bluuebunny/crossref_metadata_2025 huggingface dataset:
The Frequency of High Avidity T Cells Determines the Hierarchy of Determinant Spreading
Authors: Tian Jide , Gregori Silvia , Adorini Luciano , Kaufman Daniel L.
Published: April 2014

Autoimmunity often spreads in a predefined pattern during the progression of T cell-mediated autoimmune diseases. This progression has been well described in animal models and in man, but the basis for this phenomenon is little understood. To gain insight into the factors that determine this spreading hierarchy, we characterized the binding affinity of a panel of β cell-autoantigenic peptides to I-Ag7, as well as the precursor frequency, functional avidity, and phenotype of the T cells that recognize these peptides in type 1 diabetes-prone nonobese diabetic mice. We observed that autoimmunity gradually spreads from a β cell determinant, which had the largest precursor pool of high avidity T cells, to β cell determinants with progressively smaller and lower avidity T cell precursor pools. This correlation between the sequential development of spontaneous T cell autoimmunity and the frequency and avidity of autoantigen-reactive T cells suggests that the extent to which T cells were negatively selected by the self-determinants is the key factor determining the spreading hierarchy.
Pharmacokinetic Differences Between a T Cell-Tolerizing and a T Cell-Activating Peptide
Authors: Weijzen Sanne , Meredith Stephen C. , Velders Markwin P. , Elmishad Amira G. , Schreiber Hans , Kast W. Martin
Published: April 2014

Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.
Tolerance to Cyclosporin A-Induced Autologous Graft-Versus-Host Disease Is Mediated by a CD4+CD25+ Subset of Recent Thymic Emigrants
Authors: Wu Dianna Y. , Goldschneider Irving
Published: April 2014

Our previous studies revealed that both the autoeffector and immunoregulatory T cells in cyclosporin A (CSA)-induced autologous graft-vs-host disease are recent thymic emigrants (RTEs). The autoeffector cells appear in and are released from the thymus during the first week of CSA treatment, whereas the immunoregulatory thymocytes appear during the second week but are not released until several days after cessation of CSA treatment. In the present study, the antigenic phenotypes of these functional T cell subsets were determined by immunomagnetic separation and flow immunocytometric analysis. During CSA wk 1, the autoeffector T cells in both the thymus and lymph node (LN) expressed a CD4+8+ double-positive (DP) phenotype, after which those in the LN became CD8 single positive (SP). Timed thymectomy experiments confirmed that the CD8-SP autoeffector T cells in LN originated from these DP RTEs. During CSA wk 2, the immunoregulatory thymocytes also displayed a DP phenotype. However, they were not exported to the periphery until several days after CSA treatment had been interrupted and they had acquired a CD4-SP phenotype. In LN, these immunoregulatory RTEs expressed the CD25+ marker characteristic of anergic/suppressor T cells. Cell separation and mixing experiments demonstrated that the autoeffector T cells persist in LN after cessation of CSA treatment, but their activity is not detectable in the presence of recently exported CD4+ T cells. Hence, the results indicate that tolerance to CSA-induced autologous graft-vs-host disease is actively mediated by CD25+CD4+ RTEs that suppress the function of CD8 autoeffector T cells.
Sanglifehrin A, a Novel Cyclophilin-Binding Compound Showing Immunosuppressive Activity with a New Mechanism of Action
Authors: Zenke Gerhard , Strittmatter Ulrike , Fuchs Serge , Quesniaux Valerie F. J. , Brinkmann Volker , Schuler Walter , Zurini Mauro , Enz Albert , Billich Andreas , Sanglier Jean-Jacques , Fehr Theo
Published: April 2014

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.
The P2Y11 Receptor Mediates the ATP-Induced Maturation of Human Monocyte-Derived Dendritic Cells
Authors: Wilkin Françoise , Duhant Xavier , Bruyns Catherine , Suarez-Huerta Nathalie , Boeynaems Jean-Marie , Robaye Bernard
Published: April 2014

Recently, it has been shown that ATP and TNF-α synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-α and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5′-O-(3-thiotriphosphate) = 2′- and 3′-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.
In Vivo-Matured Langerhans Cells Continue to Take Up and Process Native Proteins Unlike In Vitro-Matured Counterparts
Authors: Ruedl Christiane , Koebel Pascale , Karjalainen Klaus
Published: April 2014

