bluuebunny/crossref_metadata_2025 huggingface dataset:
MHC Class II Derived Recombinant T Cell Receptor Ligands Protect DBA/1LacJ Mice from Collagen-Induced Arthritis
Authors: Huan Jianya , Kaler Laurie J. , Mooney Jeffery L. , Subramanian Sandhya , Hopke Corwyn , Vandenbark Arthur A. , Rosloniec Edward F. , Burrows Gregory G. , Offner Halina
Published: April 2014

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the α1 and β1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257–270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257–270 molecule could systemically reduce proinflammatory IL-17 and IFN-γ production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257–270 molecule could also selectively inhibit IL-1β, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.
Recognition and Degradation of Myelin Basic Protein Peptides by Serum Autoantibodies: Novel Biomarker for Multiple Sclerosis
Authors: Belogurov Alexey A. , Kurkova Inna N. , Friboulet Alain , Thomas Daniel , Misikov Viktor K. , Zakharova Maria Yu. , Suchkov Sergey V. , Kotov Sergey V. , Alehin Alexander I. , Avalle Bérangère , Souslova Ekaterina A. , Morse Herbert C. , Gabibov Alexander G. , Ponomarenko Natalia A.
Published: April 2014

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant “epitope library” covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48–70 and 85–170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43–68 and 146–170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81–103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.
Expanded Population of Activated Antigen-Engaged Cells within the Naive B Cell Compartment of Patients with Systemic Lupus Erythematosus
Authors: Chang Nan-Hua , McKenzie Tamara , Bonventi Gabriel , Landolt-Marticorena Carolina , Fortin Paul R. , Gladman Dafna , Urowitz Murray , Wither Joan E.
Published: April 2014

Polyclonal B cell activation is a well-described feature of systemic lupus erythematosus (SLE), but the immune mechanisms leading to this activation are unclear. To gain insight into these processes, we extensively characterized the activated peripheral blood B cell populations in SLE. PBMC from lupus patients and healthy controls were stained with various combinations of conjugated Ab to identify distinct peripheral B cell subsets, and activation was assessed by measurement of forward scatter and CD80 or CD86 expression using flow cytometry. SLE patients had altered proportions of several B cell subsets, many of which demonstrated increased activation as assessed by forward scatter. This activation occurred at an early developmental stage, as B cells in the transitional (T2) stage were already significantly larger than those seen in controls. Increased proportions of CD80- or CD86-expressing cells were also seen in multiple B cell subsets, with the most striking differences observed in the naive CD27−CD23+ population. Within the CD23+ subset, increased costimulatory molecule expression was most pronounced in an IgD+IgMlow population, suggesting that activation follows Ag engagement. Although controls also had IgD+IgMlowCD23+ cells, they were reduced in number and not activated. Thus, there is an altered response to Ig receptor engagement with self-Ags in lupus.
Blockade of CD200 in the Presence or Absence of Antibody Effector Function: Implications for Anti-CD200 Therapy
Authors: Kretz-Rommel Anke , Qin Fenghua , Dakappagari Naveen , Cofiell Roxanne , Faas Susan J. , Bowdish Katherine S.
Published: April 2014

CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.
The Kidney-Renal Lymph Node-System Contributes to Cross-Tolerance against Innocuous Circulating Antigen
Authors: Lukacs-Kornek Veronika , Burgdorf Sven , Diehl Linda , Specht Sabine , Kornek Miroslaw , Kurts Christian
Published: April 2014

Soluble Ags devoid of inflammatory stimuli, derived for example from self-serum or food proteins, induce T cell tolerance, predominantly in the spleen. In this study, we describe an additional role of the kidney-renal LN (rLN) system in tolerogenic presentation of circulating soluble Ags. Protein below albumin molecular mass constitutively passed the kidney glomerular filter and was concentrated in the tubular compartment. Enriched filterable Ag was endocytosed by kidney dendritic cells (kDCs). Simultaneously, it was transported cell independently within 2 min to DCs resident in rLNs. These DC phenotypically differed from kDCs carrying filterable Ag, and used a distinct mechanism, mannose receptor-mediated endocytosis, to internalize Ag. They activated specific CD8+ T cells, which subsequently proliferated without producing effector cytokines or developing cytotoxic activity, showed a curtailed lifespan and signs of apoptosis. Such T cell tolerization was independent of steady-state migratory kDC, because it occurred also when nephrectomy was performed soon after Ag injection. These findings demonstrate that the kidney dispatches concentrated blood-borne Ags to the rLNs, where they are captured by resident DCs, resulting in CD8+ T cell cross-tolerance. This mechanism may contribute to avoiding immunity against innocuous circulating protein Ags below albumin size.
Prostaglandin D2 Inhibits the Production of IFN-γ by Invariant NK T Cells: Consequences in the Control of B16 Melanoma
Authors: Torres David , Paget Christophe , Fontaine Josette , Mallevaey Thierry , Matsuoka Toshiyuki , Maruyama Takayuki , Narumiya Shuh , Capron Monique , Gosset Philippe , Faveeuw Christelle , Trottein François
Published: April 2014

