bluuebunny/crossref_metadata_2025 huggingface dataset:
Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation
Authors: Serreze David V. , Chapman Harold D. , Post Cristina M. , Johnson Ellis A. , Suarez-Pinzon Wilma L. , Rabinovitch Alex
Published: April 2014

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic β-cell autoreactive T cells from a Th1 (IFN-γ) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-γ, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-γ to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-γ. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among β-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.
Cutting Edge: C3, a Key Component of Complement Activation, Is Not Required for the Development of Myelin Oligodendrocyte Glycoprotein Peptide-Induced Experimental Autoimmune Encephalomyelitis in Mice
Authors: Calida Divina M. , Constantinescu Cris , Purev Enkhmaa , Zhang Guang-Xian , Ventura Elvira S. , Lavi Ehud , Rostami Abdolmohamad
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, is regarded as an experimental model for multiple sclerosis. The complement has been implicated in the pathogenesis of multiple sclerosis. To clarify the role of C in mouse EAE, we immunized mice deficient in C3 (C3−/−) and their wild-type (C3+/+) littermates with myelin oligodendrocyte glycoprotein peptide 35–55. C3−/− mice were susceptible to EAE as much as the C3+/+ mice were. No differences were found for the production of IL-2, IL-4, IL-12, TNF-α, and IFN-γ between C3+/+ and C3−/− mice. This finding shows that C3, a key component in C activation, is not essential in myelin oligodendrocyte glycoprotein peptide-induced EAE in mice.
Regulation of the CTL Response by Macrophage Migration Inhibitory Factor
Authors: Abe Riichiro , Peng Tina , Sailors Joseph , Bucala Richard , Metz Christine N.
Published: April 2014

Macrophage migration inhibitory factor (MIF) has been shown to be a pivotal cytokine that mediates host inflammatory and immune responses. Recently, immunoneutralization of MIF has been found to inhibit tumor growth in mice; however, the contributing mechanisms underlying this effect have not been well defined. We investigated whether MIF plays a regulatory role in the expression of CTL activity. In a mouse model of the CTL response using the OVA-transfected tumor cell line EL4 (EG.7), we found that cultures of splenocytes obtained from EG.7-primed mice secrete high levels of MIF following Ag stimulation in vitro. Notably, parallel splenocyte cultures treated with neutralizing anti-MIF mAb showed a significant increase in the CTL response directed against EG.7 cells compared with control mAb-treated cultures. This effect was accompanied by elevated expression of IFN-γ. Histological examination of the EG.7 tumors from anti-MIF-treated animals showed a prominent increase in both CD4+ and CD8+ T cells as well as apoptotic tumor cells, consistent with the observed augmentation of CTL activity in vivo by anti-MIF. This increased CTL activity was associated with enhanced expression of the common γc-chain of the IL-2R that mediates CD8+ T cell survival. Finally, CD8+ T lymphocytes obtained from the spleens of anti-MIF-treated EG.7 tumor-bearing mice, when transferred into recipient tumor-bearing mice, showed increased accumulation in the tumor tissue. These data provide the first evidence of an important role for MIF in the regulation and trafficking of anti-tumor T lymphocytes in vivo.
A Molecular Framework for Two-Step T Cell Signaling: Lck Src Homology 3 Mutations Discriminate Distinctly Regulated Lipid Raft Reorganization Events
Authors: Patel Viresh P. , Moran Miriana , Low Teresa A. , Miceli M. Carrie
Published: April 2014

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require “partial” TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained “complete” TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.
Mutation of the Hematopoietic Cell Phosphatase (Hcph) Gene Is Associated with Resistance to γ-Irradiation-Induced Apoptosis in Src Homology Protein Tyrosine Phosphatase (SHP)-1-Deficient “Motheaten” Mutant Mice
Authors: Hsu Hui-Chen , Shultz Leonard D. , Su Xiao , Shi Jian , Yang Ping-Ar , Relyea Melissa J. , Zhang Huang-Ge , Mountz John D.
Published: April 2014