We have been able to identify the cell subset derived from Langerhans cells in the total dendritic cell population of the peripheral lymph node and hence to follow their trafficking under normal physiological conditions as well as upon skin irritation. As expected, the rapid mobilization of Langerhans cells triggered by inflammatory signals into the draining lymph node correlated with an up-regulation of costimulatory molecules and with an enhanced immunostimulatory capacity. Surprisingly, however, these cells, instead of shutting down, maintain the capacity to capture and process protein Ags during the couple of days they stay alive in stark contrast to in vitro-matured dendritic cells.
Progesterone Up-Regulates Anandamide Hydrolase in Human Lymphocytes: Role of Cytokines and Implications for Fertility
Authors: Maccarrone Mauro , Valensise Herbert , Bari Monica , Lazzarin Natalia , Romanini Carlo , Finazzi-Agrò Alessandro
Published: April 2014

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes. At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th2 cytokines. In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the Th1 cytokines IL-12 and IFN-γ reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and 2-arachidonoylglycerol inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.
Production of TNF-α by Human Vγ9Vδ2 T Cells Via Engagement of FcγRIIIA, the Low Affinity Type 3 Receptor for the Fc Portion of IgG, Expressed upon TCR Activation by Nonpeptidic Antigen
Authors: Lafont Virginie , Liautard Janny , Liautard Jean Pierre , Favero Jean
Published: April 2014

Human lymphocytes expressing the γδ TCR represent a minor T cell subpopulation found in blood. The majority of these cells express Vγ9Vδ2 determinants and respond to nonpeptidic phosphoantigens. Several studies have shown that, in vivo, the percentage of Vγ9Vδ2 T cells dramatically increases during pathological infection, leading to the hypothesis that they play an important role in the defense against pathogens. However, the specific mechanisms involved in this response remain poorly understood. It has been established that Vγ9Vδ2 T cells display potent cytotoxic activity against virus-infected and tumor cells, thereby resembling NK cells. In this study, we show that, upon stimulation by nonpeptidic Ags, Vγ9Vδ2 T cells express FcγRIIIA (CD16), a receptor that is constitutively expressed on NK cells. CD16 appears to be an activation Ag for Vγ9Vδ2 T cells. Indeed, ligation of CD16 on Vγ9Vδ2 T cells leads to TNF-α production. This TNF-α production, which is dependent (like that induced via the TCR-CD3 complex) on the activation of the p38 and extracellular signal-regulated kinase-2 mitogen-activated protein kinases, can be modulated by CD94 NK receptors. Therefore, it appears that Vγ9Vδ2 T cells can be physiologically activated by two sequential steps via two different cell surface Ags: the TCR-CD3 complex and the FcγRIIIA receptor, which are specific cell surface Ags for T lymphocytes and NK cells, respectively. This strongly suggests that, in the general scheme of the immune response, Vγ9Vδ2 T cells represent an important subpopulation of cells that play a key role in the defense against invading pathogens.
Differential Survival of Transferred CD8 T Cells and Host Reconstitution Depending on TCR Avidity for Host-Expressed Alloantigen
Authors: Auphan-Anezin Nathalie , Schmitt-Verhulst Anne-Marie
Published: April 2014

We transferred naive alloreactive CD8 T cells from TCR transgenic mice to irradiated recipients expressing a partial (H-2Kbm8) or a full (H-2Kb) agonist alloantigen (alloAg). The consequences were strikingly distinct, resulting in acceleration of host lymphopoiesis in the former group, but in strong graft-vs-host reaction, preventing host lymphocyte reconstitution in the latter group. This was correlated, respectively, with long-term persistence and with rapid disappearance of the transferred CD8 T cells. Analysis of transferred T cells showed that initial T cell expansion and modulation of expression of activation markers CD44 and CD62L, as well as induction of cytotoxic function, were similar in both groups. However, IL-2 production and subsequent up-regulation of CD25, early perforin-independent cytolysis, and early down-regulation of Bcl-2 expression were detected only in T cells transferred in hosts expressing full agonist alloAg. Expansion of transferred CD8 T cells was not dependent on either IL-2 or CD25 expression. This expansion could lead to either accelerated host reconstitution or to strong graft-vs-host, depending on the nature of the alloAg. Thus, the extent of Ag stimulation may be a crucial parameter in protocols of alloreactive T cell immunotherapy.
CD45 Function Is Regulated by an Acidic 19-Amino Acid Insert in Domain II That Serves as a Binding and Phosphoacceptor Site for Casein Kinase 2
Authors: Greer Susanna F. , Wang Yan-ni , Raman Chander , Justement Louis B.
Published: April 2014