Invariant NK T (iNKT) cells are a subset of innate/memory lymphocytes that recognize lipid Ags presented by CD1d-expressing APCs such as dendritic cells (DCs). Upon primary stimulation through their TCR, iNKT cells promptly produce large amounts of IFN-γ and/or IL-4 that play critical roles in the regulation of innate and adaptive immune responses. To date, the role of environmental factors on iNKT cell functions has been poorly investigated. In this study, we addressed the question of whether PGD2, a potent eicosanoid lipid mediator involved in immune responses and inflammation, could be important in DC/iNKT cell cross-talk. We show that PGD2 dramatically reduced the production of IFN-γ, but not IL-4, by iNKT cells in response to the superagonist α-galactosylceramide (α-GalCer) both in vitro and in vivo. This effect is mediated by the D prostanoid receptor 1 (DP1) expressed by DCs and iNKT cells and requires protein kinase A activation. We also report that PGD2 and BW245C (a selective DP1 agonist) reduce the protective effects of α-GalCer in B16F10-induced melanoma metastasis, an effect that depends on IFN-γ production by iNKT cells. As a whole, these data reveal novel pathways regulating iNKT cell biologic functions and confirm the immunoregulatory roles of PGD2 on the innate response.
CpG Drives Human Transitional B Cells to Terminal Differentiation and Production of Natural Antibodies
Authors: Capolunghi Federica , Cascioli Simona , Giorda Ezio , Rosado Maria Manuela , Plebani Alessandro , Auriti Cinzia , Seganti Giulio , Zuntini Roberta , Ferrari Simona , Cagliuso Maria , Quinti Isabella , Carsetti Rita
Published: April 2014

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.
Endosomal Translocation of CpG-Oligodeoxynucleotides Inhibits DNA-PKcs-Dependent IL-10 Production in Macrophages
Authors: Yotsumoto Satoshi , Saegusa Kazuharu , Aramaki Yukihiko
Published: April 2014

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODNs) function as powerful immune adjuvants by activating macrophages, dendritic cells, and B cells. However, the molecular recognition mechanism that initiates signaling in response to CpG-ODN has not fully been identified. We show in this study that peritoneal macrophages from SCID mice having mutations in the catalytic subunit of DNA-protein kinase (DNA-PKcs) were almost completely defective in the production of IL-10 and in ERK activation when treated with CpG-ODN. In contrast, IL-12 p70 production significantly increased. Furthermore, small interfering RNA (siRNA)-mediated knockdown of DNA-PKcs expression in the mouse monocyte/macrophage cell line RAW264.7 led to reduced IL-10 production and ERK activation by CpG-ODN. IL-10 and IL-12 p70 production, but not ERK activation, are blocked by chloroquine, an inhibitor of endosomal acidification. Endosomal translocation of CpG-ODN in a complex with cationic liposomes consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (CpG-DOTAP-liposomes) decreased IL-10 production and ERK activation, whereas the endosomal escape of CpG-ODN in a complex with cationic liposomes consisting of DOTAP and dioleyl-phosphatidylethanolamine (DOPE) (CpG-DOTAP/DOPE-liposomes) increased. In contrast, IL-12 p70 production was increased by CpG-DOTAP-liposomes and decreased by CpG-DOTAP/DOPE-liposomes. IL-10 production induced by CpG-DOTAP/DOPE-liposomes was not observed in macrophages from SCID mice. Thus, our findings suggest that DNA-PKcs in the cytoplasm play an important role in CpG-ODN-induced production of IL-10 in macrophages. In addition, DNA-PKcs-mediated production of IL-10 and IL-12 p70 can be regulated by manipulating the intracellular trafficking of CpG-ODN in macrophages.
Polymorphonuclear Neutrophil-Derived Ectosomes Interfere with the Maturation of Monocyte-Derived Dendritic Cells
Authors: Eken Ceylan , Gasser Olivier , Zenhaeusern Gabriela , Oehri Ineke , Hess Christoph , Schifferli Jürg A.
Published: April 2014

