bluuebunny/crossref_metadata_2025 huggingface dataset:
Rap1-GTP Is a Negative Regulator of Th Cell Function and Promotes the Generation of CD4+CD103+ Regulatory T Cells In Vivo
Authors: Li Lequn , Greenwald Rebecca J. , Lafuente Esther M. , Tzachanis Dimitrios , Berezovskaya Alla , Freeman Gordon J. , Sharpe Arlene H. , Boussiotis Vassiliki A.
Published: April 2014

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.
HIV Nef-Mediated CD4 Down-Regulation Is Adaptor Protein Complex 2 Dependent
Authors: Jin Yong-Jiu , Cai Catherine Yi , Zhang Xiaoping , Zhang Hui-Tang , Hirst John A. , Burakoff Steven J.
Published: April 2014

Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the α subunit of AP-2 complex and a small interference RNA that is specific for the μ2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.
Cytokine Induction by the Hepatitis B Virus Capsid in Macrophages Is Facilitated by Membrane Heparan Sulfate and Involves TLR2
Authors: Cooper Arik , Tal Guy , Lider Ofer , Shaul Yosef
Published: April 2014

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not ΗΒc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-α, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-κB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-κB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.
Mannan-Binding Protein Blocks the Activation of Metalloproteases Meprin α and β
Authors: Hirano Makoto , Ma Bruce Yong , Kawasaki Nana , Okimura Kazumichi , Baba Makoto , Nakagawa Tomoaki , Miwa Keiko , Kawasaki Nobuko , Oka Shogo , Kawasaki Toshisuke
Published: April 2014

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin α and β (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.
Upper Respiratory Tract Resistance to Influenza Infection Is Not Prevented by the Absence of Either Nasal-Associated Lymphoid Tissue or Cervical Lymph Nodes
Authors: Wiley James A. , Tighe Michael P. , Harmsen Allen G.
Published: April 2014

The murine nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) are involved in the generation of local immune responses within the upper respiratory tract (URT). However, their involvement in these responses does not imply the necessity for resistance to URT infections. We surgically removed NALT or CLN to address the necessity of these lymphatic tissues for the development of a local protective immune response after a URT influenza infection. No histological evidence of the re-establishment of either tissue was detected after surgery and the subsequent infection. Removal of NALT did not elicit changes in serum or nasal mucosa-associated influenza-specific Ig levels. However, increases in PR8-specific serum IgG and nasal mucosa-associated IgA were detected after removal of CLN. Recruitment of influenza-specific CD4 T cells into the nasal mucosa was not altered by removal of NALT. The removal of NALT or CLN did not alter the recruitment of influenza-specific CD8 T cells into the URT. However, increased levels of influenza-specific CD8 T cells were observed in the tracheal-bronchial lymph nodes after CLN surgery. The rate of viral clearance from nasal mucosa and lungs was not altered by removal of NALT or CLN. These studies demonstrate that despite the participation of NALT and CLN in the generation of local immunity to influenza infections, neither tissue is essential for the development of protective immunity and viral clearance in URT.
NK T Cell Activation Promotes Chlamydia trachomatis Infection In Vivo
Authors: Bilenki Laura , Wang Shuhe , Yang Jie , Fan Yijun , Joyee Antony George , Yang Xi
Published: April 2014

We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using α-galactosylceramide (α-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using α-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the α-GalCer-treated BALB/c mice significantly reduced the promoting effect of α-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.
Infection of Dendritic Cells by a γ2-Herpesvirus Induces Functional Modulation
Authors: Flaño Emilio , Kayhan Basak , Woodland David L. , Blackman Marcia A.
Published: April 2014

