bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of CD166 in the Activation of Human γδT Cells by Tumor Cells Sensitized with Nonpeptide Antigens
Authors: Kato Yu , Tanaka Yoshimasa , Hayashi Mikihito , Okawa Katsuya , Minato Nagahiro
Published: April 2014

We previously reported that human Vγ2Vδ2-γδT cells were activated by many human tumor cell lines treated with pamidronate (PAM) in a γδTCR-dependent manner. In the present study, we indicated that a synthetic pyrophosphomonoester Ag, 2-methy-3-butenyl-1-pyrophosphate, could directly “sensitize” the tumor cells to activate γδT cells independently of the host metabolism, while the sensitizing effect of PAM was reported to be dependent on the pharmacological activity. Some exceptional tumor cells that failed to be sensitized by PAM were incapable of activating γδT cells by the treatment with 2-methy-3-butenyl-1-pyrophosphate either, suggesting a requirement of host factor(s) for the effective γδT cell activation in addition to the nonpeptide Ags. By screening mAbs against a large panel of tumor cell lines, we found that the expression of CD166 closely paralleled the capacity of activating γδT cells upon PAM treatment. The transfection of a CD166-negative tumor cell line with CD166 cDNA caused a marked enhancement of the capacity to activate γδ T cells following PAM treatment. On the contrary, down-regulation of the CD166 expression in a CD166-bearing tumor cell line by short hairpin RNA resulted in a significant reduction of PAM-induced γδΤ cell-stimulatory activity. γδT cells expressed CD6, a receptor of CD166, and CD6 and CD166 were recruited together to the center of synapse between γδ T cells and PAM-treated tumor cells, colocalizing with γδTCR/CD3. The results suggested that the engagement of CD6 with CD166 on tumor cells played an important role in the γδT cell activation by the tumor cells loaded with nonpeptide Ags either endogenously or exogenously.
Suppression of Antitumor Immunity by IL-10 and TGF-β-Producing T Cells Infiltrating the Growing Tumor: Influence of Tumor Environment on the Induction of CD4+ and CD8+ Regulatory T Cells
Authors: Jarnicki Andrew G. , Lysaght Joanne , Todryk Stephen , Mills Kingston H. G.
Published: April 2014

We examined the hypothesis that a failure of the immune system to eradicate tumors is due to the immunosuppressive environment created by the growing tumor, which is influenced by the site of tumor growth. We demonstrated that T cell responses to a bystander Ag in mice were suppressed by a growing CT26 tumor. T cells purified from the growing tumor expressed mRNA for IL-10, TGF-β, and Foxp3. Intracellular cytokine staining revealed a high frequency of IL-10-secreting macrophages, dendritic cells, and CD4+ and CD8+ T cells infiltrating the tumor. In contrast, T cell IFN-γ production was weak and CD8+ CTL responses were undetectable in mice with CT26 lung metastases and weak and transient following s.c. injection of CT26 cells, but were enhanced in the presence of anti-IL-10 and anti-TGF-β. Consistent with this, removal of CD8+ T cells abrogated CTL responses and promoted progression of the s.c. tumor. However, in the lung model, depletion of CD8+ T cells significantly reduced the tumor burden. Furthermore, depletion of CD4+ or CD25+ T cells in vivo reduced tumor burden in s.c. and lung models, and this was associated with significantly enhanced IFN-γ production by CD8+ T cells. These findings suggest that tumor growth facilitates the induction or recruitment of CD4+ regulatory T cells that secrete IL-10 and TGF-β and suppress effector CD8+ T cell responses. However, CD8+ T regulatory cells expressing IL-10 and TGF-β are also recruited or activated by the immunosuppressive environment of the lung, where they may suppress the induction of antitumor immunity.
Impaired Generation of CD8+ Thymocytes in Ets-1-Deficient Mice
Authors: Clements James L. , John Shinu A. , Garrett-Sinha Lee Ann
Published: April 2014