Polymorphonuclear neutrophils (PMNs) are a key component of the innate immune system. Their activation leads to the release of potent antimicrobial agents through degranulation. Simultaneously, PMNs release cell surface-derived microvesicles, so-called ectosomes (PMN-Ect). PMN-Ect are rightside-out vesicles with a diameter of 50–200 nm. They expose phosphatidylserine in the outer leaflet of their membrane and down-modulate monocyte/macrophage-activation in vitro. In this study, we analyzed the effects of PMN-Ect on maturation of human monocyte-derived dendritic cells (MoDCs). Intriguingly, exposing immature MoDCs to PMN-Ect modified their morphology, reduced their phagocytic activity, and increased the release of TGF-β1. When immature MoDCs were incubated with PMN-Ect and stimulated with the TLR4 ligand LPS, the maturation process was partially inhibited as evidenced by reduced expression of cell surface markers (CD40, CD80, CD83, CD86, and HLA-DP DQ DR), inhibition of cytokine-release (IL-8, IL-10, IL-12, and TNF-α), and a reduced capacity to induce T cell proliferation. Together these data provide evidence that PMN-Ect have the ability to modify MoDC maturation and function. PMN-Ect may thus represent an as yet unidentified host-factor influencing MoDC maturation at the site of injury, thereby possibly impacting on downstream MoDC-dependent immunity.
IFN-γ Mediates the Death of Th1 Cells in a Paracrine Manner
Authors: Foulds Kathryn E. , Rotte Masashi J. , Paley Michael A. , Singh Babu , Douek Daniel C. , Hill Brenna J. , O’Shea John J. , Watford Wendy T. , Seder Robert A. , Wu Chang-You
Published: April 2014

Th1 cells have different capacities to develop into memory cells based on their production of IFN-γ. In this study, the mechanism by which a homogenous population of IFN-γ-producing CD4 T cells was eliminated in vivo was assessed. When such cells were transferred into naive mice and activated with Ag, a striking decrease in the frequency of cells in the spleen and lung was observed. However, administration of neutralizing anti-IFN-γ Ab at the time of Ag challenge largely prevented the elimination of such cells. To determine whether IFN-γ was mediating its effects directly and/or indirectly, the ability of IFN-γ to effectively signal in such cells was assessed in vitro. Indeed, there was reduced phosphorylation of STAT1 in response to IFN-γ as well as markedly reduced expression of the IFN-γR β-chain. Furthermore, transfer of such cells into IFN-γR-deficient mice limited their death following activation with Ag. Together, these data suggest that IFN-γ acts in a paracrine manner to mediate the death of activated IFN-γ-producing Th1 cells. In contrast to Ag stimulation, administration of CpG alone resulted in the elimination of Th1 cells in IFN-γR−/− mice. These results show that in response to Ag stimulation, the death of IFN-γ-producing effector Th1 cells is controlled in an IFN-γ-dependent manner, whereas in response to innate activation, the death of IFN-γ-producing Th1 cells can occur through an IFN-γ-independent pathway. Collectively, these data show the multiple mechanisms by which Th1 effector cells are efficiently eliminated in vivo.
Regulatory NK Cells Suppress Antigen-Specific T Cell Responses
Authors: Deniz Gunnur , Erten Gaye , Kücüksezer Umut Can , Kocacik Dilara , Karagiannidis Christian , Aktas Esin , Akdis Cezmi A. , Akdis Mubeccel
Published: April 2014

The immune system has a variety of regulatory/suppressive processes, which are decisive for the development of a healthy or an allergic immune response to allergens. NK1 and NK2 subsets have been demonstrated to display counterregulatory and provocative roles in immune responses, similar to Th1 and Th2 cells. T regulatory cells suppressing both Th1 and Th2 responses have been the focus of intensive research during the last decade. In this study, we aimed to investigate regulatory NK cells in humans, by characterization of NK cell subsets according to their IL-10 secretion property. Freshly purified IL-10-secreting NK cells expressed up to 40-fold increase in IL-10, but not in the FoxP3 and TGF-β mRNAs. PHA and IL-2 stimulation as well as vitamin D3/dexamethasone and anti-CD2/CD16 mAbs are demonstrated to induce IL-10 expression in NK cells. The effect of IL-10+ NK cells on Ag-specific T cell proliferation has been examined in bee venom major allergen, phospholipase A2- and purified protein derivative of Mycobecterium bovis-induced T cell proliferation. IL-10+ NK cells significantly suppressed both allergen/Ag-induced T cell proliferation and secretion of IL-13 and IFN-γ, particularly due to secreted IL-10 as demonstrated by blocking of the IL-10 receptor. These results demonstrate that a distinct small fraction of NK cells display regulatory functions in humans.
Transcription Factor FOXO3a Mediates Apoptosis in HIV-1-Infected Macrophages
Authors: Cui Min , Huang Yunlong , Zhao Yong , Zheng Jialin
Published: April 2014