In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B cells. However, yeast two-hybrid analysis failed to demonstrate a physical interaction between the individual CK2 α, α′, or β subunits and CD45. In contrast, a yeast three-hybrid assay in which either CK2 α and β or α′ and β subunits were coexpressed with the cytoplasmic domain of CD45, demonstrated that both CK2 subunits are necessary for the interaction with CD45. Experiments using the yeast three-hybrid assay also revealed that a 19-aa acidic insert in domain II of CD45 mediates the physical interaction between CK2 and CD45. Structure/function experiments in which wild-type or mutant CD45RA and CD45RO isoforms were expressed in CD45-deficient Jurkat cells revealed that the 19-aa insert is important for optimal CD45 function. The ability of both CD45RA and CD45RO to reconstitute CD3-mediated signaling based on measurement of calcium mobilization and mitogen-activated protein kinase activation was significantly decreased by deletion of the 19-aa insert. Mutation of four serine residues within the 19-aa insert to alanine affected CD45 function to a similar extent compared with that of the deletion mutants. These findings support the hypothesis that a physical interaction between the CD45 cytoplasmic domain and CK2 is important for post-translational modification of CD45, which, in turn, regulates its catalytic function.
Regulation of NK Cell-Mediated Cytotoxicity by the Adaptor Protein 3BP2
Authors: Jevremovic Dragan , Billadeau Daniel D. , Schoon Renee A. , Dick Christopher J. , Leibson Paul J.
Published: April 2014

Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95vav and phospholipase C-γ isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.
Quantitative Relationship Between MHC Class II-Superantigen Complexes and the Balance of T Cell Activation Versus Death
Authors: Lavoie Pascal M. , McGrath Helen , Shoukry Naglaa H. , Cazenave Pierre-André , Sékaly Rafick-Pierre , Thibodeau Jacques
Published: April 2014

The binding of bacterial superantigens (SAgs) is profoundly affected by the nature of the MHC class II-associated antigenic peptide. It was proposed that this limitation in the density of SAgs displayed at the surface of APCs is important for efficient TCR serial triggering as well as for preventing apoptosis of the responding T lymphocytes. Here, we have addressed quantitatively the size of this SAg-receptive pool of HLA-DR molecules that are available to bind and present staphylococcal enterotoxin A (SEA) at the surface of B lymphocytes. Our binding curves, depletion experiments, and quantitative immunoprecipitations show that about half the HLA-DR class II molecules on B cells are refractory to SEA binding. Yet, as compared with typical nominal Ags, an unusually high amount of class II-SAg complexes can be presented to T cells. This characteristic appears to be necessary for SAg-induced T cell apoptosis. When <0.3% of the total cell surface MHC class II molecules are occupied by SEA, T cells undergo a normal sequence of early activation events. However, presentation of a ligand density beyond this threshold results in T cell activation that is readily aborted by apoptosis but only after a few cell divisions. Thus, we confirm the existence of MHC class II subsets that are structurally unable to present SEA and provide a quantitative framework to account for the ability of bacterial SAgs to induce peripheral activation vs tolerance in the host.
Processing of Immunosuppressive Pro-TGF-β1,2 by Human Glioblastoma Cells Involves Cytoplasmic and Secreted Furin-Like Proteases
Authors: Leitlein Jens , Aulwurm Steffen , Waltereit Robert , Naumann Ulrike , Wagenknecht Bettina , Garten Wolfgang , Weller Michael , Platten Michael
Published: April 2014