The Ets family of transcription factors function as key regulators of multiple aspects of immune cell development and function. To date, Ets-1 has been implicated in regulating early stages of thymic maturation and lymphocyte function and homeostasis. This report describes a novel role for Ets-1 in supporting later stages of thymic selection, in that positive selection of MHC class I-restricted CD4+CD8+ double-positive thymocytes is markedly inhibited in mice expressing a hypomorphic allele of Ets-1. This effect is thymocyte intrinsic, as Ets-1 mutant thymocytes fail to efficiently generate CD8+ single-positive thymocytes in mixed bone marrow chimeric backgrounds. Although peripheral CD8+ T cells are present in Ets-1 mutant mice, both CD4+ and CD8+ subsets contain an elevated proportion of cells with an effector memory (CD62L−CD44+) phenotype. In addition, while thymic expression of Thy1 is relatively normal, peripheral T cells isolated from Ets-1 mutant mice display a striking loss of Thy1 expression. These data identify Ets-1 as a key transcription factor regulating thymocyte positive selection and lineage commitment of MHC class I-restricted thymocytes.
Influence of Mucosal Adjuvants on Antigen Passage and CD4+ T Cell Activation during the Primary Response to Airborne Allergen
Authors: Wikstrom Matthew E. , Batanero Eva , Smith Miranda , Thomas Jennifer A. , von Garnier Christophe , Holt Patrick G. , Stumbles Philip A.
Published: April 2014

Ag delivery via the nasal route typically induces tolerance or fails to polarize CD4+ T cell responses unless an adjuvant is provided. To better understand this process, we assessed the effects of two mucosal adjuvants, Escherichia coli LPS and cholera toxin (CT), on Ag passage and T cell activation in the draining lymph nodes (DLN) of BALB/c mice following per nasal administration of the model protein allergen, OVA. We found a range of cell types acquired small amounts of fluorescent OVA in the DLN 4 h after per nasal administration. However, this early uptake was eclipsed by a wave of OVA+CD8αlow dendritic cells that accumulated in the DLN over the next 20 h to become the dominant OVA-processing and -presenting population. Both LPS and CT stimulated increases in CD80 and CD86 expression on OVA+CD8αlow DC. LPS also increased the number of OVA+CD8αlow dendritic cells accumulating in the DLN. When the primary T cell response was examined after adoptive transfer of CD4+ T cells from DO11.10 mice, CT and LPS stimulated surprisingly similar effects on T cell activation and proliferation, IL-4 and IFN-γ priming, and memory T cell production. Despite these similarities, T cell recipients immunized with CT, but not LPS, developed lung eosinophilia upon secondary OVA challenge. Thus, we found no bias within the DLN in Ag handling or the primary T cell response associated with the eventual Th2 polarization induced by CT, and suggest that additional tissue-specific factors influence the development of allergic disease in the airways.
CCR5 in T Cell-Mediated Liver Diseases: What’s Going On?
Authors: Ajuebor Maureen N. , Carey Jillian A. , Swain Mark G.
Published: April 2014

The chemokine receptor CCR5 came into worldwide prominence a decade ago when it was identified as one of the major coreceptors for HIV infectivity. However, subsequent studies suggested an important modulatory role for CCR5 in the inflammatory response. Specifically, CCR5 has been reported to directly regulate T cell function in autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and type 1 diabetes. Moreover, T cell-mediated immune responses are proposed to be critical in the pathogenesis of autoimmune and viral liver diseases, and recent clinical and experimental studies have also implicated CCR5 in the pathogenesis of autoimmune and viral liver diseases. Therefore, in this brief review, we highlight the evidence that supports an important role of CCR5 in the pathophysiology of T cell-mediated liver diseases with specific emphasis on autoimmune and viral liver diseases.
Cutting Edge: Epstein-Barr Virus Transactivates the HERV-K18 Superantigen by Docking to the Human Complement Receptor 2 (CD21) on Primary B Cells
Authors: Hsiao Francis C. , Lin Miao , Tai Albert , Chen Gang , Huber Brigitte T.
Published: April 2014

EBV, a ubiquitous human herpesvirus, is the causative agent of infectious mononucleosis and is associated with many carcinomas. We have previously shown that the EBV latent genes LMP-1 and LMP-2A (for latent membrane proteins 1 and 2A), transactivate a human endogenous retrovirus (HERV), HERV-K18, in infected B lymphocytes. The envelope (Env) protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. In this study we report that HERV-K18 env is transactivated even earlier in the infection process, before the establishment of latency; namely, we found that EBV, through its interaction with its cellular receptor CD21, induces the HERV-K18 env gene in resting B lymphocytes. This transactivation is direct and immediate, as up-regulation of transcripts can be detected within 30 min after EBV exposure. Thus, EBV binding to human CD21 on resting B cells triggers the expression of an endogenous superantigen. The biological significance of this superantigen expression for the EBV life cycle is discussed.
1-Methyl-Tryptophan Can Interfere with TLR Signaling in Dendritic Cells Independently of IDO Activity
Authors: Agaugué Sophie , Perrin-Cocon Laure , Coutant Frédéric , André Patrice , Lotteau Vincent
Published: April 2014