Macrophages serve as a major reservoir for HIV-1 because a large number of macrophages in the brain and lung are infected with HIV-1 during late stage disease. Recent evidence suggests that those HIV-1-infected macrophages play a key role in contributing to tissue damage in AIDS pathogenesis. Macrophages undergo apoptosis upon HIV-1 infection; however, the mechanisms of this process are not well-defined. Previously, we demonstrated that HIV-1 infection inhibits Akt-1, a critical protein for cell survival of macrophages. In the present study, we investigated the involvement of transcription factor FOXO3a in the regulation of HIV-1-mediated apoptosis in macrophages. HIV-1 infection significantly decreased phosphorylation of FOXO3a and promoted FOXO3a translocation to the nucleus in human monocyte-derived macrophages (MDM). Overexpression of a constitutively active FOXO3a increased DNA fragmentation with decreased cell viability in MDM, whereas a dominant-negative mutant of FOXO3a or small interfering RNA for FOXO3a to knockdown the function of FOXO3a in HIV-1-infected MDM decreased DNA fragmentation and protected macrophages from death in HIV-1-infected MDM. Overexpression of constitutively active Akt-1 increased FOXO3a phosphorylation, suggesting that FOXO3a phosphorylation in human MDM is dependent on Akt-1. We therefore conclude that FOXO3a plays an important role in HIV-1-induced cell death of human macrophage. Understanding the PI3K/Akt-1/FOXO3a pathway and its associated death mechanism in macrophages during HIV-1 infection would lead to identification of potential therapeutic avenues for the treatment of HIV-1 infection.
T Cell Development from Kit-Negative Progenitors in the Foxn1Δ/Δ Mutant Thymus
Authors: Xiao Shiyun , Su Dong-ming , Manley Nancy R.
Published: April 2014

Foxn1 Δ is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1Δ/Δ adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1−/− mice were able to differentiate to the DN2 or DN3 stage in the Foxn1Δ/Δ thymus. Our data suggest that thymopoiesis in the Foxn1Δ/Δ adult thymus proceeds from CD117− atypical progenitors, while CD117+ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1Δ/Δ adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.
IL-27 Induces a Th1 Immune Response and Susceptibility to Experimental Arthritis
Authors: Cao Yanxia , Doodes Paul D. , Glant Tibor T. , Finnegan Alison
Published: April 2014

IL-27 is the newest member of the cytokine family comprised of IL-12 and IL-23. IL-27 was originally described as a cytokine that along with IL-12 induces the differentiation of naive precursor T cells into Th1 effector cells. This activity has been called into question based on evidence in infectious disease and autoimmune models in which IL-27 is not absolutely required for the generation of IFN-γ, and IL-27 plays a regulatory role in controlling inflammation. We have previously reported in proteoglycan-induced arthritis (PGIA), a model of rheumatoid arthritis, that severe arthritis is dependent on the production of IFN-γ. In this study, we report that IL-27 was expressed in spleen and joint tissues of arthritic mice. We determined the involvement of IL-27 in PGIA by assessing the progression of arthritis in IL-27R−/− mice. Development of arthritis in IL-27R−/− mice was delayed and severity reduced in comparison with IL-27R+/+ littermate controls. Histology confirmed a reduction in joint cellularity, cartilage destruction, and bone erosion. Diminished arthritis was associated with fewer T cells producing IFN-γ and decreased IFN-γ secretion overtime. Moreover, the frequency of IL-4- and IL-17-expressing T cells and the production of IL-4 and IL-17 were similar in IL-27R−/− mice and controls. Our results indicate that IL-27 is critically involved in the induction of inflammation in PGIA. IL-27 functions by inducing the differentiation of IFN-γ-producing T cells in vivo that are essential for the development of arthritis.
Tax-Inducible Production of CC Chemokine Ligand 22 by Human T Cell Leukemia Virus Type 1 (HTLV-1)-Infected T Cells Promotes Preferential Transmission of HTLV-1 to CCR4-Expressing CD4+ T Cells
Authors: Hieshima Kunio , Nagakubo Daisuke , Nakayama Takashi , Shirakawa Aiko-Konno , Jin Zhe , Yoshie Osamu
Published: April 2014

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.
Structural Determinants of Herpesvirus Entry Mediator Recognition by Murine B and T Lymphocyte Attenuator
Authors: Nelson Christopher A. , Fremont Marcel D. , Sedy John R. , Norris Paula S. , Ware Carl F. , Murphy Kenneth M. , Fremont Daved H.
Published: April 2014