TGF-β is a putative mediator of immunosuppression associated with malignant glioma and other types of cancer. Subtilisin-like proprotein convertases such as furin are thought to mediate TGF-β processing. Here we report that human malignant glioma cell lines express furin mRNA and protein, exhibit furin-like protease (FLP) activity, and release active furin into the cell culture supernatant. FLP activity is not modulated by exogenous TGF-β or neutralizing TGF-β Abs. Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the TGF-β1 and TGF-β2 precursor molecules and, consequently, the release of mature bioactive TGF-β molecules. Ectopic expression of PDX, a synthetic antitrypsin analog with antifurin activity, in the glioma cells inhibits FLP activity, TGF-β processing, and TGF-β release. Thus, subtilisin-like proprotein convertases may represent a novel target for the immunotherapy of malignant glioma and other cancers or pathological conditions characterized by enhanced TGF-β bioactivity.
Extrathymic Development of Vα11 T Cells in Placenta During Pregnancy and Their Possible Physiological Role
Authors: Yamasaki Masahiko , Sasho Takahisa , Moriya Hideshige , Kanno Masamoto , Harada Michishige , Kamada Noriaki , Shimizu Eiko , Nakayama Toshinori , Taniguchi Masaru
Published: April 2014

The molecular and cellular mechanisms of the feto-maternal immune responses in the placenta in connection with natural abortion remain unclear. In this report we provide evidence that Vα11 T cells developed in the placenta may be responsible for the induction of natural abortion. The majority of Vα11 TCRs detected during pregnancy showed a consensus motif in the CDR3 region, similar to that of anti-GM3 TCR clones, and were of maternal origin. Vα11 TCRs were found in the middle to late stages of gestation due to de novo generation in the placenta, not to migration from the maternal side, as evidenced by the significant increases in the out-of-frame Vα11 TCR mRNA and the copy number of circular DNA generated by Vα11 gene rearrangements. Furthermore, administration of anti-Vα11 Ab to pregnant mice resulted in a significant decrease in the incidence of fetal demise, suggesting that Vα11 T cells detected in the placenta develop extrathymically and are involved in natural abortion.
Novel G Protein-Coupled Responses in Leukocytes Elicited by a Chemotactic Bacteriophage Displaying a Cell Type-Selective Binding Peptide
Authors: Jaye David L. , Edens Heather A. , Mazzucchelli Luca , Parkos Charles A.
Published: April 2014

Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils. In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels. Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses. Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation. Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins. However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors. These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.
Recognition of HLA-Cw4 but Not HLA-Cw6 by the NK Cell Receptor Killer Cell Ig-Like Receptor Two-Domain Short Tail Number 4
Authors: Katz Gil , Markel Gal , Mizrahi Sa’ar , Arnon Tal I. , Mandelboim Ofer
Published: April 2014

NK cells are cytotoxic to virus-infected and tumor cells that have lost surface expression of class I MHC proteins. Target cell expression of class I MHC proteins inhibits NK cytotoxicity through binding to inhibitory NK receptors. In contrast, a similar family of activating NK receptors, characterized by the presence of a charged residue in their transmembrane portion and a truncated cytoplasmic tail, augment lysis by NK cells when ligated by an appropriate class I MHC protein. However, the class I MHC specificity of many of these activating NK receptors is still unknown. Here, we show enhanced lysis of HLA-Cw4 but not HLA-Cw6-expressing cells, by a subset of NK clones. This subset may express killer cell Ig-like receptor two-domain short tail number 4 (KIR2DS4), as suggested by staining with various mAb. It is still possible, however, that these clones may express receptors other than KIR2DS4 that might recognize HLA-Cw4. Binding of KIR2DS4-Ig fusion protein to cells expressing HLA-Cw4 but not to those expressing HLA-Cw6 was also observed. The binding of KIR2DS4-Ig to HLA-Cw4 is weaker than that of killer cell Ig-like receptor two-domain long tail number 1 (KIR2DL1)-Ig fusion protein; however, such weak recognition is capable of inhibiting lysis by an NK transfectant expressing a chimeric molecule of KIR2DS4 fused to the transmembrane and cytoplasmic portion of KIR2DL1. Residue α14 is shown to be important in the KIR2DS4 binding to HLA-Cw4. Implications of the role of the activating NK receptors in immunosurveillance are discussed.
Factors Affecting the Efficiency of CD8+ T Cell Cross-Priming with Exogenous Antigens
Authors: Maecker Holden T. , Ghanekar Smita A. , Suni Maria A. , He Xiao-Song , Picker Louis J. , Maino Vernon C.
Published: April 2014