The murine γ-herpesvirus-68 (γHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by γHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of γHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas γHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, γHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn’t interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased γHV68 long-term latency. Taken together, these observations indicate that γ2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.
Wild-Type Measles Virus Infection in Human CD46/CD150-Transgenic Mice: CD11c-Positive Dendritic Cells Establish Systemic Viral Infection
Authors: Shingai Masashi , Inoue Naokazu , Okuno Tsuyoshi , Okabe Masaru , Akazawa Takashi , Miyamoto Yasuhide , Ayata Minoru , Honda Kenya , Kurita-Taniguchi Mitsue , Matsumoto Misako , Ogura Hisashi , Taniguchi Tadatsugu , Seya Tsukasa
Published: April 2014

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-β was higher in mDC than the profile of IFN-α. Wild-type IC induced markedly high levels of IFN-β compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)−/− mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.
Regulation of Aged Humoral Immune Defense against Pneumococcal Bacteria by IgM Memory B Cell
Authors: Shi Yuhui , Yamazaki Takashi , Okubo Yoshio , Uehara Yoshio , Sugane Kazuo , Agematsu Kazunaga
Published: April 2014

Elderly persons have a high incidence of lethal infections by encapsulated bacteria. However, mechanisms involved in their poor defense and maintenance of immunological memory have been poorly understood. The present study characterized the population of B cells known as IgM memory B cell compartment and their response by pneumococcal vaccine in elderly people. CD27+ memory B cells, particularly IgD+IgM+CD27+ IgM memory B cells, had dramatically declined in the aged. Their Ig syntheses by B cells and the differentiation into plasma cells were diminished in vitro compared with those in adults. A rise of anti-pneumococcal IgM in sera of elderly persons was found with lower levels compared with those in adults after pneumococcal vaccination. Although diminished function itself of aged B cells surely exist, decline of the IgM memory B cell pool is expected to result in a poor humoral immunity against pneumococcal infection in elderly people.
In Vivo Depletion of CD11c+ Cells Delays the CD4+ T Cell Response to Mycobacterium tuberculosis and Exacerbates the Outcome of Infection
Authors: Tian Tian , Woodworth Joshua , Sköld Markus , Behar Samuel M.
Published: April 2014

Although dendritic cells (DC) are potent APC that prime T cells against many pathogens, there is no direct evidence that DC are required for immunity to Mycobacterium tuberculosis (Mtb) infection. The requirement for DC to prime the CD4+ T cell response following Mtb infection was investigated using pCD11c-diptheria toxin receptor/GFP transgenic mice, in which DC can be transiently ablated in vivo. We show a critical role for DC in initiation of the CD4+ T cell response to the mycobacterial Ag early secretory Ag of tuberculosis 6. The delay in initiating the Ag-specific T cell response led to impaired control of Mtb replication. Interestingly, DC were not required for the secondary CD4+ T cell response following Mtb infection in peptide-vaccinated mice. Thus, this study shows that DC are essential for the initiation of the adaptive T cell response to the human pathogen Mtb.
Monocyte-Astrocyte Networks and the Regulation of Chemokine Secretion in Neurocysticercosis
Authors: Uddin Jasim , Garcia Hector H. , Gilman Robert H. , Gonzalez Armando E. , Friedland Jon S.
Published: April 2014