To determine the role of Src homology protein tyrosine phosphatase (SHP-1) in the ionizing radiation-induced stress response, we analyzed the apoptotic response and cell cycle function in irradiated spleen cells of motheaten (me/me) mice. The defect in me/me mice has been attributed to mutations of the Hcph gene, which encodes SHP-1. Homozygotes develop severe systemic autoimmune and inflammatory disease, whereas heterozygotes live longer and develop hematopoietic and lymphoid malignance. Spleen cells from C57BL/6 (B6)-me/me and B6-+/+ controls were analyzed after γ-irradiation from a 137Cs source. B6-me/me cells were significantly more resistant than B6-+/+ cells to γ-irradiation-induced apoptosis exhibiting a higher LD50. The defective apoptosis response of the B6-me/me cells was exhibited by T and B cells and macrophages. Of the Bcl-2 family members analyzed, a significant difference was observed in the transcription of Bax mRNA, which was up-regulated early after irradiation in B6-+/+ cells, but not B6-me/me cells. Analysis of 3,3′-dihexyloxacarbocyanine iodide revealed resistance to the γ-irradiation-induced mitochondrial transmembrane permeability transition in the B6-me/me cells. The blocking of the cell cycle in the G0/G1 phase characteristic of the irradiated B6-+/+ cells was not observed in the B6-me/me cells. There was decreased phosphorylation of p38 mitogen-activated protein kinase and increased phosphorylation of p53 from spleen cell lysates of irradiated B6-me/me mice compared with wild-type mice. These data suggest that SHP-1 plays an important role in regulation of apoptosis and cell cycle arrest after a γ-irradiation-induced stress response.
Uterine Macrophages Express the gp49B Inhibitory Receptor in Midgestation
Authors: Matsumoto Yukie , Wang Lawrence L. , Yokoyama Wayne M. , Aso Takeshi
Published: April 2014

Mouse gp49B is an immunoreceptor tyrosine-based inhibitory motif-bearing receptor identified on mast cells and NK cells. In this report, however, we show that this receptor is expressed on macrophages accumulating in the uterine metrial gland in midgestation, along with gp49A that has a very homologous extracellular domain with gp49B but has a short cytoplasmic domain without ITIM. Culture of bone marrow cells in the conditioned medium of the metrial gland resulted in the selective proliferation of macrophages expressing both Fcγ-activating receptors and gp49B inhibitory receptor. Stimulation of macrophages with immobilized IgG, but not with anti-FcγRII/III, induced a considerable amount of TNF-α and IL-10 production, suggesting that the high-affinity receptor for IgG (FcγRI) can transmit activating signals in cytokine production of macrophages. Furthermore, coligation of gp49B with FcγRI resulted in the inhibition of TNF-α production. Thus, our data provide evidence that gp49B is an endogenous negative regulator of macrophage activation and may regulate the function of macrophages during pregnancy.
Amino Acid Substitutions in the Putative MHC Class II “Dimer of Dimers” Interface Inhibit CD4+ T Cell Activation
Authors: Lindstedt Ragnar , Monk Nicola , Lombardi Giovanna , Lechler Robert
Published: April 2014