The B and T lymphocyte attenuator (BTLA) appears to act as a negative regulator of T cell activation and growth. BTLA specifically interacts with herpesvirus entry mediator (HVEM), a member of the TNFR family. Herein, we have undertaken surface plasmon resonance studies to quantitatively assess BTLA and HVEM ectodomain interactions. We find that soluble BALB/cJ BTLA engages HVEM with an equilibrium affinity of 0.97 ± 0.19 μM while the C57BL/6 BTLA binds slightly better with an equilibrium affinity of 0.42 ± 0.06 μM. Despite its lower affinity for HVEM, the kinetic half-life of BALB/cJ BTLA complexes are twice as long as observed for C57BL/6 BTLA (4 vs 2 s). To further explore these interactions, we solved the crystal structure of a murine BTLA (BALB/cJ) ectodomain at 1.8-Å resolution, revealing a β sandwich fold with strong similarity to I-set members of the Ig superfamily. Using a structure-based mutagenesis strategy, we then examined the individual contributions of 26 BTLA surface-exposed residues toward HVEM binding. Four single-site substitutions were identified that decrease HVEM binding below detectable levels and two that decrease binding by more than half. All six of these cluster at the edge of the β sandwich in a membrane distal patch formed primarily from the A and G strands. This patch falls within the contacting surface recently revealed in the crystal structure of the human BTLA-HVEM cocomplex. The critical binding residues identified here are highly conserved across species, suggesting that BTLA employs a conserved binding mode for HVEM recognition.
Dissecting the Structural Determinants of the Interaction between the Human Cytomegalovirus UL18 Protein and the CD85j Immune Receptor
Authors: Occhino Marzia , Ghiotto Fabio , Soro Simonetta , Mortarino Mimosa , Bosi Stefania , Maffei Massimo , Bruno Silvia , Nardini Marco , Figini Mariangela , Tramontano Anna , Ciccone Ermanno
Published: April 2014

UL18 is a glycoprotein encoded by the human cytomegalovirus genome and is thought to play a pivotal role during human cytomegalovirus infection, although its exact function is still a matter of debate. UL18 shares structural similarity with MHC class I and binds the receptor CD85j on immune cells. Besides UL18, CD85j binds MHC class I molecules. The binding properties of CD85j to MHC class I molecules have been thoroughly studied. Conversely, very little information is available on the CD85j/UL18 complex, namely that UL18 binds CD85j through its α3 domain with an affinity that is ∼1000-fold higher than the MHC class I affinity for CD85j. Deeper knowledge of features of the UL18/CD85j complex would help to disclose the function of UL18 when it binds to CD85j. In this study we first demonstrated that the UL18α3 domain is not sufficient per se for binding and that β2-microglobulin is necessary for UL18–CD85j interaction. We then dissected structural determinants of binding UL18 to CD85j. To this end, we constructed a three-dimensional model of the complex. The model was used to design mutants in selected regions of the putative interaction interface, the effects of which were measured on binding. Six regions in both the α2 and α3 domains and specific amino acids within them were identified that are potentially involved in the UL18–CD85j interaction. The higher affinity of UL18 to CD85j, compared with MHC class I, seems to be due not to additional interaction regions but to an overall better fit of the two molecules.
Human Cytomegalovirus Regulates Surface Expression of the Viral Protein UL18 by Means of Two Motifs Present in the Cytoplasmic Tail
Authors: Maffei Massimo , Ghiotto Fabio , Occhino Marzia , Bono Maria , De Santanna Amleto , Battini Lorenzo , Gusella G. Luca , Fais Franco , Bruno Silvia , Ciccone Ermanno
Published: April 2014

UL18 is a trans-membrane viral protein expressed on human cytomegalovirus (HCMV)-infected cells, and its surface expression determines the interaction of infected cells with lymphocytes expressing the CD85j (LIR-1/ILT2) receptor. We previously showed that the UL18–CD85j interaction elicits activation of T lymphocytes. However, in in vitro cell models UL18 displays mostly undetectable surface expression. Thus, we asked how surface expression of UL18 is regulated. Domain-swapping experiments and construction of specific mutants demonstrated that two motifs on its cytoplasmic tail, homologous to YXXΦ and KKXX consensus sequences, respectively, are responsible for impairing UL18 surface expression. However, the presence of the whole HCMV genome, granted by HCMV infection of human fibroblasts, restored surface expression of either UL18 or chimeric proteins carrying the UL18 cytoplasmic tail, starting from the third day after infection. It is of note that the two motifs responsible for cytoplasmic retention are identical in all 17 HCMV strains examined. We disclosed a control mechanism used by the HCMV to regulate the availability of UL18 on the infected-cell surface to allow interaction with its ligand on T and NK cells.
Regulation of CD1a Surface Expression and Antigen Presentation by Invariant Chain and Lipid Rafts
Authors: Sloma Ivan , Zilber Marie-Thérèse , Vasselon Thierry , Setterblad Niclas , Cavallari Marco , Mori Lucia , De Libero Gennaro , Charron Dominique , Mooney Nuala , Gelin Catherine
Published: April 2014