Neurocysticercosis, caused by infection with larval Taenia solium, is a major cause of epilepsy worldwide. Larval degeneration, which is symptomatic, results in inflammatory cell influx. Astrocytes, the most abundant cell type and major cytokine-producing cell within the CNS, may be important in orchestrating inflammatory responses after larval degeneration. We investigated the effects of direct stimulation and of conditioned medium from T. solium larval Ag (TsAg)-stimulated monocytes (CoMTsAg) on neutrophil and astrocyte chemokine release. CoMTsAg, but not control conditioned medium, stimulated astrocyte CCL2/MCP-1 (161.5 ± 16 ng/ml), CXCL8/IL-8 (416 ± 6.2 ng/ml), and CXCL10/IFN-γ-inducible protein (9.07 ± 0.6 ng/ml) secretion after 24 h, whereas direct astrocyte or neutrophil stimulation with TsAg had no effect. There was rapid accumulation of CCL2 and CXCL8 mRNA within 1 h, with somewhat delayed expression of CXCL10 mRNA initially detected 8 h poststimulation. Neutralizing anti-TNF-α inhibited CoMTsAg-induced CCL2 mRNA accumulation by up to 99%, causing total abolition of CXCL10 and up to 77% reduction in CXCL8 mRNA. CoMTsAg induced maximal nuclear binding of NF-κB p65 and p50 by 1 h, with IκBα and IκBβ decay within 15 min. In addition, CoMTsAg induced transient nuclear binding of AP-1, which peaked 4 h poststimulation. In NF-κB blocking experiments using pyrrolidine dithiocarbamate, CoMTsAg-induced CCL2 secretion was reduced by up to 80% (p = 0.0006), whereas CXCL8 was inhibited by up to 75% (p = 0.0003). In summary, the data show that astrocytes are an important source of chemokines following larval Ag stimulation. Such chemokine secretion is NF-κB dependent, likely to involve AP-1, and is regulated in a paracrine loop by monocyte-derived TNF-α.
CD11c+ Dendritic Cells Are Required for Survival in Murine Polymicrobial Sepsis
Authors: Scumpia Philip O. , McAuliffe Priscilla F. , O’Malley Kerri A. , Ungaro Ricardo , Uchida Takefumi , Matsumoto Tadashi , Remick Daniel G. , Clare-Salzler Michael J. , Moldawer Lyle L. , Efron Philip A.
Published: April 2014

CD11c+ dendritic cells (DCs) are APCs that link innate and adaptive immunity. Although DCs are lost from spleen and lymph nodes in sepsis, their role in outcome remains unclear. Transgenic mice (B6.FVB-Tg.Itgax-DTR/EGFP.57Lan/J) expressing the diphtheria toxin (DT) receptor on the CD11c promoter (DCKO mice) received 4 ng/kg DT, which resulted in depletion of 88–95% of mature myeloid and lymphoid DCs, with less depletion (75%) of plasmacytoid DCs. Pretreatment of DCKO mice with DT resulted in reduced survival in sepsis compared with saline-pretreated DCKO mice (0 vs 54%; p < 0.05) or DT-treated wild-type littermates (0 vs 54%; p < 0.05). This increased mortality was not associated with either increased bacteremia or plasma cytokine concentrations. Intravenous injection of 107 wild-type DCs improved survival in DCKO mice (42 vs 0%; p = 0.05). These data confirm that DCs are essential in the septic response and suggest that strategies to maintain DC numbers or function may improve outcome.
Monocytes Stimulated by 110-kDa Fibronectin Fragments Suppress Proliferation of Anti-CD3-Activated T Cells
Authors: Birdsall Holly H. , Porter Wendy J. , Trial JoAnn , Rossen Roger D.
Published: April 2014

One hundred ten to 120-kDa fragments of fibronectin (FNf), generated by proteases released in the course of tissue injury and inflammation, stimulate monocytes to produce proinflammatory cytokines, promote mononuclear leukocytes (MNL) transendothelial migration, up-regulate monocyte CD11b and CD86 expression, and induce monocyte-derived dendritic cell differentiation. To investigate whether the proinflammatory consequences of FNf are offset by responses that can suppress proliferation of activated T lymphocytes, we investigated the effect of FNf-treated MNL on autologous T lymphocytes induced to proliferate by substrate-immobilized anti-CD3. FNf-stimulated MNL suppressed anti-CD3-induced T cell proliferation through both contact-dependent and contact-independent mechanisms. Contact-independent suppression was mediated, at least in part, by IL-10 and TGF-β released by the FNf-stimulated MNL. After 24–48 h exposure to FNf, activated T cells and monocytes formed clusters displaying CD25, CD14, CD3, and CD4 that were not dissociable by chelation of divalent cations. Killing monocytes with l-leucine methyl ester abolished these T cell-monocyte clusters and the ability of the FNf-stimulated MNL to suppress anti-CD3 induced T cell proliferation. Thus, in addition to activating MNL and causing them to migrate to sites of injury, FNf appears to induce suppressor monocytes.
Requirement of IL-17 Receptor Signaling in Radiation-Resistant Cells in the Joint for Full Progression of Destructive Synovitis
Authors: Lubberts Erik , Schwarzenberger Paul , Huang Weitao , Schurr Jill R. , Peschon Jacques J. , van den Berg Wim B. , Kolls Jay K.
Published: April 2014