The compound 1-methyl-tryptophan (1-MT) is a competitive inhibitor of IDO that can break tolerance and induce fetus, graft, and tumor rejection. Because of its broad effect on immune-related mechanisms, the direct action of 1-MT on human monocyte-derived dendritic cells (DC) was analyzed. It is shown here that the effect of 1-MT on DC is dependent on the maturation pathway. Although 1-MT had no effect on DC stimulated by the TLR3 ligand poly(I:C), it strongly enhanced the Th1 profile of DC stimulated with TLR2/1 or TLR2/6 ligands. Drastic changes in the function of DC stimulated by the TLR4 ligand LPS were induced by 1-MT. These cells could still activate allogeneic and syngeneic T cells but stimulation yielded T cells secreting IL-5 and IL-13 rather than IFN-γ. This action of 1-MT correlated with an increased phosphorylation of p38 and ERK MAPKs and sustained activation of the transcription factor c-Fos. Inhibiting p38 and ERK phosphorylation with synthetic inhibitors blocked the effect of 1-MT on LPS-stimulated DC. Thus, 1-MT can modulate DC function depending on the maturation signal and independently of its action on IDO. This is consistent with previous observations and will help further understanding the mechanisms of DC polarization.
Endogenous Myelin Basic Protein Is Presented in the Periphery by Both Dendritic Cells and Resting B Cells with Different Functional Consequences
Authors: Seamons Audrey , Perchellet Antoine , Goverman Joan
Published: April 2014

Multiple sclerosis is an inflammatory disease believed to be triggered by erroneous activation of self-reactive T cells specific for myelin proteins such as myelin basic protein (MBP). Inflammation is limited to the CNS, suggesting that the myelin-specific T cells encounter their Ags only after they cross the blood-brain barrier. However, our previous studies in mice showed that MBP epitopes are constitutively presented in lymphoid tissues. Here we identified which APCs in lymph nodes present endogenous MBP epitopes and determined the functional consequences of this presentation for both naive and activated MBP-specific T cells. Both CD8α+ and CD8α− dendritic cells were potent stimulators of proliferation for both naive and previously activated/memory MBP-specific T cells. Surprisingly, resting B cells also presented endogenous MBP that was acquired using a BCR-independent mechanism. Interaction with resting B cells triggered proliferation of both naive and activated MBP-specific T cells. Activated/memory MBP-specific T cells proliferating in response to resting B cells presenting endogenous MBP did not produce cytokines and became more refractory to subsequent stimulation. Interestingly, cytokine production by activated/memory T cells was triggered by resting B cells if the number of MBP epitopes presented was increased by adding exogenous MBP peptide. These results suggest that activated MBP-specific T cells may become less pathogenic in vivo following encounter with resting B cells presenting steady-state levels of endogenous MBP but can expand and remain pathogenic if the amount of MBP presented by B cells is increased, which could occur during chronic demyelinating disease.
Type 2 Cytotoxic T Lymphocytes Modulate the Activity of Dendritic Cells Toward Type 2 Immune Responses
Authors: Iezzi Giandomenica , Boni Andrea , Degl’Innocenti Elena , Grioni Matteo , Bertilaccio Maria T. S. , Bellone Matteo
Published: April 2014