In immature dendritic cells (DCs), CD1a is almost exclusively expressed at the cell surface and its membrane organization is poorly understood. In this study, we report that MHC class II, invariant chain (Ii), and CD9 molecules are coimmunoprecipitated with CD1a in immature DCs, and that CD1a/Ii colocalization is dependent on lipid raft integrity. In HeLa-CIITA cells CD1a expression leads to increased Ii trafficking to the cell surface, confirming the relevance of this association. Furthermore, silencing of Ii in DCs induces significant CD1a accumulation on the plasma membrane whereas the total CD1a expression remains similar to that of control cells. These data suggest that CD1a recycling is facilitated by the association with the Ii. The CD1a localization in lipid rafts has functional relevance as demonstrated by inhibition of CD1a-restricted presentation following raft disruption. Overall, these findings identify Ii and lipid rafts as key regulators of CD1a organization on the surface of immature DCs and of its immunological function as Ag-presenting molecule.
Effector CD8 T Cell Development: A Balancing Act between Memory Cell Potential and Terminal Differentiation
Authors: Joshi Nikhil S. , Kaech Susan M.
Published: April 2014

Immune responses to infection are optimally designed to generate large numbers of effector T cells while simultaneously minimizing the collateral damage of their potentially lethal actions and generating memory T cells to protect against subsequent encounter with pathogens. Much remains to be discovered about how these equally essential processes are balanced to enhance health and longevity and, more specifically, what factors control effector T cell expansion, differentiation, and memory cell formation. The innate immune system plays a prominent role in the delicate balance of these decisions. Insights into these questions from recent work in the area of effector CD8 T cell differentiation will be discussed.
Fast Progression of Recombinant Human Myelin/Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis in Marmosets Is Associated with the Activation of MOG34–56-Specific Cytotoxic T Cells
Authors: Kap Yolanda S. , Smith Paul , Jagessar S. Anwar , Remarque Ed , Blezer Erwin , Strijkers Gustav J. , Laman Jon D. , Hintzen Rogier Q. , Bauer Jan , Brok Herbert P. M. , ‘t Hart Bert A.
Published: April 2014

The recombinant human (rh) myelin/oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) model in the common marmoset is characterized by 100% disease incidence, a chronic disease course, and a variable time interval between immunization and neurological impairment. We investigated whether monkeys with fast and slow disease progression display different anti-MOG T or B cell responses and analyzed the underlying pathogenic mechanism(s). The results show that fast progressor monkeys display a significantly wider specificity diversification of anti-MOG T cells at necropsy than slow progressors, especially against MOG34–56 and MOG74–96. MOG34–56 emerged as a critical encephalitogenic peptide, inducing severe neurological disease and multiple lesions with inflammation, demyelination, and axonal injury in the CNS. Although EAE was not observed in MOG74–96-immunized monkeys, weak T cell responses against MOG34–56 and low grade CNS pathology were detected. When these cases received a booster immunization with MOG34–56 in IFA, full-blown EAE developed. MOG34–56-reactive T cells expressed CD3, CD4, or CD8 and CD56, but not CD16. Moreover, MOG34–56-specific T cell lines displayed specific cytotoxic activity against peptide-pulsed B cell lines. The phenotype and cytotoxic activity suggest that these cells are NK-CTL. These results support the concept that cytotoxic cells may play a role in the pathogenesis of multiple sclerosis.
In Vivo Polarization of IFN-γ at Kupfer and Non-Kupfer Immunological Synapses during the Clearance of Virally Infected Brain Cells
Authors: Barcia Carlos , Wawrowsky Kolja , Barrett Robert J. , Liu Chunyan , Castro Maria G. , Lowenstein Pedro R.
Published: April 2014