IL-17 is a proinflammatory cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. In the present study, we report that IL-17R signaling is required in radiation-resistant cells in the joint for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (streptococcal cell wall) directly into the knee joint of naive IL-17R-deficient (IL-17R−/−) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Microarray analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, cytokines, and collagenase-3 in the synovium of IL-17R−/− mice. Bone marrow (BM) chimeric mice revealed the need for IL-17R expression on radiation-resistant joint cells for destructive inflammation. Chimeric mice of host wt and donor IL-17R−/− BM cells developed destructive synovitis in this chronic reactivated streptococcal cell wall arthritis model similar to wt→wt chimeras. In contrast, chimeric mice of host IL-17R−/− and donor wt BM cells were protected from chronic destructive arthritis similar as IL-17R−/−→IL-17R−/− chimeras. These data strongly indicate that IL-17R signaling in radiation-resistant cells in the joint is required for turning an acute macrophage-mediated inflammation into a chronic destructive synovitis.
Gene Expression Profiling Reveals Multiple Protective Influences of the Peptide α-Melanocyte-Stimulating Hormone in Experimental Heart Transplantation
Authors: Colombo Gualtiero , Gatti Stefano , Turcatti Flavia , Sordi Andrea , Fassati Luigi R. , Bonino Ferruccio , Lipton James M. , Catania Anna
Published: April 2014

Novel therapies are sought to increase efficiency and survival of transplanted organs. Previous research on experimental heart transplantation showed that treatment with the anti-inflammatory peptide α-melanocyte-stimulating hormone (α-MSH) prolongs allograft survival. The aim of the present research was to determine the molecular mechanism of this protective activity. Gene expression profile was examined in heart grafts removed on postoperative days 1 and 4 from rats treated with saline or the synthetic α-MSH analog Nle4DPhe7 (NDP)-α-MSH. On postoperative day 1, the peptide induced expression of cytoskeleton proteins, intracellular kinases, transcription regulators, metallopeptidases, and protease inhibitors. Conversely, NDP-α-MSH repressed immune, inflammatory, cell cycle, and protein turnover mediators. Later effects of α-MSH treatment included down-regulation of oxidative stress response and up-regulation of ion channels, calcium regulation proteins, phosphatidylinositol signaling system, and glycolipidic metabolism. NDP-α-MSH exerted its effects on both Ag-dependent and -independent injury. The results indicate that NDP-α-MSH preserves heart function through a broad effect on multiple pathways and suggest that the peptide could improve the outcome of organ transplantation in combination with immunosuppressive treatments.
Cutting Edge: CD8 T Cells Specific for Lymphocytic Choriomeningitis Virus Require Type I IFN Receptor for Clonal Expansion
Authors: Aichele Peter , Unsoeld Heike , Koschella Marie , Schweier Oliver , Kalinke Ulrich , Vucikuja Smiljka
Published: April 2014

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.
Cutting Edge: Insufficient Perforin Expression in CD8+ T Cells in Response to Hemagglutinin from Avian Influenza (H5N1) Virus
Authors: Hsieh Szu-Min , Chang Shan-Chwen
Published: April 2014