Activated CD8+ T cells can differentiate into type 1 (Tc1) cells, producing mainly IFN-γ, and type 2 (Tc2) cells, producing mostly IL-4, IL-5, and IL-10. Tc1 cells are potent CTL involved in the defense against intracellular pathogens and cancer cells. The role of Tc2 cells in the immune response is largely unknown, although their presence in chronic infections, cancer, and autoimmune diseases is associated with disease severity and progression. Here, we show that mouse Tc2 cells modify, through a cell-to-cell contact mechanism, the function of bone marrow-derived dendritic cells (DC). Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and costimulatory molecules, produced IL-10 instead of IL-12, and favored the differentiation of both naive CD4+ and CD8+ T cells toward type 2 cells in the absence of added polarizing cytokines. The novel function for Tc2 cells suggests a type 2 loop in which Tc2 cells modify DC function and favor differentiation of naive T cells to type 2 cells. The type 2 loop may at least in part explain the unexpected high frequency of type 2 cells during a chronic exposure to the Ag.
Porcine UL16-Binding Protein 1 Expressed on the Surface of Endothelial Cells Triggers Human NK Cytotoxicity through NKG2D
Authors: Lilienfeld Benjamin G. , Garcia-Borges Carmen , Crew Mark D. , Seebach Jörg D.
Published: April 2014

Cellular rejection mechanisms, including NK cells, remain a hurdle for successful pig-to-human xenotransplantation. Human anti-pig NK cytotoxicity depends on the activating receptor NKG2D. Porcine UL16-binding protein 1 (pULBP1) and porcine MHC class I chain-related protein 2 (pMIC2) are homologues of the human NKG2D ligands ULBP 1–4 and MICA and B, respectively. Although transcribed in porcine endothelial cells (pEC), it is not known whether pULBP1 and pMIC2 act as functional ligands for human NKG2D. In this study, surface protein expression of pULBP1 was demonstrated by flow cytometry using a novel pULBP1-specific polyclonal Ab and by cellular ELISA using NKG2D-Fc fusion protein. Reciprocally, pULBP1-Fc bound to primary human NK cells, whereas pMIC2-Fc did not. Transient and stable down-regulation of pULBP1 mRNA in pEC using short-interfering RNA oligonucleotide duplexes and short hairpin RNA, respectively, resulted in a partial inhibition of xenogeneic NK cytotoxicity through NKG2D in 51Cr release assays. In contrast, down-regulation of pMIC2 mRNA did not inhibit NK cytotoxicity. Human NK cytotoxicity against pEC mediated by freshly isolated or IL-2-activated NK cells through NKG2D was completely blocked using anti-pULBP1 polyclonal Ab. In conclusion, this study suggests that pULBP1 is the predominant, if not only, functional porcine ligand for human NKG2D. Thus, the elimination of pULBP1 on porcine tissues represents an attractive target to protect porcine xenografts from human NK cytotoxicity.
Induction of Allospecific Tolerance by Immature Dendritic Cells Genetically Modified to Express Soluble TNF Receptor
Authors: Wang Quanxing , Liu Yushan , Wang Jianli , Ding Guoshan , Zhang Weiping , Chen Guoyou , Zhang Minghui , Zheng Shusen , Cao Xuetao
Published: April 2014

The ability of dendritic cells (DC) to initiate immune responses or induce immune tolerance is strictly dependent on their maturation state. TNF-α plays a pivotal role in the differentiation and maturation of DC. Blockade of TNF-α action may arrest DC in an immature state, prolonging their window of tolerogenic opportunity. Immature DC (imDC) were transfected with recombinant adenovirus to express soluble TNF-α receptor type I (sTNFRI), a specific inhibitor of TNF-α. The capacity of sTNFRI gene-modified imDC (DC-sTNFRI) to induce immune tolerance was analyzed. sTNFRI expression renders imDC resistant to maturation induction and impairs their capacity to migrate or present Ag. This process leads to induction of allogeneic T cell hyporesponsiveness and the generation of IL-10-producing T regulatory-like cells. In vivo pretreatment of transplant recipients with DC-sTNFRI induces long-term survival of cardiac allografts in 50% of cases, and leads to a substantial increase in the generation of microchimerism and T regulatory cell numbers. Thus, blockade of TNF-α action by sTNFRI genetic modification can inhibit the maturation of DC and potentiate the in vivo capacity of imDC to induce donor-specific immune tolerance and prolong allograft survival.
Salmonella typhimurium Infection in Nonobese Diabetic Mice Generates Immunomodulatory Dendritic Cells Able to Prevent Type 1 Diabetes
Authors: Raine Tim , Zaccone Paola , Mastroeni Pietro , Cooke Anne
Published: April 2014