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, “cross-priming” can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor’s blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual’s APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.
Stat6 Is Necessary and Sufficient for IL-4’s Role in Th2 Differentiation and Cell Expansion
Authors: Zhu Jinfang , Guo Liying , Watson Cynthia J. , Hu-Li Jane , Paul William E.
Published: April 2014

IL-4 plays a critical role in the differentiation of TCR-stimulated naive CD4 T cells to the Th2 phenotype. In response to IL-4, the IL-4R activates a set of phosphotyrosine binding domain-containing proteins, including insulin receptor substrate 1/2, Shc, and IL-4R interacting protein, as well as Stat6. Stat6 has been shown to be required for Th2 differentiation. To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation, we prepared a retrovirus containing a mutant of the human (h)IL-4R α-chain, Y497F, which is unable to recruit these adaptors. The mutant hIL-4Rα, as well as the wild-type (WT) hIL-4Rα, was introduced into naive CD4 T cells. Upon hIL-4 stimulation, Y497F worked as well as the WT hIL-4Rα in driving Th2 differentiation, as measured by Gata3 up-regulation and IL-4 production. Furthermore, IL-4-driven cell expansion was also normal in the cells infected with Y497F, although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2. These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4-driven Th2 differentiation and cell expansion. Both WT and Y497F hIL-4Rα lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells. A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion. Thus, activated Stat6 is necessary and sufficient to mediate both IL-4-driven Th2 differentiation and cell expansion in CD4 T cells.
A Role for TGF-β in the Generation and Expansion of CD4+CD25+ Regulatory T Cells from Human Peripheral Blood
Authors: Yamagiwa Satoshi , Gray J. Dixon , Hashimoto Shigeo , Horwitz David A.
Published: April 2014

An elusive goal in transplanting organs across histocompatibility barriers has been the induction of specific tolerance to avoid graft rejection. A considerable body of evidence exists that the thymus produces regulatory T cells that suppress the response of other T cells to antigenic stimulation. We report that TGF-β can induce certain CD4+ T cells in the naive (CD45RA+RO−) fraction in human peripheral blood to develop powerful, contact-dependent suppressive activity that is not antagonized by anti-TGF-β or anti-IL-10 mAbs. The costimulatory effects of TGF-β on naive CD4+ T cells up-regulated CD25 and CTLA-4 expression, increased their transition to the activated phenotype, but decreased activation-induced apoptosis. Suppressive activity was concentrated in the CD25+ fraction. These CD4+CD25+ regulatory cells prevented CD8+ T cells from proliferating in response to alloantigens and from becoming cytotoxic effector cells. Moreover, these regulatory cells exerted their suppressive activities in remarkably low numbers and maintained these effects even after they are expanded. Once activated, their suppressive properties were Ag nonspecific. Although <1% of naive CD4+ T cells expressed CD25, depletion of this subset before priming with TGF-β markedly decreased the generation of suppressive activity. This finding suggests that CD4+CD25+ regulatory T cells induced ex vivo are the progeny of thymus-derived regulatory T cells bearing a similar phenotype. The adoptive transfer of these regulatory T cells generated and expanded ex vivo has the potential to prevent rejection of allogeneic organ grafts.
Evidence for Epigenetic Mechanisms That Silence Both Basal and Immune-Stimulated Transcription of the IL-8 Gene
Authors: Wen Xiaoming , Wu Gary D.
Published: April 2014