Hypercytokinemia has been considered to be responsible for hemophagocytosis and multiorgan failure in severe human cases of H5N1 viral infection. In this study, we show a recombinant hemagglutinin (H5) from a H5N1 virus may suppress the perforin expression and reduce cytotoxicity of human CD8+ T cells to kill H5-bearing cells. The persistence of H5-presenting cells provides sustained stimulation and leads to a marked lymphoproliferation and IFN-γ hyperproduction. We propose the IFN-γ hyperproduction may explain macrophage overactivation and subsequent hypercytokinemia and hemophagocytosis in severe human cases of avian influenza.
Limited Infiltration of Exogenous Dendritic Cells and Naive T Cells Restricts Immune Responses in Peripheral Lymph Nodes
Authors: Mullins David W. , Engelhard Victor H.
Published: April 2014

Primary CD8 T cell responses in lymph nodes (LN) and protective immunological tumor control are quantitatively limited following immunization with exogenous peptide-pulsed dendritic cells (DC). This arises from two constraints. First, LN are saturated by relatively small quantities of exogenous DC. Second, circulation of new naive T cells into DC-infiltrated LN during the functional lifespan of the DC is negligible. Limits on DC and T cellularity in, and flux through, LN constrain the magnitude of both primary and subsequent recall responses. Enhanced immune responses and tumor control can be achieved using maneuvers to augment LN retention of DC or availability of naive T cells to Ag-presenting DC. These data offer an increased understanding of LN function in general and provide a practical basis for improvements in tumor immunotherapy.
Bruton’s Tyrosine Kinase and SLP-65 Regulate Pre-B Cell Differentiation and the Induction of Ig Light Chain Gene Rearrangement
Authors: Kersseboom Rogier , Ta Van B. T. , Zijlstra A. J. Esther , Middendorp Sabine , Jumaa Hassan , Fokko van Loo Pieter , Hendriks Rudolf W.
Published: April 2014

Bruton’s tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig λ L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both κ and λ germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of κ and λ light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion.
Insufficient APC Capacities of Dendritic Cells in Gene Gun-Mediated DNA Vaccination
Authors: Lauterbach Henning , Gruber Anton , Ried Christine , Cheminay Cedric , Brocker Thomas
Published: April 2014

Gene gun-mediated DNA immunization is a powerful mode of vaccination against infectious diseases and tumors. Many studies have identified dendritic cells (DC) as the central players in inducing immunity upon biolistic DNA vaccination; however, none of these studies directly quantify DC-mediated responses in comparison with immunity triggered by all Ag- and MHC-expressing cells. In this study we use two different approaches to decipher the relative role of DC vs other cell types in gene gun-induced immunity. First, we directly compared the immunization efficacy of different DNA constructs, which allow Ag expression ubiquitously (CMV promoter) or specifically in DC (CD11c promoter) and would encode either for soluble or membrane bound forms of Ag. Second, we immunized transgenic mice in which only DC can present MHC-restricted Ag, and directly compared the magnitudes of CTL activation with those obtained in wild-type mice. Surprisingly, our combined data suggest that, although DC-specific Ag expression is sufficient to induce humoral responses, DC alone cannot trigger optimal CD4 and CD8 T cell responses upon gene gun vaccination. Therefore, we conclude that DC alone are insufficient to mediate optimal induction of T cell immunity upon gene gun DNA vaccination and that broad Ag expression rather than DC-restricted approaches are necessary for induction of complete immune responses.
MHC Class II Expression Identifies Functionally Distinct Human Regulatory T Cells
Authors: Baecher-Allan Clare , Wolf Elizabeth , Hafler David A.
Published: April 2014

It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4+CD25high regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+CD25high T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II− CD4+CD25high Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25high Treg express higher levels of Foxp3 message and protein, compared with the DR−CD25high Treg population. Direct single-cell cloning of CD4+CD25high Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25high, and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25high identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.
DOCK2 Is Required in T Cell Precursors for Development of Vα14 NK T Cells
Authors: Kunisaki Yuya , Tanaka Yoshihiko , Sanui Terukazu , Inayoshi Ayumi , Noda Mayuko , Nakayama Toshinori , Harada Michishige , Taniguchi Masaru , Sasazuki Takehiko , Fukui Yoshinori
Published: April 2014