Activation of T lymphocytes is dependent on multiple ligand-receptor interactions. The possibility that TCR dimerization contributes to T cell triggering was raised by the crystallographic analysis of MHC class II molecules. The MHC class II molecules associated as double dimers, and in such a way that two TCR (and two CD4 molecules) could bind simultaneously. Several subsequent studies have lent support to this concept, although the role of TCR cross-linking in T cell activation remains unclear. Using DRA cDNAs modified to encode two different C-terminal tags, no evidence of constitutive double dimer formation was obtained following immunoprecipitation and Western blotting from cells transiently transfected with wild-type DRB and tagged DRA constructs, together with invariant chain and HLA-DM. To determine whether MHC class II molecules contribute actively to TCR-dependent dimerization and consequent T cell activation, panels of HLA-DR1β and H2-Ek cDNAs were generated with mutations in the sequences encoding the interface regions of the MHC class II double dimer. Stable DAP.3 transfectants expressing these cDNAs were generated and characterized biochemically and functionally. Substitutions in either interface region I or III did not affect T cell activation, whereas combinations of amino acid substitutions in both regions led to substantial inhibition of proliferation or IL-2 secretion by human and murine T cells. Because the amino acid-substituted molecules were serologically indistinguishable from wild type, bound antigenic peptide with equal efficiency, and induced Ag-dependent CD25 expression indicating TCR recognition, the reduced ability of the mutants to induce full T cell activation is most likely the result of impaired double dimer formation. These data suggest that MHC class II molecules, due to their structural properties, actively contribute to TCR cross-linking.
Positive and Negative Selection of Thymocytes Depends on Lck Interaction with the CD4 and CD8 Coreceptors
Authors: Trobridge Patty A. , Forbush Katherine A. , Levin Steven D.
Published: April 2014

Considerable evidence supports a role for the Src family protein tyrosine kinase Lck in regulating multiple aspects of thymocyte development. In this report, we establish that early events in T lymphopoiesis are restored to Lck-deficient mice by provision of a transgene encoding a version of Lck that cannot interact with the coreceptors CD4 and CD8. In addition, we demonstrate that later events in thymocyte development, specifically, the processes of positive and negative selection, are compromised in mice where the only Lck available cannot associate with either CD4 or CD8. We conclude that not only is Lck activity required for positive and negative selection, but that that activity must be coupled to the CD4 and CD8 coreceptors.
Salicylates Inhibit T Cell Adhesion on Endothelium Under Nonstatic Conditions: Induction of L-Selectin Shedding by a Tyrosine Kinase-Dependent Mechanism
Authors: Gerli Roberto , Gresele Paolo , Bistoni Onelia , Paolucci Cristina , Lanfrancone Luisa , Fiorucci Stefano , Muscat Christopher , Costantini Vincenzo
Published: April 2014

Salicylates inhibit T cell adhesion to and transmigration through endothelium by preventing integrin activation induced by contact with endothelial cells. In the present study the effects of aspirin and sodium salicylate on the first steps of T cell adhesion have been analyzed in a nonstatic in vitro system. Salicylates partially reduced adhesion to activated endothelium and, in parallel, L-selectin expression on resting T cells by inducing shedding of the molecule without affecting its mRNA transcript. The role of L-selectin down-regulation in reducing T cell adhesion in this system was supported by the fact that aspirin inhibited T cell adhesion also on plastic-immobilized L-selectin ligand or when α4 integrin-mediated adhesion to endothelium was blocked by specific mAbs. In addition, preincubation of T cells with inhibitors of L-selectin shedding prevented both functional and phenotypic inhibitory effects of salicylates. The decrease in T cell adhesion and L-selectin expression seems to be dependent on intracellular calcium increase and tyrosine kinase activation, because these effects could be reversed by preincubating salicylate-treated T cells with EGTA, genistein, or tyrphostin. Finally, the infusion of aspirin into healthy volunteers induced down-regulation of L-selectin on circulating T cells. These results suggest that salicylates interfere not only with integrin activation, but also with the L-selectin-mediated first steps of T cell binding to endothelium.
Role of CD40 in a T Cell-Mediated Negative Regulation of Ig Production
Authors: Majlessi Laleh , Bordenave Guy
Published: April 2014