Infection, commencing across a wide age range, with a live, attenuated strain of Salmonella typhimurium, will halt the development of type 1 diabetes in the NOD mouse. The protective mechanism appears to involve the regulation of autoreactive T cells in a manner associated with long lasting changes in the innate immune compartment of these mice. We show in this study that autoreactive T cell priming and trafficking are altered in mice that have been infected previously by S. typhimurium. These changes are associated with sustained alterations in patterns of chemokine expression. We find that small numbers of dendritic cells from mice that have been previously infected with, but cleared all trace of a S. typhimurium infection are able to prevent the development of diabetes in the highly synchronized and aggressive cyclophosphamide-induced model. The effects we observe on autoreactive T cell trafficking are recapitulated by the immunomodulatory dendritic cell transfers in the cyclophosphamide model.
Pre-B Cell Receptor Assesses the Quality of IgH Chains and Tunes the Pre-B Cell Repertoire by Delivering Differential Signals
Authors: Kawano Yohei , Yoshikawa Soichiro , Minegishi Yoshiyuki , Karasuyama Hajime
Published: April 2014

It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that μH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of μH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their μH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their μH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of μH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of μH chains.
FcRγ Presence in TCR Complex of Double-Negative T Cells Is Critical for Their Regulatory Function
Authors: Thomson Christopher W. , Teft Wendy A. , Chen Wenhao , Lee Boris P.-L. , Madrenas Joaquin , Zhang Li
Published: April 2014

TCRαβ+CD4−CD8− double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRγ was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRγ-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRγ-sufficient DN T cells, FcRγ-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRγ, DN Treg cell clones also express higher levels of TCRβ, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRγ associates with the TCR complex and that both FcRγ and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRγ expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRγ deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRγ plays a role in mediating TCR signaling in DN Treg cells.
Sialoadhesin Promotes the Inflammatory Response in Experimental Autoimmune Uveoretinitis
Authors: Jiang Hui-Rong , Hwenda Lenias , Makinen Kimmo , Oetke Cornelia , Crocker Paul R. , Forrester John V.
Published: April 2014

Macrophages are a prominent component of the effector cell compartment in a number of CD4+ T cell-mediated organ-specific autoimmune diseases. In this study, we investigated the role of the sialic acid binding Ig-like lectin sialoadhesin (Sn, Siglec-1) in a model of interphotoreceptor retinal binding protein peptide-induced experimental autoimmune uveoretinitis in mice with targeted deletion of Sn. Our data show that compared with wild-type mice, experimental autoimmune uveoretinitis is reduced in severity in the initial stages in the Sn knockout (KO) mice. In addition, there is a reduction in the proliferative capacity of T cells from the KO mice draining lymph nodes after immunization with interphotoreceptor retinal binding protein peptides, which is manifest some days before disease onset and persists for the duration of disease. Furthermore, activated T cells from the draining lymph nodes of Sn KO mice secrete lower levels of IFN-γ. The data suggest a role for Sn in “fine tuning” the immune response to autoantigens by modulating T cell priming.
Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes
Authors: Arruda Luciana B. , Sim Del , Chikhlikar Priya R. , Maciel Milton , Akasaki Kenji , August J. Thomas , Marques Ernesto T. A.
Published: April 2014

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.
Preferential Induction of CD4+ T Cell Responses through In Vivo Targeting of Antigen to Dendritic Cell-Associated C-Type Lectin-1
Authors: Carter Robert W. , Thompson Clare , Reid Delyth M. , Wong Simon Y. C. , Tough David F.
Published: April 2014