It is becoming increasingly clear that epigenetic silencing of gene transcription plays a critical role in the regulation of gene expression in many biological processes. Tight regulation of immunomodulatory substances that are important for the initiation of the inflammatory cascade, such as chemoattractive cytokines, is essential to prevent initiation of unrestrained immune activation. Using the Caco-2 intestinal cell line as a model, we reveal two distinctly different mechanisms by which the gene for the neutrophil chemoattractive cytokine IL-8 is silenced. Nuclear run-on studies, as well as stably transfected reporter and marked minigene constructs, demonstrate that cellular differentiation inhibits immune-activated transcription of the IL-8 gene, a mechanism that is dependent on histone deacetylase activity. Unexpectedly, this silencing mechanism does not involve previously described regulatory elements in the IL-8 promoter but rather cis-acting regions located at a distance from the IL-8 gene locus. Genomic elements distant to the immediate IL-8 locus are also required to silence aberrant basal transcriptional activity of the IL-8 promoter in the absence of immune activation. However, in this case, silencing occurs in a histone deacetylase-independent fashion. These findings were confirmed in transgenic mice in which, in the absence of these elements, aberrant IL-8 gene activity was present primarily in the intestinal tract. Epigenetic silencing of cytokine gene transcription through distant genomic elements is an important level of gene regulation that may be relevant to the pathogenesis of immunologic disease states.
Differential Expression of Inducible Costimulator-Ligand Splice Variants: Lymphoid Regulation of Mouse GL50-B and Human GL50 Molecules
Authors: Ling Vincent , Wu Paul W. , Miyashiro Joy S. , Marusic Suzana , Finnerty Heather F. , Collins Mary
Published: April 2014

The process of immunological costimulation between APC and T cells is mediated by protein ligand:receptor interactions. To date, costimulatory receptors known to be expressed by T cells include the structurally related proteins CD28 and the inducible costimulator (ICOS). The ligands to human and mouse ICOS, human GL50 (hGL50), and mouse GL50 (mGL50) were recently cloned and demonstrated to have sequence similarity to the CD28 ligands B7-1 and B7-2. Examination of mGL50 cDNA transcripts by 3′RACE revealed an alternatively spliced form, mGL50-B, that encoded a protein product with a divergent 27-aa intracellular domain. Both mGL50- and mGL50-B-transfected cells exhibited binding to human and mouse ICOS-Ig fusion protein, indicating that the alternate cytoplasmic domain of mGL50-B does not interfere with extracellular interactions with ICOS receptor. Flow cytometric and RT-PCR analysis of BALB/c and RAG1−/− mice splenocytes demonstrate that freshly isolated B cells, T cells, macrophages, and dendritic cells express both splice variant forms of ICOS ligand. Comparative analyses with the human ICOS ligand splice variants hGL50 and B7-H2 indicate that differential splicing at the junction of cytoplasmic exon 6 and exon 7 may be a common method by which GL50-ICOS immunological costimulatory processes are regulated in vivo.
Proteomic Analysis of Dendritic Cell-Derived Exosomes: A Secreted Subcellular Compartment Distinct from Apoptotic Vesicles
Authors: Théry Clotilde , Boussac Muriel , Véron Philippe , Ricciardi-Castagnoli Paola , Raposo Graça , Garin Jerôme , Amigorena Sebastian
Published: April 2014

Dendritic cells constitutively secrete a population of small (50–90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1α) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.
Characterization of a Pattern Recognition Protein, a Masquerade-Like Protein, in the Freshwater Crayfish Pacifastacus leniusculus
Authors: Lee So Young , Söderhäll Kenneth
Published: April 2014