To investigate the possible role of CD40 in a negative regulation of Ig production, we used the mouse Ig allotype suppression model. T splenocytes from Igha/a mice are able in vivo to totally and chronically inhibit the production of IgG2ab (IgG2a from the Ighb haplotype). Accordingly, postnatal transfer of Igha/a T splenocytes into histocompatible Igha/b F1 or congenic Ighb/b mice leads to a characteristic IgG2ab suppression. The helper action of anti-IgG2ab CD4+ T cells is required for the recruitment of anti-IgG2ab CD8+ T suppression effectors. The latter use perforin (pore-forming protein, Pfp)- and/or Fas-dependent cytotoxic pathways to continuously eliminate B cells recently committed to IgG2ab production. In the present study we first showed that in vivo agonistic anti-CD40 mAb treatment of Igha/a mice, deprived of their CD4+ T cell compartment, could bypass the help of Ig allotype-specific CD4+ T cells and generate CD8+ T effector cells able to strongly inhibit IgG2ab production. This result demonstrates the usefulness of CD40 triggering in setting up an immune regulatory mechanism. Furthermore, with regard to the suppression-effector mechanism, we demonstrated that B cell CD40 expression was required for full suppression establishment via the Fas-dependent pathway. Indeed, Igha/a Pfp°/° T cells (using exclusively the Fas pathway) induced full IgG2ab suppression against Ighb/b CD40+/+ B cells, but only partial inhibition of IgG2ab production against Ighb/b CD40°/° B cells. This finding provides the first demonstration of direct involvement of B cell CD40 expression in in vivo negative control of an Ig production.
VH Gene Replacement in Thymocytes
Authors: Golub Rachel , Martin Denise , Bertrand Fred E. , Cascalho Marilia , Wabl Matthias , Wu Gillian E.
Published: April 2014

The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, VH gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for VH gene replacement. In normal mice, thymic DJH rearrangements are common, but VDJH rearrangements are not. We found intermediate products of VH replacement in double-positive CD4+CD8+ cells of the QM thymus, demonstrating that the inserted VH gene was accessible and ruling out the possibility that a VH gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no μ H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by VH gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate VH-to-VDJH joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to VH joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.
Attenuation of Very Late Antigen-5-Mediated Adhesion of Bone Marrow-Derived Mast Cells to Fibronectin by Peptides with Inverted Hydropathy to EF-Hands
Authors: Houtman René , Ten Broeke Robert , Blalock J. Edwin , Villain Matteo , Koster Andries S. , Nijkamp Frans P.
Published: April 2014

Release of allergic mediators from mast cells is enhanced by very late Ag (VLA)-5-mediated interaction of these cells with fibronectin. In this report, we show that VLA-5-mediated adhesion of bone marrow-derived mast cells to fibronectin can be induced by two different pathways: first, FcεRI clustering, which depends on calmodulin activation and extracellular Ca2+, and, second, by Mn2+ stimulation, which is independent of calmodulin activation and antagonized by Ca2+. Previous studies have shown the presence of several cation-binding domains in VLA-5 that are homologous to the calcium-binding EF-hands of calmodulin. To show a role for EF-hands of different proteins in VLA-5-mediated adhesion, we used calcium-like peptides (CALP), CALP1 and CALP2, designed to bind to EF-hands based on inverted hydropathy. CALP1 and, more potently, CALP2 inhibited FcεRI-induced adhesion to fibronectin via different mechanisms. The target for the effects of CALP1 and 2 on FcεRI-induced adhesion and degranulation was intracellular and likely involved calmodulin. Interestingly only CALP2 was able to inhibit Mn2+-induced calmodulin-independent adhesion by interfering with an extracellular target, which is probably VLA-5. We conclude that CALP1 and 2 can inhibit VLA-5-mediated adhesion of mast cells to fibronectin through binding to EF-hands of multiple proteins, and that these peptides can be used as lead compounds for the development of future therapy against allergy.
Mast Cell-Dependent B and T Lymphocyte Activation Is Mediated by the Secretion of Immunologically Active Exosomes
Authors: Skokos Dimitris , Le Panse Sophie , Villa Irène , Rousselle Jean-Claude , Peronet Roger , David Bernard , Namane Abdelkader , Mécheri Salaheddine
Published: April 2014