Targeting of Ags and therapeutics to dendritic cells (DCs) has immense potential for immunotherapy and vaccination. Because DCs are heterogeneous, optimal targeting strategies will require knowledge about functional specialization among DC subpopulations and identification of molecules for targeting appropriate DCs. We characterized the expression of a fungal recognition receptor, DC-associated C-type lectin-1 (Dectin-1), on mouse DC subpopulations and investigated the ability of an anti-Dectin-1 Ab to deliver Ag for the stimulation of immune responses. Dectin-1 was shown to be expressed on CD8α−CD4−CD11b+ DCs found in spleen and lymph nodes and dermal DCs present in skin and s.c. lymph nodes. Injection of Ag-anti-Dectin-1 conjugates induced CD4+ and CD8+ T cell and Ab responses at low doses where free Ag failed to elicit a response. Notably, qualitatively different immune responses were generated by targeting Ag to Dectin-1 vs CD205, a molecule expressed on CD8α+CD4−CD11b− DCs, dermal DCs, and Langerhans cells. Unlike anti-Dectin-1, anti-CD205 conjugates failed to elicit an Ab response. Moreover, when conjugates were injected i.v., anti-Dectin-1 stimulated a much stronger CD4+ T cell response and a much weaker CD8+ T cell response than anti-CD205. The results reveal Dectin-1 as a potential targeting molecule for immunization and have implications for the specialization of DC subpopulations.
The Th2 Lymphoproliferation Developing inLatY136FMutant Mice Triggers Polyclonal B Cell Activation and Systemic Autoimmunity
Authors: Genton Céline , Wang Ying , Izui Shozo , Malissen Bernard , Delsol Georges , Fournié Gilbert J. , Malissen Marie , Acha-Orbea Hans
Published: April 2014

Lat Y136F knock-in mice harbor a point mutation in Tyr136 of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the LatY136F mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of LatY136F mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In LatY136F mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in LatY136F serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in LatY136F mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.
Constitutively Active β-Catenin Promotes Expansion of Multipotent Hematopoietic Progenitors in Culture
Authors: Baba Yoshihiro , Yokota Takafumi , Spits Hergen , Garrett Karla P. , Hayashi Shin-Ichi , Kincade Paul W.
Published: April 2014

This study was designed to investigate one component of the Wnt/β-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable β-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-KitlowSca-1low/−CD19− CD11b/Mac-1−Flk-2−CD43+AA4.1+NK1.1−CD3−CD11c−Gr-1−CD45R/B220+ cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a β-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.
TRAIL-Mediated Apoptosis in HIV-1-Infected Macrophages Is Dependent on the Inhibition of Akt-1 Phosphorylation
Authors: Huang Yunlong , Erdmann Nathan , Peng Hui , Herek Shelley , Davis John S. , Luo Xu , Ikezu Tsuneya , Zheng Jialin
Published: April 2014

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.
Antigen Chemically Coupled to the Surface of Liposomes Are Cross-Presented to CD8+ T Cells and Induce Potent Antitumor Immunity
Authors: Taneichi Maiko , Ishida Hideaki , Kajino Kiichi , Ogasawara Kazumasa , Tanaka Yuriko , Kasai Michiyuki , Mori Masahito , Nishida Mitsuhiro , Yamamura Hiroyuki , Mizuguchi Junichiro , Uchida Tetsuya
Published: April 2014

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA257–264-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA257–264 peptides in the context of H-2Kb was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.
Homeostatic Lymphoid Chemokines Synergize with Adhesion Ligands to Trigger T and B Lymphocyte Chemokinesis
Authors: Stachowiak Agnieszka N. , Wang Yana , Huang Yen-Chen , Irvine Darrell J.
Published: April 2014

Homeostatic chemokines such as CCL19, CCL21, and CXCL13 are known to elicit chemotaxis from naive T and B cells and play a critical role in lymphocyte homing to appropriate zones within secondary lymphoid organs (SLO). Here we tested whether CCL21 and CXCL13 modulate murine lymphocyte motility in the absence of concentration gradients, using videomicroscopy to directly observe the migration of single cells. CCL21 treatment of T cells induced rapid polarization and sustained random migration with average speeds of 5.16 ± 2.08 μm/min; B cell migration (average velocity 4.10 ± 1.58 μm/min) was similarly induced by CXCL13. Migration required the presence of both chemokine and adhesion ligands and was sustained for >24 h. Furthermore, in in vitro assays modeling the relative infrequency of Ag-specific T cell-dendritic cell (DC) encounters during primary immune responses, we found that CCL21 addition to T-DC cocultures accelerated the kinetics of CD69 up-regulation and enhanced by 2-fold the proliferation of Ag-specific T cells in a manner dependent on G-protein-coupled receptor signaling in T cells. These results suggest that homeostatic chemokines could substantially impact the dynamics and priming of lymphocytes within SLO even in the absence of significant concentration gradients.
ICOS-Induced B7h Shedding on B Cells Is Inhibited by TLR7/8 and TLR9
Authors: Logue Eric C. , Bakkour Sonia , Murphy Michael M. , Nolla Hector , Sha William C.
Published: April 2014