Mouse CD1d-restricted Vα14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in Vα14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of Vα14 NKT cells in the thymus, liver, and spleen. When α-galactosylceramide (α-GalCer), a ligand for Vα14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of Vα14 NKT cells. Supporting this idea, staining with CD1d/α-GalCer tetramers revealed that CD44−NK1.1− Vα14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of Vα14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of Vα14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.
Direct Stimulation of T Cells by Type I IFN Enhances the CD8+ T Cell Response during Cross-Priming
Authors: Le Bon Agnes , Durand Vanessa , Kamphuis Elisabeth , Thompson Clare , Bulfone-Paus Silvia , Rossmann Cornelia , Kalinke Ulrich , Tough David F.
Published: April 2014

Type I IFN (IFN-αβ), which is produced rapidly in response to infection, plays a key role in innate immunity and also acts as a stimulus for the adaptive immune response. We have investigated how IFN-αβ induces cross-priming, comparing CD8+ T cell responses generated against soluble protein Ags in the presence or absence of IFN-αβ. Injection of IFN-α was found to prolong the proliferation and expansion of Ag-specific CD8+ T cells, which was associated with marked up-regulation of IL-2 and IL-15 receptors on Ag-specific cells and expression of IL-15 in the draining lymph node. Surprisingly, neither IL-2 nor IL-15 was required for IFN-α-induced cross-priming. Conversely, expression of the IFN-αβR by T cells was shown to be necessary for effective stimulation of the response by IFN-α. The finding that T cells represent direct targets of IFN-αβ-mediated stimulation reveals an additional mechanism by which the innate response to infection promotes adaptive immunity.
Identification of Memory B Cells Using a Novel Transgenic Mouse Model
Authors: Chappell Craig P. , Jacob Joshy
Published: April 2014

Memory B cells help to protect the host from invading pathogens by maintaining persistent levels of Ag-specific serum Ab and generating rapid Ab responses upon re-exposure to Ag. Unambiguous identification of memory B cells has been a major obstacle to furthering our knowledge concerning both the development of B cell memory and secondary Ab responses due to an absence of specific cell surface markers. Germinal centers (GCs) are thought to be the major site of Ig hypermutation and Ag-driven selection of memory B cells. To develop a model that would identify GC-derived memory B cells, we generated transgenic mice that expressed cre recombinase in a GC-specific fashion. Interbreeding these mice with the cre-reporter strain, ROSA26R, produced progeny in which β-galactosidase (β-gal) was permanently expressed in B cells of the GC-memory pathway. Analysis following immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken γ globulin showed that long-lived β-gal+ B cells exclusively contained somatically mutated λ1 V regions and were capable of producing Ag-specific Ab-forming cell (AFC) responses that were >100-fold higher than those afforded by β-gal− B cells following adoptive transfer to naive hosts. Secondary challenge of immune mice showed that only ∼20% of secondary AFCs expressed β-gal. Interestingly, we found that somatic hypermutation of rearranged λ1 V regions within secondary AFCs showed a strong correlation with β-gal expression, suggesting that nonmutated B cells contribute significantly to secondary Ab responses. This model should provide useful insights into memory B cell development, maintenance, and differentiation following immunization or pathogenic infection.
IFN-γ Acts on T Cells to Induce NK Cell Mobilization and Accumulation in Target Organs
Authors: Wald Ori , Weiss Ido D. , Wald Hanna , Shoham Hadas , Bar-Shavit Yochay , Beider Katia , Galun Eithan , Weiss Lola , Flaishon Liat , Shachar Idit , Nagler Arnon , Lu Bao , Gerard Craig , Gao Ji-Liang , Mishani Eyal , Farber Joshua , Peled Amnon
Published: April 2014