Mitogenic activity of bone marrow-derived mouse mast cells and mast cell lines P815 and MC/9 on B and T lymphocytes is present in their culture supernatants. To identify this activity, mast cells were incubated in serum-free medium and the supernatant was subjected to differential centrifugation, which resulted in two fractions, the hypodense and dense fraction (pellet). When analyzed for their mitogenic activity on spleen cells, all activity was found to be associated with the dense fraction. Electron microscopy studies revealed the presence in this fraction of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induced blast formation, proliferation, as well as IL-2 and IFN-γ production, but no detectable IL-4. Similar data were obtained by injecting exosomes into naive mice. In contrast to mast cell lines, a pretreatment with IL-4 is required for bone marrow-derived mast cells to secrete active exosomes. Structurally, exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These findings indicate that mast cells can represent a critical component of the immunoregulatory network through secreted exosomes that display mitogenic activity on B and T lymphocytes both in vitro and in vivo.
CCR7 Expression and Memory T Cell Diversity in Humans
Authors: Campbell James J. , Murphy Kristine E. , Kunkel Eric J. , Brightling Christopher E. , Soler Dulce , Shen Zhimin , Boisvert Judie , Greenberg Harry B. , Vierra Mark A. , Goodman Stuart B. , Genovese Mark C. , Wardlaw Andrew J. , Butcher Eugene C. , Wu Lijun
Published: April 2014

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag α4β7 integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.
Prostaglandin E2 Suppressed IL-15-Mediated Human NK Cell Function Through Down-Regulation of Common γ-Chain
Authors: Joshi Pratibha C. , Zhou Xinchun , Cuchens Marvin , Jones Quintus
Published: April 2014

NK cell function is regulated by cytokines and certain biochemical mediators in a positive or negative manner. This study was performed to investigate the suppressive effects of PGE2 on IL-15-activated human NK cell function. Purified NK cells were cultured with 200 ng/ml IL-15 for 2 days in the presence or absence of 10–200 ng/ml PGE2. PGE2 significantly suppressed NK cell-mediated cytotoxicity and IFN-γ production at the secretional and the transcriptional levels. We also evaluated the effect of PGE2 on the IL-15R complex that consists of IL-2Rβ, common γ-chain (γc-chain), and a specific chain IL-15Rα. Percentage of positive cells and number of binding sites for γc-chain were significantly increased after IL-15 treatment; however, a substantial decrease was observed with PGE2 cotreatment. In contrast, constitutive expression of IL-2Rβ was significantly decreased after IL-15 treatment, with no change detected in the presence of PGE2. At the transcriptional level, neither IL-15 nor PGE2 had significant effects on the expression of β- or γc-chains. There was a 3-fold increase in the expression of IL-15Rα at the transcriptional level that peaked at 8 h after IL-15 treatment; however, PGE2 had no significant effect. Suppression of NK function by PGE2 was not due to the endogenous production of IL-4, IL-10, or TGF-β1 by NK cells. These results suggest that down-regulation of surface expression of γc-chain on NK cells may be one mechanism through which PGE2 mediates suppression of IL-15-activated NK cell function.
The Targeting of Somatic Hypermutation Closely Resembles That of Meiotic Mutation
Authors: Oprea Mihaela , Cowell Lindsay G. , Kepler Thomas B.
Published: April 2014

