Yong-Jun , Hwu Patrick , Wang Gang
Published: April 2014

Plasmacytoid dendritic cells (pDC) are capable of producing high levels of type I IFNs upon viral stimulation, and play a central role in modulating innate and adaptive immunity against viral infections. Whereas many studies have assessed myeloid dendritic cells (mDC) in the induction of antitumor immune responses, the role of pDC in antitumor immunity has not been addressed. Moreover, the interaction of pDC with other dendritic cell subsets has not been evaluated. In this study, we analyzed the capacity of pDC in stimulating an Ag-specific T cell response. Immunization of mice with Ag-pulsed, activated pDC significantly augmented Ag-specific CD8+ CTL responses, and protected mice from a subsequent tumor challenge. Immunization with a mixture of activated pDC plus mDC resulted in increased levels of Ag-specific CD8+ T cells and an enhanced antitumor response compared with immunization with either dendritic cell subset alone. Synergy between pDC and mDC in their ability to activate T cells was dependent on MHC I expression by mDC, but not pDC, suggesting that pDC enhanced the ability of mDC to present Ag to T cells. Our results demonstrate that pDC and mDC can interact synergistically to induce an Ag-specific antitumor immune response in vivo.
B7-Deficient Autoreactive T Cells Are Highly Susceptible to Suppression by CD4+CD25+ Regulatory T Cells
Authors: May Kenneth F. , Chang Xing , Zhang Huiming , Lute Kenneth D. , Zhou Penghui , Kocak Ergun , Zheng Pan , Liu Yang
Published: April 2014

CD4+CD25+ regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4+CD25+ T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.
Combined TLR/CD40 Stimulation Mediates Potent Cellular Immunity by Regulating Dendritic Cell Expression of CD70 In Vivo
Authors: Sanchez Phillip J. , McWilliams Jennifer A. , Haluszczak Catherine , Yagita Hideo , Kedl Ross M.
Published: April 2014

We previously showed that immunization with a combination of TLR and CD40 agonists (combined TLR/CD40 agonist immunization) resulted in an expansion of Ag-specific CD8 T cells exponentially greater than the expansion observed to immunization with either agonist alone. We now show that the mechanism behind this expansion of T cells is the regulated expression of CD70 on dendritic cells. In contrast to previous results in vitro, the expression of CD70 on dendritic cells in vivo requires combined TLR/CD40 stimulation and is not significantly induced by stimulation of either pathway alone. Moreover, the exponential expansion of CD8+ T cells following combined TLR/CD40 agonist immunization is CD70 dependent. Thus, the transition from innate stimuli (TLRs) to adaptive immunity is controlled by the regulated expression of CD70.
Slamf1, the NKT Cell Control Gene Nkt1
Authors: Jordan Margaret A. , Fletcher Julie M. , Pellicci Daniel , Baxter Alan G.
Published: April 2014

Invariant NKT cells play a critical role in controlling the strength and character of adaptive immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. In this study, we report the production and characterization of a NOD.Nkrp1b.Nkt1b congenic mouse strain, apply microarray expression analyses to limit candidate genes within the 95% confidence region, identify Slamf1 (encoding signaling lymphocyte activation molecule) and Slamf6 (encoding Ly108) as potential candidates, and demonstrate retarded signaling lymphocyte activation molecule expression during T cell development of NOD mice, resulting in reduced expression at the CD4+CD8+ stage, which is consistent with decreased NKT cell production and deranged tolerance induction in NOD mice.
Role of CD40-Dependent Down-Regulation of CD154 in Impaired Induction of CD154 in CD4+ T Cells from HIV-1-Infected Patients
Authors: Subauste Carlos S. , Subauste Angela , Wessendarp Matthew
Published: April 2014

CD40-CD154 interaction is pivotal for cell-mediated immunity. There are contradictory reports on whether HIV-1 infection impairs CD154 induction. The interaction between CD40 and CD154 is important not only because it results in activation of APCs but also because it controls CD154 by diminishing expression of this molecule. Compared with healthy controls, CD4+ T cells from HIV-1+ patients had impaired induction of CD154 when T cell activation was mediated by CD40+ APCs. In contrast, T cell activation in the absence of these cells resulted in normal CD154 expression. CD154 induction in HIV-1+ patients and controls were similar upon blockade of CD40-CD154 binding. Defective regulation of CD154 appeared to occur downstream of the control of mRNA levels because up-regulation of CD154 mRNA was not impaired by HIV-1 infection. This work identifies CD40 as a mediator of impaired CD154 induction in HIV-1 infection and explains why this defect was not detected by studies where T cell activation was triggered independently of CD40+ APCs. In addition, dysregulation of CD154 in HIV-1 infection likely contributes to immunodeficiency because diminished expression of CD154 induced by CD40 is of functional relevance, resulting in decreased dendritic cell maturation.