We report in this study that B7h, the ligand for the ICOS costimulatory receptor, is rapidly shed from mouse B cells following either ICOS binding or BCR engagement. Shedding occurs through proteolytic cleavage that releases the extracellular ICOS-binding region of B7h. Prior exposure of B7h-expressing APCs to ICOS-expressing cells inhibits their subsequent ability to costimulate IFN-γ and IL-4 production from CD4+ T cells. Shedding is regulated as TLR7/8 and TLR9 ligands inhibit B7h shedding. A shedding-resistant B7h mutant elicits greater costimulation of IFN-γ production from CD4+ T cells than does wild-type B7h. These data define shedding of B7h as a novel mechanism for controlling costimulatory signaling by B7-CD28 family members that is regulated on B cells by TLR signaling.
Activation of NFAT Signal In Vivo Leads to Osteopenia Associated with Increased Osteoclastogenesis and Bone-Resorbing Activity
Authors: Ikeda Fumiyo , Nishimura Riko , Matsubara Takuma , Hata Kenji , Reddy Sakamuri V. , Yoneda Toshiyuki
Published: April 2014

The transcription factor family member NFAT plays an important role in the regulation of osteoclast differentiation. However, the role of NFAT in osteoclasts in vivo is still not fully understood. Thus, we generated transgenic mice in which constitutively active-NFAT1/NFATc2 (CA-NFAT1) is specifically expressed in the osteoclast lineage, using the tartrate-resistant acid phosphatase gene promoter. Both x-ray and histological analyses demonstrated an osteopenic bone phenotype in the CA-NFAT1 transgenic mice, whereas the number of tartrate-resistant acid phosphatase-positive osteoclasts was markedly higher in the long bones of these mice. Furthermore, the bone-resorbing activity of mature osteoclasts derived from the transgenic mice was much higher than that of wild-type mice. Interestingly, the introduction of CA-NFAT1 into osteoclasts or RAW264 cells increased the expression and activity of c-Src and stimulated actin ring formation. In contrast, CA-NFAT1 or GFP-tagged VIVIT peptide, a specific inhibitor of NFAT, did not affect the survival of mature osteoclasts. Collectively, our data indicate that NFAT controls bone resorption in vivo by stimulating the differentiation and functioning of osteoclasts but not their survival.
A Pathogenic Role for CD8+ T Cells in a Spontaneous Model of Demyelinating Disease
Authors: Brisebois Marcel , Zehntner Simone P. , Estrada José , Owens Trevor , Fournier Sylvie
Published: April 2014

Transgenic (Tg) mice that overexpress the costimulatory ligand B7.2/CD86 on microglia spontaneously develop a T cell-mediated demyelinating disease. Characterization of the inflammatory infiltrates in the nervous tissue revealed a predominance of CD8+ T cells, suggesting a prominent role of this T cell subset in the pathology. In this study, we show that the same neurological disease occurred in Tg mice deficient in the generation of CD4+ T cells, with an earlier time of onset. Analysis of the CD8+ T cell repertoire at early stage of disease revealed the presence of selected clonal expansions in the CNS but not in peripheral lymphoid organs. We further show that Tg animals deficient in IFN-γ receptor expression were completely resistant to disease development. Microglia activation that is an early event in disease development is IFN-γ dependent and thus appears as a key element in disease pathogenesis. Collectively, our data indicate that the spontaneous demyelinating disease in this animal model occurs as a consequence of an inflammatory response initiated through the activation of CNS-specific CD8+ T cells by Tg expression of B7.2 within the target organ. Thus, autoreactive CD8+ T cells can contribute directly to the pathogenesis of neuroinflammatory diseases such as multiple sclerosis.
Identification of PP1α as a Caspase-9 Regulator in IL-2 Deprivation-Induced Apoptosis
Authors: Dessauge Frédéric , Cayla Xavier , Albar Juan Pablo , Fleischer Aarne , Ghadiri Ata , Duhamel Marianne , Rebollo Angelita
Published: April 2014

One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylates caspase-9 at Thr125, and this phosphorylation is crucial for caspase-9 inhibition. Until now, the phosphatase responsible for Thr125 dephosphorylation has not been described. Here, we demonstrate that in IL-2-proliferating cells, phosphorylated serine/threonine phosphatase type 1α (PP1α) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1α dephosphorylation, which leads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of both molecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we show that in this process PP1α is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1α associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylation and activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1α consensus motifs, and we demonstrate that these novel sites mediate the interaction of PP1α with caspase-9.
Diverse Immunoglobulin Light Chain Organizations in Fish Retain Potential to Revise B Cell Receptor Specificities
Authors: Hsu Ellen , Criscitiello Michael F.
Published: April 2014

We have characterized the genomic organization of the three zebrafish L chain isotypes and found they all differed from those reported in other teleost fishes. Two of the zebrafish L chain isotypes are encoded by two loci, each carrying multiple V gene segments. To understand the derivation of these L chain genes and their organizations, we performed phylogenetic analyses and show that IgL organization can diverge considerably among closely related species. Except in zebrafish, the teleost fish IgL each contain only two to four recombinogenic components (one to three V, one J) and exist in multiple copies. BCR heterogeneity can be generated, but this arrangement apparently provides neither combinatorial diversification nor an opportunity for the secondary rearrangements that, in mammals, take place during receptor editing, a process crucial to the promotion of tolerance in developing lymphocytes. Examination of the zebrafish IgL recombination possibilities gave insight into how the suppression of self-reactivity by receptor editing might be managed, including in miniloci. We suggest that, despite the diverse IgL organizations in early and higher vertebrates, two elements essential to generating the Ab repertoire are retained: the numerous genes/loci for ligand-binding diversification and the potential for correcting unwanted specificities that arise.
TCR-α CDR3 Loop Audition Regulates Positive Selection
Authors: Ferreira Cristina , Furmanski Anna , Millrain Maggie , Bartok Istvan , Guillaume Philippe , Lees Rosemary , Simpson Elizabeth , MacDonald H. Robson , Dyson Julian
Published: April 2014

How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-β-transgenic mice in which MHC shapes TCR-α use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of αβ pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the α CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.
Identification and Characterization of a FcR Homolog in an Ectothermic Vertebrate, the Channel Catfish (Ictalurus punctatus)
Authors: Stafford James L. , Wilson Melanie , Nayak Deepak , Quiniou Sylvie M. , Clem L. W. , Miller Norman W. , Bengtén Eva
Published: April 2014

An FcR homolog (IpFcRI), representing the first such receptor from an ectothermic vertebrate, has been identified in the channel catfish (Ictalurus punctatus). Mining of the catfish expressed sequence tag databases using mammalian FcR sequences for CD16, CD32, and CD64 resulted in the identification of a teleost Ig-binding receptor. IpFcRI is encoded by a single-copy gene containing three Ig C2-like domains, but lacking a transmembrane segment and cytoplasmic tail. The encoded Ig domains of IpFcRI are phylogenetically and structurally related to mammalian FcR and the presence of a putative Fc-binding region appears to be conserved. IpFcRI-related genomic sequences are also present in both pufferfish and rainbow trout, indicating the likely presence of a soluble FcR in other fish species. Northern blot and qualitative PCR analyses demonstrated that IpFcRI is primarily expressed in IgM-negative leukocytes derived from the lymphoid kidney tissues and PBL. Significantly lower levels of IpFcRI expression were detected in catfish clonal leukocyte cell lines. Using the native leader, IpFcRI was secreted when transfected into insect cells and importantly the native IpFcRI glycoprotein was detected in catfish plasma using a polyclonal Ab. Recombinant IpFcRI binds catfish IgM as assessed by both coimmunoprecipation and cell transfection studies and it is presumed that it functions as a secreted FcR akin to the soluble FcR found in mammals. The identification of an FcR homolog in an ectothermic vertebrate is an important first step toward understanding the evolutionary history and functional importance of vertebrate Ig-binding receptors.
NK Cells Use NKG2D to Recognize a Mouse Renal Cancer (Renca), yet Require Intercellular Adhesion Molecule-1 Expression on the Tumor Cells for Optimal Perforin-Dependent Effector Function
Authors: Abdool Karen , Cretney Erika , Brooks Alan D. , Kelly Janice M. , Swann Jeremy , Shanker Anil , Bere Earl W. , Yokoyama Wayne M. , Ortaldo John R. , Smyth Mark J. , Sayers Thomas J.
Published: April 2014

The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1γ, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.