The mechanism(s) that regulates NK cell mobilization and the significance of this process to NK cell activity are unknown. After Con A-induced hepatitis, NK cells are mobilized from the spleen and bone marrow into the periphery in an IFN-γ-dependent fashion. Intraperitoneal administration of IFN-γ stimulates the mobilization of NK cells into the circulation, but not their cell death or proliferation. Increased number of circulating NK cells was coupled with their accumulation in the peritoneum, liver, and tumor-bearing lung tissue. Furthermore, increased number of NK cells in the lung reduced metastasis of Lewis lung carcinoma cells (3LL cell line) resulting in significantly extended NK-dependent survival. Mobilization of NK cells was specific and required the presence of T cells. Moreover, mobilization and migration of spleen NK cells in response to IFN-γ treatment is dependent on the chemokine receptor CXCR3. Mechanistic insights regarding the role of IFN-γ in the regulation of NK cell mobilization and their accumulation at sites of tumor metastasis may lead to the development of novel immunotherapy for cancer.
Identification of Novel Th2-Associated Genes in T Memory Responses to Allergens
Authors: Bosco Anthony , McKenna Kathy L. , Devitt Catherine J. , Firth Martin J. , Sly Peter D. , Holt Patrick G.
Published: April 2014

Atopic diseases are associated with hyperexpression of Th2 cytokines by allergen-specific T memory cells. However, clinical trials with recently developed Th2 inhibitors in atopics have proven disappointing, suggesting underlying complexities in atopy pathogenesis which are not satisfactorily explained via the classical Th1/Th2 paradigm. One likely possibility is that additional Th2-associated genes which are central to disease pathogenesis remain unidentified. The aim of the present study was to identify such novel Th2-associated genes in recall responses to the inhalant allergen house dust mite. In contrast to earlier human microarray studies in atopy which focused on mitogen-activated T cell lines and clones, we concentrated on PBMC-derived primary T cells stimulated under more physiological conditions of low dose allergen exposure. We screened initially for allergen-induced gene activation by microarray, and validated novel genes in independent panels of subjects by quantitative RT-PCR. Kinetic analysis of allergen responses in PBMC revealed an early wave of novel atopy-associated genes involved in signaling which were coexpressed with IL-4 and IL-4R, followed by a later wave of genes encoding the classical Th2 effector cytokines. We further demonstrate that these novel activation-associated Th2 genes up-regulate in response to another atopy-associated physiological stimulus bacterial superantigen, but remain quiescent in nonphysiological responses in primary T cells or cell lines driven by potent mitogens, which may account for their failure to be detected in earlier microarray studies.
In Vivo Recognition of Ovalbumin Expressed by Transgenic Leishmania Is Determined by Its Subcellular Localization
Authors: Prickett Sara , Gray Peter M. , Colpitts Sara L. , Scott Phillip , Kaye Paul M. , Smith Deborah F.
Published: April 2014

The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity.
SUMOylation Interferes with CCAAT/Enhancer-Binding Protein β-Mediated c-myc Repression, but Not IL-4 Activation in T Cells
Authors: Berberich-Siebelt Friederike , Berberich Ingolf , Andrulis Mindaugas , Santner-Nanan Brigitte , Jha Mithilesh K. , Klein-Hessling Stefan , Schimpl Anneliese , Serfling Edgar
Published: April 2014

The transcription factor C/EBPβ transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPβ also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPβ represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPβ-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPβ. Whereas sumoylation does not affect the C/EBPβ-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPβ and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPβ.
The Bovine CD1 Family Contains Group 1 CD1 Proteins, but No Functional CD1d
Authors: Van Rhijn Ildiko , Koets Ad P. , Im Jin Seon , Piebes Diewertje , Reddington Faye , Besra Gurdyal S. , Porcelli Steven A. , van Eden Willem , Rutten Victor P. M. G.
Published: April 2014

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-γ and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, α-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.
Novel Immunostimulatory Agent Based on CpG Oligodeoxynucleotide Linked to the Nontoxic B Subunit of Cholera Toxin
Authors: Adamsson Jenni , Lindblad Marianne , Lundqvist Annika , Kelly Denise , Holmgren Jan , Harandi Ali M.
Published: April 2014

In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-κB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.