Kupfer-type immunological synapses are thought to mediate intercellular communication between antiviral T cells and virally infected target Ag-presenting brain cells in vivo during an antiviral brain immune response. This hypothesis predicts that formation of Kupfer-type immunological synapses is necessary for polarized distribution of effector molecules, and their directed secretion toward the target cells. However, no studies have been published testing the hypothesis that cytokines can only form polarized clusters at Kupfer-type immunological synapses. Here, we show that IFN-γ and granzyme-B cluster in a polarized fashion at contacts between T cells and infected astrocytes in vivo. In some cases these clusters were found in Kupfer-type immunological synapses between T cells and infected astrocytes, but we also detected polarized IFN-γ at synaptic immunological contacts which did not form Kupfer-type immunological synaptic junctions, i.e., in the absence of polarization of TCR or LFA-1. This indicates that TCR signaling, which leads to the production, polarization, and eventual directed secretion of effector molecules such as IFN-γ, occurs following the formation of both Kupfer-type and non-Kupfer type immunological synaptic junctions between T cells and virally infected target astrocytes in vivo.
Cross Presentation of Antigen on MHC Class II via the Draining Lymph Node after Corneal Transplantation in Mice
Authors: Kuffová Lucia , Netuková Magdaléna , Duncan Linda , Porter Andrew , Stockinger Brigitta , Forrester John V.
Published: April 2014

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45+, CD11b+, CD11c- cells, with a few MHC class II+ cells, and F4/80+ cells. However, MHC class II+ passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c+ and F4/80+ APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4–6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4+ T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.
Helper B Cells Promote Cytotoxic T Cell Survival and Proliferation Independently of Antigen Presentation through CD27/CD70 Interactions
Authors: Deola Sara , Panelli Monica C. , Maric Dragan , Selleri Silvia , Dmitrieva Natalia I. , Voss Ching Y. , Klein Harvey , Stroncek David , Wang Ena , Marincola Francesco M.
Published: April 2014

CD8-expressing cytotoxic T cell (CTL) interactions with APCs and helper T cells determine their function and ability to survive. In this study, we describe a novel interaction independent of Ag presentation between activated CTLs and bystander CD19-expressing B lymphocytes. Ag-stimulated CTLs serially engage autologous B lymphocytes through CD27/CD70 contact that promotes their survival and proliferation. Moreover, these interactions induce the release of proinflammatory cytokines that follows two general patterns: 1) an epitope-dependent enhancement of cytokine release, and 2) a previously undiscovered coordinate release of cytokines independent of epitope exposure. The latter includes chemoattractants targeting activated T cells. As a result, activated T cells are attracted to B cells, which exert a “helper” role in lymphatic organs or in areas of inflammation. This observation provides a mechanistic explanation to previously reported experimental observations suggesting that B cells are required for T cell priming in vivo.
Stimulation by TLR5 Modulates Osteoclast Differentiation through STAT1/IFN-β
Authors: Ha Hyunil , Lee Jong-Ho , Kim Ha-Neui , Kwak Han Bok , Kim Hyun-Man , Lee Shee Eun , Rhee Joon Haeng , Kim Hong-Hee , Lee Zang Hee
Published: April 2014

Osteoclasts are bone-resorbing cells that are differentiated from hemopoietic precursors of the monocyte-macrophage lineage. Stimulation of TLRs has been shown to positively or negatively modulate osteoclast differentiation, depending on the experimental condition. However, the molecular mechanism by which this modulation takes place remains unclear. In the present study, we examined the effects of flagellin, a specific microbial ligand of TLR5, on the receptor activator of NF-κB ligand (RANKL)-stimulated osteoclastogenesis. Flagellin suppressed RANKL induction of c-Fos protein expression in bone marrow-derived macrophages without affecting c-Fos mRNA expression. Ectopic overexpression of c-Fos and a constitutively active form of NFATc1 reversed the flagellin-induced anti-osteoclastogenic effect. The inhibitory effect of flagellin was mediated by IFN-β production. Flagellin stimulated IFN-β expression and release in bone marrow-derived macrophages, and IFN-β-neutralizing Ab prevented the flagellin-induced c-Fos down-regulation and the anti-osteoclastogenic effect. IFN-β gene induction by flagellin, LPS, or RANKL was dependent on STAT1 activation. Treatment with flagellin or RANKL stimulated STAT1 activation, and STAT1 deficiency or the JAK2 inhibitor AG490 dramatically prevented IFN-β induction in response to flagellin or RANKL. In addition, STAT1 deficiency abolished the anti-osteoclastogenic effect induced by flagellin or LPS. In contrast, flagellin stimulated osteoclast differentiation in cocultures of osteoblasts and bone marrow cells without inducing IFN-β. Thus, IFN-β acts as a critical modulator of osteoclastogenesis in response to TLR5 activation.
Human Follicular Dendritic Cells Interact with T Cells via Expression and Regulation of Cyclooxygenases and Prostaglandin E and I Synthases
Authors: Lee In Yong , Cho Whajung , Kim Jini , Park Chan-Sik , Choe Jongseon
Published: April 2014