We have compared the microsequence specificity of mutations introduced during somatic hypermutation (SH) and those introduced meiotically during neutral evolution. We have minimized the effects of selection by studying nonproductive (hence unselected) Ig V region genes for somatic mutations and processed pseudogenes for meiotic mutations. We find that the two sets of patterns are very similar: the mutabilities of nucleotide triplets are positively correlated between the somatic and meiotic sets. The major differences that do exist fall into three distinct categories: 1) The mutability is sharply higher at CG dinucleotides under meiotic but not somatic mutation. 2) The complementary triplets AGC and GCT are much more mutable under somatic than under meiotic mutation. 3) Triplets of the form WAN (W = T or A) are uniformly more mutable under somatic than under meiotic mutation. Nevertheless, the relative mutabilities both within this set and within the SAN (S = G or C) triplets are highly correlated with those under meiotic mutation. We also find that the somatic triplet specificity is strongly symmetric under strand exchange for A/T triplets as well as for G/C triplets in spite of the strong predominance of A over T mutations. Thus, we suggest that somatic mutation has at least two distinct components: one that specifically targets AGC/GCT triplets and another that acts as true catalysis of meiotic mutation.
Identification of MHC Class II-Restricted Peptide Ligands, Including a Glutamic Acid Decarboxylase 65 Sequence, that Stimulate Diabetogenic T Cells from Transgenic BDC2.5 Nonobese Diabetic Mice
Authors: Judkowski Valeria , Pinilla Clemencia , Schroder Kim , Tucker Lee , Sarvetnick Nora , Wilson Darcy B.
Published: April 2014

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet β cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR α- and β-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528–539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC50 > 100 μM), a larger 16-mer fragment, 526–541, shows activity in the low micromolar range (EC50 = 2.3 μM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.
The Serine/Threonine Phosphatase, PP2A: Endogenous Regulator of Inflammatory Cell Signaling
Authors: Shanley Thomas P. , Vasi Niti , Denenberg Alvin , Wong Hector R.
Published: April 2014

We have investigated the regulation of kinases and phosphatases in early gene activation in monocytes because these cells are implicated in the pathogenesis of acute inflammatory states, such as sepsis and acute lung injury. One early gene up-regulated by endotoxin is c-Jun, a member of the activating protein (AP) family. C-Jun is phosphorylated by c-Jun N-terminal kinase (JNK) and associates with c-Fos to form the AP-1 transcriptional activation complex that can drive cytokine expression. Inhibition of the serine/threonine phosphatase, PP2-A, with okadaic acid resulted in a significant increase in JNK activity. This finding was associated with increased phosphorylation of c-Jun, AP-1 transcriptional activity, and IL-1β expression. Activation of PP2A inhibited JNK activity and JNK coprecipitated with the regulatory subunit, PP2A-Aα, supporting the conclusion that PP2A is a key regulator of JNK in the context of an inflammatory stimulus.
Chemokine Receptor Expression and Function in CD4+ T Lymphocytes with Regulatory Activity
Authors: Sebastiani Silvia , Allavena Paola , Albanesi Cristina , Nasorri Francesca , Bianchi Giancarlo , Traidl Claudia , Sozzani Silvano , Girolomoni Giampiero , Cavani Andrea
Published: April 2014

We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4+ T cells (ThIL-10) releasing high levels of IL-10, low IFN-γ, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting ThIL-10, including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting ThIL-10, in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1β, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1α, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated ThIL-10 exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently ThIL-10 than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory ThIL-10 lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated ThIL-10 cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.
Cutting Edge: Granzyme B Proteolysis of a Neuronal Glutamate Receptor Generates an Autoantigen and Is Modulated by Glycosylation
Authors: Gahring Lorise C. , Carlson Noel G. , Meyer Erin L. , Rogers Scott W.
Published: April 2014

