Chemokines and their receptors: trafficking cues for Th1 and Th2 cells.
Explore the source record for details and available documents.
Chemokines↗
Biomedical subjects
F Sinigaglia
Publications and source records attributed to F Sinigaglia.
Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments.
The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.
Actins↗
Upregulation of integrin alpha6/beta1 and chemokine receptor CCR1 by interleukin-12 promotes the migration of human type 1 helper T cells.
CD4(+) T helper 1 (Th1) cells and Th2 cells are distinguished based on the pattern of cytokines they are able to produce. Selectin ligands and chemokine receptors are differentially expressed in Th1 and Th2 cells, providing a basis for tissue-specific recruitment of helper T-cell subsets. However, the modes and mechanisms regulating tissue-specific localization of Th1 and Th2 cells are still largely unknown. Here, we show the preferential expression on Th1 cells of the integrin alpha6/beta1, which is distinctly regulated by the Th1-inducing cytokines interleukin-12 (IL-12) and interferon-alfa (IFN-alpha). The pattern of integrin alpha6/beta1 regulation closely mirrors that of the chemokine receptor CCR1. Analysis of signal transducer and activator of transcription 4 (Stat4) activation by IL-12 and IFN-alpha shows distinct signaling kinetics by these cytokines, correlating with the pattern of CCR1 and integrin alpha6/beta1 expression. Unlike IFN-alpha, the ability of IL-12 to generate prolonged intracellular signals appears to be critical for inducing integrin alpha6/beta1 upregulation in Th1 cells. The expression and upregulation of CCR1 and alpha6/beta1 integrin promotes the migration of Th1 cells. These findings suggest that the exquisite regulation of integrin alpha6/beta1 and CCR1 may play an important role in tissue-specific localization of Th1 cells.
Adjuvants, Immunologic↗
Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction.
Although atopic allergy affects </=20% of the total population, the relationship between the protein structure and immunogenic activity of the allergens is still largely unknown. We observed that group 5 grass allergens are characterized by repeated structural motifs. Using a new algorithm, TEPITOPE, we predicted promiscuous HLA-DR ligands within the repeated motifs of the Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.
Allergens↗
Hydrolysis of NADP+ by platelet CD38 in the absence of synthesis and degradation of cyclic ADP-ribose 2'-phosphate.
CD38 is a multifunctional cell surface ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose from NAD+ and its hydrolysis to ADP-ribose. In this work, we investigated the metabolism of NADP+ by CD38 expressed on human platelets. Incubation of either platelet membranes or intact cells with NADP+ resulted in the rapid and time-dependent accumulation of ADP-ribose 2'-phosphate that paralleled the consumption of the substrate. However, under the same conditions, synthesis of cyclic ADP-ribose 2'-phosphate was not observed. By immunoprecipitation experiments, we identified CD38 as the enzyme responsible for the observed NADP+ glycohydrolase activity. The lack of detection of cyclic ADP-ribose 2'-phosphate was not due to its rapid hydrolysis, since direct incubation of platelet membranes with cyclic ADP-ribose 2'-phosphate did not result in the formation of ADP-ribose 2'-phosphate. By contrast, the same membrane samples expressed a significant ability to hydrolyze cyclic ADP-ribose to ADP-ribose. The absence of cyclic ADP-ribose 2'-phosphate hydrolase activity was also confirmed using high concentrations of substrate and by analysing both intact Jurkat T-lymphocytes and immunoprecipitated CD38. These results indicate that CD38, which is a multifunctional enzyme towards NAD+, displays exclusively a NADP+ glycohydrolase activity and is unable to catalyze both the synthesis and the hydrolysis of cyclic ADP-ribose 2'-phosphate.
ADP-ribosyl Cyclase↗
Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves FcgammaII receptor-mediated protein tyrosine phosphorylation.
Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.
Blood Platelets↗
Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo.
Great attention has been placed on the possibility of distinguishing Th1 from Th2 cells on the basis of differential expression of surface receptors. We have recently shown that the differential expression of the IL-12R beta 2 chain in Th1 and Th2 cells, as measured at the mRNA level, accounts for an important regulatory mechanism in the differentiation of the two cell subsets. In this study, we identify IL-12R expression at the protein level. We have generated an anti-IL-12R beta 2-specific mAb and analyzed IL-12R beta 2 expression on polarized Th cell populations generated in vitro and on T cells derived from patients with Th1- or Th2-mediated inflammatory conditions. Although IL-12R beta 2 was absent in freshly isolated PBMC and in cord blood cells, we were able to detect IL-12R beta 2 expression selectively in differentiated Th1 and T cytotoxic 1, but not Th2 or T cytotoxic 2 cells. In the presence of IL-12, cell surface expression of the IL-12R beta 2 subunit was readily detected on T cells after 24 h, reached the maximum at day 5, and declined thereafter. Most importantly, the anti-IL-12R beta 2 mAb recognizes lung T cells from patients with sarcoidosis, a disease characterized by a typical cell-mediated, Th1-type inflammatory response. In contrast, IL-12R beta 2 was absent in lung T cells from patients with allergic asthma, a disease characterized by a Th2-type inflammatory response. The mAb reported in this study should represent a powerful tool to investigate the role of Th1 and Th2 cells in inflammatory conditions and to monitor therapies aimed at altering the balance of Th cell subsets.
Adult↗
Interleukin-12 induces expression of interferon regulatory factor-1 via signal transducer and activator of transcription-4 in human T helper type 1 cells.
IRF-1-deficient mice show a striking defect in the development of T helper 1 (Th1) cells. In the present report, we investigate the expression of IRF-1 during differentiation of human T helper cells. No significant differences of IRF-1 mRNA expression were found in established Th1 and Th2 cells; however, interleukin 12 (IL-12) induced a strong up-regulation of IRF-1 transcripts in Th1 but not in Th2 cells. We demonstrate that IL-12-induced up-regulation of IRF-1 is mediated by signal transducer and activator of transcription-4, which binds to the interferon (IFN)-gamma-activated sequence present in the promoter of the IRF-1 gene. Strong IL-12-dependent activation of a reporter gene construct containing the IRF-1 IFN-gamma-activated sequence element provides further evidence for the key role of signal transducer and activator of transcription-4 in the IL-12-induced up-regulation of IRF-1 transcripts in T cells. IRF-1 expression was strongly induced after stimulation of naive CD4(+) T cells via the T cell receptor, irrespective of the cytokines present at priming, indicating that this transcription factor does not play a major role in initiating a Th1-specific transcriptional cascade in differentiating helper T cells. However, our finding that IRF-1 is a target gene of IL-12 suggests that some of the IL-12-induced effector functions of Th1 cells may be mediated by IRF-1.
Activating Transcription Factor 4↗
Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11.
In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4(+) T cells from the blood of a healthy donor. CD4(+) T cells strongly recognized MAGE-3281-295 and, to a lesser extent, MAGE-3141-155 and MAGE-3146-160. Moreover, CD4(+) T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4(+) T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3-positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4(+) T cells recognized MAGE-3281-295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4(+) T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4(+) T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4(+) T cell epitopes.
Amino Acid Sequence↗
Identification of destabilizing residues in HLA class II-selected bacteriophage display libraries edited by HLA-DM.
HLA-DM (DM) functions as a peptide editor by catalyzing the release of class II-associated invariant chain peptides (CLIP) and other unstable peptides, thus supporting the formation of stable class II-peptide complexes for presentation. To investigate the general features that determine the DM susceptibility of HLA-DR1/peptide complexes, we generated a large DM-sensitive peptide repertoire from an M13 bacteriophage display library using a novel double selection protocol: we selected bacteriophage capable of binding to DR1 molecules and, subsequently, we enriched DR1-bound bacteriophage susceptible to elution by purified DM molecules. Sequence and mutational analyses of the DR1/DM double-selected peptides revealed that the amino acids Gly and Pro play a destabilizing role in the dissociation kinetics of DR1 ligands. This observation was confirmed also in natural peptide sequences such as CLIP 89-101, HA 307-319 and bovine collagen II (CII) 261-273. Our results demonstrate that DM susceptibility does not only depend on the number and nature of anchor residues, or the peptide length. Instead, less obvious sequence characteristics play a major role in the DM editing process and ultimately in the composition of peptide repertoires presented to T cells.
Amino Acid Sequence↗
Molecular mechanisms of T helper cell differentiation and tissue-specific migration.
Explore the source record for details and available documents.
Animals↗
Substitution of aspartic acid at beta57 with alanine alters MHC class II peptide binding activity but not protein stability: HLA-DQ (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303).
In class II major histocompatibility complex (MHC) proteins, residue beta57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Asp(beta)57 participates in a conserved salt bridge that bridges the alpha and beta subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Asp(beta)57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303) differing only in having aspartic acid or alanine at position beta57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alabeta57 proteins display slightly different secondary structures relative to their Aspbeta57 counterparts. A set of three peptides shows different binding affinities for DQ(alpha1*0201, beta1*0302) relative to DQ(alpha1*0201, beta1*0303). We propose that substitution of Asp(beta)57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Asp(beta)57 DQ proteins.
Alanine↗
Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices.
Most pockets in the human leukocyte antigen-group DR (HLA-DR) groove are shaped by clusters of polymorphic residues and, thus, have distinct chemical and size characteristics in different HLA-DR alleles. Each HLA-DR pocket can be characterized by "pocket profiles," a quantitative representation of the interaction of all natural amino acid residues with a given pocket. In this report we demonstrate that pocket profiles are nearly independent of the remaining HLA-DR cleft. A small database of profiles was sufficient to generate a large number of HLA-DR matrices, representing the majority of human HLA-DR peptide-binding specificity. These virtual matrices were incorporated in software (TEPITOPE) capable of predicting promiscuous HLA class II ligands. This software, in combination with DNA microarray technology, has provided a new tool for the generation of comprehensive databases of candidate promiscuous T-cell epitopes in human disease tissues. First, DNA microarrays are used to reveal genes that are specifically expressed or upregulated in disease tissues. Second, the prediction software enables the scanning of these genes for promiscuous HLA-DR binding sites. In an example, we demonstrate that starting from nearly 20,000 genes, a database of candidate colon cancer-specific and promiscuous T-cell epitopes could be fully populated within a matter of days. Our approach has implications for the development of epitope-based vaccines.
Alleles↗
Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.
We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.
Amino Acid Sequence↗
Regulation of the IL-12/IL-12R axis: a critical step in T-helper cell differentiation and effector function.
Interleukin (IL)-12 is required for the development of T-helper (Th) 1 cells, which have been shown to be important for protective cell-mediated immune responses against a variety of intracellular pathogens. Recent studies have clarified the sources and the regulation of IL-12 production leading to Th1 development against microbes. Expression of IL-12R is necessary for maintaining IL-12 responsiveness and controlling Th1 lineage commitment. Advances in this area have included a broader understanding of the factors involved in the regulation of the IL-12R beta 2 signaling component. Expression of this receptor subunit in humans is critically influenced by IL-12 and type I interferons. IL-12 signaling results in STAT4 activation and interferon (IFN)-gamma production. Recent evidence suggests that IL-12 also modulates a number of genes involved in leukocyte trafficking. Thus, IL-12 is not only an important proinflammatory cytokine, which induces production of IFN-gamma and subsequent activation of phagocytic cells but also plays a major role in regulating the migration and proper positioning of effector cells.
Animals↗
Envelope-dependent restriction of human immunodeficiency virus type 1 spreading in CD4(+) T lymphocytes: R5 but not X4 viruses replicate in the absence of T-cell receptor restimulation.
The human immunodeficiency virus (HIV) replicates in activated CD4(+) T lymphocytes. However, only CD4(+) Th2 and Th0, but not Th1, CD4(+) T-cell clones have been reported to efficiently support HIV-1 replication. This dichotomous pattern was further investigated in the present study in Th1, Th2, or Th0 cell lines derived from umbilical human cord blood and in T-cell clones obtained from the peripheral blood mononuclear cells (PBMC) of healthy adults. Both primary and laboratory-adapted HIV-1 strains with CCR5 as the exclusive entry coreceptor (R5 viruses) efficiently replicated in Th1, Th2, and Th0 cells. In sharp contrast, CXCR4-dependent (X4) viruses poorly replicated in both polarized and unpolarized CD4(+) T cells, including adults' PBMC infected several days after mitogenic stimulation. Unlike the X4 HIV-1(NL4-3), a chimera in which the env gene had been replaced with that of the R5 HIV-1(NL(AD8)), efficiently replicated in both Th1 and Th2 cells. This X4-dependent restriction of HIV replication was not explained by either the absence of functional CXCR4 on the cell surface or by the inefficient viral entry and reverse transcription. T-cell receptor stimulation by anti-CD3 monoclonal antibodies fully rescued X4 HIV-1 replication in both Th1 and Th2 cells, whereas it did not alter the extent and kinetics of R5 HIV-1 spreading. Thus, R5 HIVs show a replicative advantage in comparison to X4 viruses in their ability to efficiently propagate among suboptimally activated T lymphocytes, regardless of their polarized or unpolarized functional profiles. This observation may help to explain the absolute predominance of R5 HIVs over X4 viruses observed after viral transmission and during early-stage disease.
Adult↗
Clustering of integrin alphaIIb-beta3 differently regulates tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK in concanavalin A-stimulated platelets.
Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.
Blood Platelets↗
The role of Stat4 in species-specific regulation of Th cell development by type I IFNs.
Type I IFNs (IFN-alpha/beta), in addition to IL-12, have been shown to play an important role in the differentiation of human, but not mouse, Th cells. We show here that IFN-alpha/beta act directly on human T cells to drive Th1 development, bypassing the need for IL-12-induced signaling, whereas IFN-alpha cannot substitute IL-12 for mouse Th1 development. The molecular basis for this species specificity is that IFN-alpha/beta activate Stat4 in differentiating human, but not mouse, Th cells. Unlike IL-12, which acts only on Th1 cells, IFN-alpha/beta can activate Stat4 not only in human Th1, but also in Th2 cells. However, restimulation of human Th2 lines and clones in the presence of IFN-alpha does not induce the production of IFN-gamma. These results suggest that activation of Stat4, which is necessary for the differentiation of naive T cells into polarized Th1 cells, is not sufficient to induce phenotype reversal of human Th2 cells.
Animals↗