A multifunctional masquerade-like protein has been isolated, purified, and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. It was isolated by its Escherichia coli binding property, and it binds to formaldehyde-treated Gram-negative bacteria as well as to yeast, Saccharomyces cerevisiae, whereas it does not bind to formaldehyde-fixed Gram-positive bacteria. The intact masquerade (mas)-like protein is present in crayfish hemocytes as a heterodimer composed of two subunits with molecular masses of 134 and 129 kDa. Under reducing conditions the molecular masses of the intact proteins are not changed. After binding to bacteria or yeast cell walls, the mas-like protein is processed by a proteolytic enzyme. The 134 kDa of the processed protein yields four subunits of 65, 47, 33, and 29 kDa, and the 129-kDa protein results in four subunits of 63, 47, 33, and 29 kDa in 10% SDS-PAGE under reducing conditions. The 33-kDa protein could be purified by immunoaffinity chromatography using an Ab to the C-terminal part of the mas-like protein. This subunit of the mas-like protein has cell adhesion activity, whereas the two intact proteins, 134 and 129 kDa, have binding activity to LPSs, glucans, Gram-negative bacteria, and yeast. E. coli coated with the mas-like protein were more rapidly cleared in crayfish than only E. coli, suggesting this protein is an opsonin. Therefore, the cell adhesion and opsonic activities of the mas-like protein suggest that it plays a role as an innate immune protein.
MHC Class I Recognition by NK Receptors in the Ly49 Family Is Strongly Influenced by the β2-Microglobulin Subunit
Authors: Michaëlsson Jakob , Achour Adnane , Rölle Alexander , Kärre Klas
Published: April 2014

NK cell recognition of targets is strongly affected by MHC class I specific receptors. The recently published structure of the inhibitory receptor Ly49A in complex with H-2Dd revealed two distinct sites of interaction in the crystal. One of these involves the α1, α2, α3, and β2-microglobulin (β2m) domains of the MHC class I complex. The data from the structure, together with discrepancies in earlier studies using MHC class I tetramers, prompted us to study the role of the β2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the β2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine β2m to human β2m in three different MHC class I molecules, H-2Db, H-2Kb, and H-2Dd, resulted in a loss of binding to the receptors Ly49A and Ly49C. Analysis of the amino acids involved in the binding of Ly49A to H-2Dd in the published crystal structure, and differing between the mouse and the human β2m, suggests the cluster formed by residues Lys3, Thr4, Thr28, and Gln29, as a potentially important domain for the Ly49A-H-2Dd interaction. Another possibility is that the change of β2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the α1 and α2 domains of the heavy chain.
Augmentation in Expression of Activation-Induced Genes Differentiates Memory from Naive CD4+ T Cells and Is a Molecular Mechanism for Enhanced Cellular Response of Memory CD4+ T Cells
Authors: Liu Kebin , Li Yu , Prabhu Vinayakumar , Young Lynn , Becker Kevin G. , Munson Peter J. , Weng Nan-ping
Published: April 2014

In an attempt to understand the molecular basis for the immunological memory response, we have used cDNA microarrays to measure gene expression of human memory and naive CD4+ T cells at rest and after activation. Our analysis of 54,768 cDNA clones provides the first glimpse into gene expression patterns of memory and naive CD4+ T cells at the genome-scale and reveals several novel findings. First, memory and naive CD4+ T cells expressed similar numbers of genes at rest and after activation. Second, we have identified 14 cDNA clones that expressed higher levels of transcripts in memory cells than in naive cells. Third, we have identified 135 (130 known genes and 5 expressed sequence tags) up-regulated and 68 (42 known genes and 26 expressed sequence tags) down-regulated cDNA clones in memory CD4+ T after in vitro stimulation with anti-CD3 plus anti-CD28. Interestingly, the increase in mRNA levels of up-regulated genes was greater in memory than in naive CD4+ T cells after in vitro stimulation and was higher with anti-CD3 plus anti-CD28 than with anti-CD3 alone in both memory and naive CD4+ T cells. Finally, the changes in expression of actin and cytokine genes identified by cDNA microarrays were confirmed by Northern and protein analyses. Together, we have identified ∼200 cDNA clones whose expression levels changed after activation and suggest that the level of expression of up-regulated genes is a molecular mechanism that differentiates the response of memory from naive CD4+ T cells.
Bcl-2 Targets Protein Phosphatase 1α to Bad
Authors: Ayllón Verónica , Cayla Xavier , García Alphonse , Roncal Fernando , Fernández Raul , Albar Juan Pablo , Martínez-A. Carlos , Rebollo Angelita
Published: April 2014

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1α is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1α and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1α to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1α. Disruption of Bcl-2/PP1α association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1α to Bad.
Role of Chemokines and Formyl Peptides in Pneumococcal Pneumonia-Induced Monocyte/Macrophage Recruitment
Authors: Fillion Isabelle , Ouellet Nathalie , Simard Marie , Bergeron Yves , Sato Sachiko , Bergeron Michel G.
Published: April 2014

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1α, MIP-1β, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1α with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1α alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-d-Leu-Phe-d-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.
The Differential Roles of LFA-1 and Mac-1 in Host Defense Against Systemic Infection with Streptococcus pneumoniae
Authors: Prince Joseph E. , Brayton Cory F. , Fossett Milligan C. , Durand Jennifer A. , Kaplan Sheldon L. , Smith C. Wayne , Ballantyne Christie M.
Published: April 2014

Mice deficient in CD18, which lack all four CD11 integrins, have leukocytosis and increased susceptibility to bacterial infection. To determine the effect of deficiencies in LFA-1 (CD11a/CD18) or Mac-1 (CD11b/CD18) on host defense against systemic bacterial infection, knockout mice were inoculated i.p. with Streptococcus pneumoniae. Increased mortality occurred in both LFA-1−/− (15 of 17 vs 13 of 35 in wild type (WT), p < 0.01) and Mac-1−/− (17 of 34 vs 6 of 25, p < 0.01) mice. All deaths in LFA-1−/− mice occurred after 72 h, whereas most deaths in Mac-1−/− mice occurred within 24–48 h. At 24 h, 21 of 27 Mac-1−/− mice were bacteremic, vs 15 of 25 WT (p = 0.05); no difference was observed between LFA-1−/− and WT. Increased bacteria were recovered from Mac-1−/− spleens at 2 h (p = 0.03) and 6 h (p = 0.002) and from livers (p = 0.001) by 6 h. No difference was observed at 2 h in LFA-1−/− mice, but by 6 h increased bacteria were recovered from spleens (p = 0.008) and livers (p = 0.04). Baseline and peak leukocyte counts were similar between Mac-1−/− and WT, but elevated in LFA-1−/−. At 8 h, peritoneal neutrophils were increased in Mac-1−/−, but not significantly different in LFA-1−/−. Histopathologically, at 24 h Mac-1−/− animals had bacteremia and lymphoid depletion, consistent with sepsis. LFA-1−/− mice had increased incidence of otitis media and meningitis/encephalitis vs WT at 72 and 96 h. Both Mac-1 and LFA-1 play important but distinct roles in host defense to S. pneumoniae.
Immunotherapy of Melanoma: A Dichotomy in the Requirement for IFN-γ in Vaccine-Induced Antitumor Immunity Versus Adoptive Immunotherapy
Authors: Winter Hauke , Hu Hong-Ming , McClain Kimberly , Urba Walter J. , Fox Bernard A.
Published: April 2014

The mechanism by which tumors are rejected following the adoptive transfer of tumor-specific T cells is not well characterized. Recent work has challenged the requirement for cytotoxicity mediated by either the perforin/granzyme or Fas/Fas ligand pathway in T cell-mediated tumor regression. Many reports, including ours, suggest that tumor-specific production of IFN-γ is critical for T cell-mediated tumor regression. However, in most of these studies the evidence to support the role for IFN-γ is only indirect. We have directly examined the requirement for IFN-γ using IFN-γ knockout (GKO) mice. The results show an interesting dichotomy in the requirement for IFN-γ: Antitumor immunity induced by active-specific immunotherapy (vaccination) required IFN-γ, whereas adoptive immunotherapy did not. In GKO mice vaccination with the GM-CSF gene-modified B16BL6-D5 tumor (D5-G6) failed to induce protective immunity against parental D5 tumor. However, adoptive transfer of effector T cells from GKO mice cured 100% of GKO mice with established pulmonary metastases and induced long term antitumor immunity and depigmentation of skin. Furthermore, in vivo neutralization of IFN-γ by mAb treatment or adoptive transfer into IFN-γ receptor knockout mice failed to block the therapeutic efficacy of effector T cells generated from wild-type or perforin knockout mice. Analysis of regressing metastases revealed similar infiltrates of macrophages and granulocytes in both wild-type and GKO mice. These results indicate that in this adoptive immunotherapy model, neither a direct effect on the tumor nor an indirect effect of IFN-γ through activation of myeloid or lymphoid cells is critical for therapeutic efficacy.