Autoimmune processes are initiated when tolerance to self-proteins fails to be established or maintained and immune cells are stimulated by self-Ags. Although intracellular autoantigens are common, the origin of extracellular autoantigens is poorly defined and possibly more dangerous. In this study, we considered a mechanism for the origin of an extracellular autoantigen from the neuronal glutamate receptor subunit 3 (GluR3) in Rasmussen’s encephalitis, a severe form of pediatric epilepsy. We demonstrate that specific cleavage of GluR3 by granzyme B (GB), a serine protease released by activated immune cells, can generate the GluR3B autoantigenic peptide, but only if an internal N-linked glycosylation sequon within the GluR3-GB recognition sequence (ISND*S) is not glycosylated. However, this N-glycon sequon while glycosylated normally is inefficiently used and glycosylation can fail. These results suggest that GB/N-glycon sites may escape normal tolerance mechanisms and contribute to autoantibody-mediated immune diseases.
Cutting Edge: Dendritic Cells Are Sufficient to Cross-Present Self-Antigens to CD8 T Cells In Vivo
Authors: Kurts Christian , Cannarile Michael , Klebba Ina , Brocker Thomas
Published: April 2014

The mechanism of cross-presentation enables professional APCs to induce CD8 T cell-mediated immune responses against exogenous Ags. Through this mechanism, APCs can induce either immunity against infectious pathogens or tolerance against self-Ag residing in extralymphatic locations. An unanswered question in this field concerns the identity of the cross-presenting APC. All major classes of professional APCs, particularly dendritic cells, macrophages, and B cells, have previously been shown to be able to cross-present Ags in vitro. In the present study, we have created transgenic mice where MHC class I expression is driven selectively in dendritic cells and provide direct in vivo evidence that dendritic cells are sufficient to cross-present exogenous self-Ags and induce Ag-specific cell division of CD8-positive T cells.
Visualization of Syk-Antigen Receptor Interactions Using Green Fluorescent Protein: Differential Roles for Syk and Lyn in the Regulation of Receptor Capping and Internalization
Authors: Ma Haiyan , Yankee Thomas M. , Hu Jianjie , Asai David J. , Harrison Marietta L. , Geahlen Robert L.
Published: April 2014

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.
Stochastic Regulation of Cell Migration from the Efferent Lymph to Oxazolone-Stimulated Skin
Authors: West Charles A. , He Chufa , Su Mei , Rawn James , Swanson Scott , Hay John B. , Mentzer Steven J.
Published: April 2014

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks ∼96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.
Murine B1 B Cells Require IL-5 for Optimal T Cell-Dependent Activation
Authors: Erickson Loren D. , Foy Teresa M. , Waldschmidt Thomas J.
Published: April 2014

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L−/− mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.
p59fyn(Fyn) Promotes the Survival of Anergic CD4−CD8− αβ TCR+ Cells but Negatively Regulates Their Proliferative Response to Antigen Stimulation
Authors: Utting Oliver , Priatel John J. , Teh Soo-Jeet , Teh Hung-Sia
Published: April 2014

T cell anergy is characterized by alterations in TCR signaling that may play a role in controlling the unresponsiveness of the anergic cell. We have addressed questions regarding the importance of the Src kinase p59fyn (Fyn) in this process by using Fyn null mice. We demonstrate that a mature population of CD4−CD8− αβ TCR+ anergic T cells lacking Fyn have a substantial recovery of their proliferation defect in response to Ag stimulation. This recovery cannot be explained by ameliorated production of IL-2, and the improved proliferation correlates with an enhanced ability of the Fyn−/− anergic T cells to up-regulate the high affinity IL-2 receptor. We also observe that anergic CD4−CD8− αβ TCR+ T cells have a heightened survival ability that is partially dependent on the elevated levels of Fyn and IL-2 receptor β-chain expressed by these cells. The enhanced survival correlates with an increased capacity of the anergic cells to respond to IL-15. We conclude that Fyn plays an important role in aspects of T cell anergy pertaining to TCR signaling and to cell survival.
CTLA4Ig-Induced Linked Regulation of Allogeneic T Cell Responses
Authors: Lee Richard S. , Rusche James R. , Maloney Michaella E. , Sachs David H. , Sayegh Mohamed H. , Madsen Joren C.
Published: April 2014