PGE2 inhibits mature T cell proliferation and protects T cells from activation-induced cell death (AICD). We have previously demonstrated that human follicular dendritic cells (FDC) strongly express PGI synthase. In this study, the hypothesis that FDC have regulatory roles on germinal center T cells by controlling production of PGE2 and PGI2 was tested. Confocal microscopic analyses of human tonsil tissues revealed that FDC indeed expressed PGE synthase in addition to PGIS. To confirm these results, we studied the regulation mechanism of PG production in FDC, using an established human FDC-like cell line, HK. Specifically in response to TNF-α, TGF-β, and LPS, protein expression of cyclooxygenase (COX)-2 and downstream PGE synthase was up-regulated with coordinate kinetics, whereas COX-1 and PGIS were constitutively expressed. The increase of these enzymes was reflected in actual production of PGE2 and PGI2. Interestingly, IL-4 almost completely abrogated the stimulatory activity of TNF-α, TGF-β, and LPS in PG production. Furthermore, the up-regulation of PGE2 and PGI2 production was markedly down-regulated by indomethacin and a selective COX-2 inhibitor. PGI2 analog and PGE2 inhibited proliferation and AICD of T cells in dose- and time-dependent manners. Finally, coculture experiments revealed that HK cells indeed inhibit proliferation and AICD of T cells. Put together, these results show an unrecognized pathway of FDC and T cell interactions and differential mechanisms for PGE2 and PGI2 production, suggesting an important implication for development and use of anti-inflammatory drugs.
Mathematical Modeling Reveals the Biological Program Regulating Lymphopenia-Induced Proliferation
Authors: Yates Andrew , Saini Manoj , Mathiot Anne , Seddon Benedict
Published: April 2014

Recognition of peptide-MHC by the TCR induces T lymphocytes to undergo cell division. Although recognition of foreign peptide induces a program of cellular division and differentiation by responding T cells, stimulation by self-peptide MHC complexes in lymphopenic conditions induces a slower burst of divisions that may or may not be accompanied by effector differentiation. Although both responses are triggered by signals from the TCR, it is not known whether they represent distinct programs of cell cycle control. In this study, we use a mathematical modeling approach to analyze the proliferative response of TCR transgenic F5 T cells to lymphopenia. We tested two fundamentally different models of cell division: one in which T cells are triggered into an “autopilot” deterministic burst of divisions, a model successfully used elsewhere to describe T cell responses to cognate Ag, and a second contrasting model in which cells undergo independent single stochastic divisions. Whereas the autopilot model provided a very poor description of the F5 T cell responses to lymphopenia, the model of single stochastic divisions fitted the experimental data remarkably closely. Furthermore, this model proved robust because specific predictions of cellular behavior made by this model concerning the onset, rate, and nature of division were successfully validated experimentally. Our results suggest cell division induced by lymphopenia involves a process of single stochastic divisions, which is best suited to a homeostatic rather than differentiation role.
17β-Estradiol Alters the Activity of Conventional and IFN-Producing Killer Dendritic Cells
Authors: Siracusa Mark C. , Overstreet Michael G. , Housseau Franck , Scott Alan L. , Klein Sabra L.
Published: April 2014

Estrogens increase aspects of innate immunity and contribute to sex differences in the prevalence of autoimmune diseases and in response to infection. The goal of the present study was to assess whether exposure to 17β-estradiol (E2) affects the development and function of bone marrow-derived dendritic cells and to determine whether similar changes are observed in CD11c+ splenocytes exposed to E2 in vivo. E2 facilitated the differentiation of BM precursor cells into functional CD11c+CD11b+MHC class II+ dendritic cells (DCs) with increased expression of the costimulatory molecules CD40 and CD86. Exposure of bone marrow-derived dendritic cells to E2 also enhanced production of IL-12 in response to the TLR ligands, CpG and LPS. In contrast, CD11c+ cells isolated from the spleens of female C57BL/6 mice that were intact, ovariectomized, or ovariectomized with E2 replacement exhibited no differences in the number or activity of CD11c+CD11b+MHC class II+ DCs. The presence of E2 in vivo, however, increased the number of CD11c+CD49b+NK1.1low cells and reduced numbers of CD11c+CD49b+NK1.1high cells, a surface phenotype for IFN-producing killer DCs (IKDCs). Ultrastructural analysis demonstrated that CD11c+NK1.1+ populations were comprised of cells that had the appearance of both DCs and IKDCs. CD11c+ splenocytes isolated from animals with supplemental E2 produced more IFN-γ in response to IL-12 and IL-18. These data illustrate that E2 has differential effects on the development and function of DCs and IKDCs and provide evidence that E2 may strengthen innate immunity by enhancing IFN-γ production by CD11c+ cells.