The mechanisms by which CTLA4Ig exerts its powerful immunomodulatory effects are not clear. We show here that CTLA4Ig can induce linked regulation of allogeneic porcine T cell responses in vitro. Naive miniature swine SLAdd T cells were rendered hyporesponsive to specific allogeneic Ag after coculturing with MHC-mismatched SLAcc stimulators in the presence of CTLA4Ig. These Ag-specific hyporesponsive T cells were subsequently able to actively inhibit the allogeneic responses of naive syngeneic T cells in an MHC-linked fashion, as the responses of naive SLAdd responders against specific SLAcc and (SLAac)F1 stimulators were inhibited, but allogeneic responses against a 1:1 mixture of SLAaa (Ia, IIa) and SLAcc (Ic, IIc) were maintained. This inhibition could be generated against either class I or class II Ags, was radiosensitive, and required cell-cell contact. Furthermore, the mechanism of inhibition was not dependent upon a deletional, apoptotic pathway, but it was reversed by anti-IL-10 mAb. These data suggest that CTLA4Ig-induced inhibition of naive allogeneic T cell responses can be mediated through the generation of regulatory T cells via an IL-10-dependent mechanism.
TNF-α-Converting Enzyme Cleaves the Macrophage Colony-Stimulating Factor Receptor in Macrophages Undergoing Activation
Authors: Rovida Elisabetta , Paccagnini Alessandro , Del Rosso Mario , Peschon Jacques , Dello Sbarba Persio
Published: April 2014

We previously reported that macrophage activators such as LPS, IL-2, and IL-4 down-modulate the M-CSFR via a mechanism involving protein kinase C and phospholipase C. In this study, we showed that M-CSFR is shed from macrophage surface and identified the protease responsible for M-CSFR cleavage and down-modulation. The shedding of M-CSFR elicited by phorbol esters (tetradecanoylphorbol myristate acetate (TPA)) or LPS in murine BAC.1-2F5 macrophages was prevented by cation chelators, as well as hydroxamate-based competitive inhibitors of metalloproteases. We found that the protease cleaving M-CSFR is a transmembrane enzyme and that its expression is controlled by furin-like serine endoproteases, which selectively process transmembrane metalloproteases. M-CSFR down-modulation was inhibited by treating cells in vivo, before TPA stimulation, with an Ab raised against the extracellular, catalytic domain of proTNF-converting enzyme (TACE). TACE expression was confirmed in BAC.1-2F5 cells and found inhibited after blocking furin-dependent processing. Using TACE-negative murine Dexter-ras-myc cell monocytes, we found that in these cells TPA is unable to down-modulate M-CSFR expression. These data indicated that TACE is required for the TPA-induced M-CSFR cleavage. The possibility that the cleavage is indirectly driven by TACE via the release of TNF was excluded by treating cells in vivo with anti-TNF Ab. Thus, we concluded that TACE is the protease responsible for M-CSFR shedding and down-modulation in mononuclear phagocytes undergoing activation. The possible physiological relevance of this mechanism is discussed.
Enhancement of Human Cord Blood CD34+ Cell-Derived NK Cell Cytotoxicity by Dendritic Cells
Authors: Yu Ying , Hagihara Masao , Ando Kiyoshi , Gansuvd Balgansuren , Matsuzawa Hideyuki , Tsuchiya Takahide , Ueda Yoko , Inoue Hiroyasu , Hotta Tomomitsu , Kato Shunichi
Published: April 2014

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34+ cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-γ production. However, NK cell cytotoxicity and IFN-γ production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-γ Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-γ production plays an important role in Fas-mediated cytotoxicity.
Extracellular ATP Induces a Distorted Maturation of Dendritic Cells and Inhibits Their Capacity to Initiate Th1 Responses
Authors: la Sala Andrea , Ferrari Davide , Corinti Silvia , Cavani Andrea , Di Virgilio Francesco , Girolomoni Giampiero
Published: April 2014

Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1α, IL-1β, TNF-α, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA+ T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-γ and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-α and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses.