bluuebunny/crossref_metadata_2025 huggingface dataset:
Critical Role for CXCR4 Signaling in Progenitor Localization and T Cell Differentiation in the Postnatal Thymus
Authors: Plotkin Jason , Prockop Susan E. , Lepique Ana , Petrie Howard T.
Published: April 2014

T cell differentiation in the thymus depends on sequential interactions between lymphoid progenitors and stromal cells in discrete regions of the cortex. Here we show that CXCL12/CXCR4 signaling is absolutely required for proper localization of early progenitors into the cortex and thus for successful steady state differentiation. All early progenitors in the thymus express CXCR4, and its ligand (CXCL12) is expressed only by stromal cells in the cortex, where early progenitors are found. Early progenitors migrate in response to CXCL12 in vitro, while thymus-specific deletion of CXCR4 in vivo results in failed cortical localization and developmental arrest. These findings indicate a crucial and nonredundant role for CXCR4 in facilitating localization of early lymphoid progenitors to tissue regions of the thymus, where lineage commitment and proliferation are controlled.
Cross-Linking of the Mannose Receptor on Monocyte-Derived Dendritic Cells Activates an Anti-Inflammatory Immunosuppressive Program
Authors: Chieppa Marcello , Bianchi Giancarlo , Doni Andrea , Del Prete Annalisa , Sironi Marina , Laskarin Gordana , Monti Paolo , Piemonti Lorenzo , Biondi Andrea , Mantovani Alberto , Introna Martino , Allavena Paola
Published: April 2014

Immature monocyte-derived dendritic cells (DC) strongly express the endocytic mannose receptor (MR). Addition of a specific anti-MR mAb (clone PAM-1) for 24 h to cultures of immature DC induced phenotypical and functional maturation of the cells, assessed as up-regulation of costimulatory molecules and CD83, and chemotactic response to CCL19. A different isotype-matched anti-MR mAb (clone 19.2) had no significant effect. Engagement of MR with mAb PAM-1 induced the production of the anti-inflammatory cytokines IL-10, IL-1R antagonist, and of the nonsignaling IL-1R type II. In contrast IL-1β, TNF, and IL-12 were not produced. PAM-1-treated DC were unable to polarize Th1 effector cells and did not secrete the chemokines CXCL10 and CCL19; in turn, they produced large amounts of CCL22 and CCL17, thus favoring the amplification of Th2 circuits. T cells cocultured with PAM-1-matured DC initially proliferated but later became anergic and behaved as suppressor/regulatory cells. Natural ligands binding to MR had differential effects. MUC III (a partially purified mucin), biglycan (a purified complex proteoglycan), and mannosylated lipoarabinomannan from Mycobacterium tuberculosis affected cytokine production with high IL-10, IL-1R antagonist, IL-1R type II, and inhibition of IL-12. In contrast, mannan, dextran, and thyroglobulin had no significant effect. In conclusion, the appropriate engagement of the MR by mAb PAM-1 and selected natural ligands elicit a secretory program in mono-derived DC characterized by a distinct profile of cytokines/chemokines with the ability to dampen inflammation and to inhibit the generation of Th1-polarized immune responses.
Dectin-1 Expression and Function Are Enhanced on Alternatively Activated and GM-CSF-Treated Macrophages and Are Negatively Regulated by IL-10, Dexamethasone, and Lipopolysaccharide
Authors: Willment Janet A. , Lin Hsi-Hsen , Reid Delyth M. , Taylor Philip R. , Williams David L. , Wong Simon Y. C. , Gordon Siamon , Brown Gordon D.
Published: April 2014

Dectin-1 is the major macrophage receptor for β-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mφ). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-γ, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-α, although some Mφ-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.
Absence of Programmed Death Receptor 1 Alters Thymic Development and Enhances Generation of CD4/CD8 Double-Negative TCR-Transgenic T Cells
Authors: Blank Christian , Brown Ian , Marks Reinhard , Nishimura Hiroyuki , Honjo Tasuku , Gajewski Thomas F.
Published: April 2014

Programmed death receptor 1 (PD-1) is expressed on thymocytes in addition to activated lymphocyte cells. Its ligation is thought to negatively regulate T cell activation, and PD-1−/− mice develop autoimmunity. To study the role of PD-1 on the development and function of a monoclonal CD8+ T cell population, 2C TCR-transgenic/recombination-activating gene 2−/−/PD-1−/− mice were generated. Unexpectedly, ∼30% of peripheral T cells in these mice were CD4/CD8 double negative (DN). Although the DN cells were not activated by Ag-expressing APCs, they functioned normally in response to anti-CD3/anti-CD28. These cells had a naive surface phenotype and lacked expression of NK1.1, B220, and γδ TCR; and the majority did not up-regulate CD8αα expression upon activation, arguing that they are not predominantly diverted γδ-lineage cells. The thymus was studied in detail to infer the mechanism of generation of DN peripheral T cells. Total thymus cellularity was reduced in 2C TCR-transgenic/recombination-activating gene 2−/−/PD-1−/− mice, and a relative increase in DN cells and decrease in double-positive (DP) cells were observed. Increased annexin V+ cells among the DP population argued for augmented negative selection in PD-1−/− mice. In addition, an increased fraction of the DN thymocytes was HSA negative, suggesting that they had undergone positive selection. This possibility was supported by decreased emergence of DN PD-1−/− 2C cells in H-2k bone marrow chimera recipients. Our results are consistent with a model in which absence of PD-1 leads to greater negative selection of strongly interacting DP cells as well as increased emergence of DN αβ peripheral T cells.
Early Events in Peripheral Regulatory T Cell Induction via the Nasal Mucosa
Authors: Unger Wendy W. J. , Hauet-Broere Femke , Jansen Wendy , van Berkel Lisette A. , Kraal Georg , Samsom Janneke N.
Published: April 2014

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4+ regulatory T cells (TR cells) from the spleen, but little is known about the induction of mucosal TR cells in vivo. To investigate the induction of TR cells in the nose-draining cervical lymph node (CLN), CD4+ T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4+ DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4+ DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4+ DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4+ DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4+ DO11.10 T cells from CLN were phenotypically similar to CD4+ DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25+ proliferating CD4+ DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal TR cells because both CD25+ and CD25− CD4+ DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of TR and effector T cells, this does not include their function. We are the first to demonstrate that functional TR cells, which reside within both CD25+ and CD25− subsets, can be isolated from CLN as early as 3 days after nasal OVA application.
CD4+CD25+ T Cells Lyse Antigen-Presenting B Cells by Fas-Fas Ligand Interaction in an Epitope-Specific Manner
Authors: Janssens Wim , Carlier Vincent , Wu Bo , VanderElst Luc , Jacquemin Marc G. , Saint-Remy Jean-Marie R.
Published: April 2014

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4+CD25+ population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4+ cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4+ T cell line derived from BALB/c mice immunized with peptide 21–35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4+CD25+ T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.
Ly49D Receptor Expressed on Immature B Cells Regulates Their IFN-γ Secretion, Actin Polymerization, and Homing
Authors: Hart Gili , Flaishon Liat , Becker-Herman Shirly , Shachar Idit
Published: April 2014

Low levels of IFN-γ secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN-γ secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN-γ transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN-γ gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN-γ and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation.
A Novel Interaction between Protein Kinase D and TNF Receptor-Associated Factor Molecules Regulates B Cell Receptor-CD40 Synergy
Authors: Haxhinasto Sokol A. , Bishop Gail A.
Published: April 2014

Signaling by Ag to the B cell Ag receptor (BCR) is enhanced by several cooperating signals, including several provided by B-T cell interactions. One of these, CD40, provides critical signals for B cell differentiation, isotype switching, and B cell memory. The molecular mechanisms by which BCR and CD40 signals synergize are not well understood. Although the BCR and CD40 share certain signaling pathways, we hypothesized that unique signals provided by each could provide mutual enhancement of their signaling pathways. The BCR, but not CD40, activates protein kinase D (PKD), while CD40, but not the BCR, employs the TNFR-associated factor (TRAF) adapter proteins in signaling. In this study, we show that genetic or pharmacologic inhibition of BCR-mediated PKD activation in B lymphocytes abrogated the synergy between the CD40 and the BCR, as measured by activation of Ig and cytokine secretion. Interestingly, the role of PKD was dependent upon the association of CD40 with TRAF2, and was inhibited by the binding of TRAF3, revealing a novel functional link between these two classes of signaling molecules.
Selection of Ig μ Heavy Chains by Complementarity-Determining Region 3 Length and Amino Acid Composition
Authors: Martin Denise A. , Bradl Harald , Collins Tara J. , Roth Edith , Jäck Hans-Martin , Wu Gillian E.
Published: April 2014

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring VH81X and JH4 gene segments from JH+/− and λ5−/−mice. VH81X-JH4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.
Silent Cleanup of Very Early Apoptotic Cells by Macrophages
Authors: Kurosaka Kahori , Takahashi Munehisa , Watanabe Naoko , Kobayashi Yoshiro
Published: April 2014

Apoptotic cells are phagocytosed as soon as they appear in vivo. In this study, we first determined precisely at what stage apoptotic cells are phagocytosed by macrophages, and then examined the subsequent cytokine production. Phagocytosis was confirmed by flow cytometry and confocal laser microscopy, whereas the subsequent response was examined by ELISA and RT-PCR for quantitative and semiquantitative measurement of the protein and mRNA levels of cytokines, respectively. Even the cell populations containing very early apoptotic cells, such as IL-2-dependent CTLL-2 cells cultured in the absence of IL-2 for 4 h and a murine leukemic cell line, P388 cells, treated with etoposide for 5 h, were phagocytosed by macrophages. Although the cell populations containing the very early apoptotic cells used in this study were FITC-Annexin V-negative and did not show a decrease in cell size as compared with untreated cells, they showed a very small increase in phosphatidylserine on the cell surface, as detected with Cy3-Annexin V, and a decrease in mitochondrial membrane potential, indicating that the cell populations had already started the apoptotic process. Phagocytosis of such populations containing very early apoptotic cells was inhibited by phospho-l-serine much more significantly than Arg-Gly-Asp-Ser. In addition, macrophages hardly produced either proinflammatory or anti-inflammatory cytokines after phagocytosis, thus being an almost null response. These results are contrary to the generally accepted concept that the phagocytosis of apoptotic cells leads to the production of anti-inflammatory cytokines, suggesting instead that cells starting to undergo apoptosis are quickly phagocytosed by macrophages without any inflammation in vivo.
TNF-α-Dependent ICAM-1- and VCAM-1-Mediated Inflammatory Responses Are Delayed in Neonatal Mice Infected with Pneumocystis carinii
Authors: Qureshi Mahboob H. , Cook-Mills Joan , Doherty Dennis E. , Garvy Beth A.
Published: April 2014

Neonatal mice have a delayed CD4-mediated inflammatory response to Pneumocystis carinii (PC) infection in the lungs that corresponds to a delayed TNF-α response and a delayed clearance of the organisms compared with adult mice. Since TNF-α is known to drive the up-regulation of adhesion molecules, we examined the expression and function of adhesion molecules in the lungs of neonatal mice. The expression of both ICAM-1 and VCAM-1 was significantly lower in the lungs of PC-infected neonatal mice compared with adults. Additionally, migration of neonatal T cells across endothelial cells expressing VCAM-1 and monocyte chemotactic protein-1 was aberrant compared with that in adult T cells, although α4β1 integrin-mediated adhesion of neonatal lymphocytes was comparable to that of adult lymphocytes. Treatment of neonatal mice with exogenous TNF-α resulted in increased expression of ICAM-1 and VCAM-1 as well as increased expression of chemokines, resulting in infiltration of CD4+ cells into the lungs. Treatment with exogenous TNF-α resulted in a trend (although not statistically significant) toward a reduction of PC organisms from the lungs. These data indicate that neonatal lung endothelial cells do not up-regulate ICAM-1 and VCAM-1 in response to PC infection, probably due to depressed TNF-α production. Additionally, neonatal T cells are defective in the ability to migrate across endothelial cells.
IL-4 Suppresses the Expression and the Replication of Hepatitis B Virus in the Hepatocellular Carcinoma Cell Line Hep3B
Authors: Lin Sue-Jane , Shu Pei-Yun , Chang Chungming , Ng Ah-Kau , Hu Cheng-po
Published: April 2014

IL-4 has been known as a Th2 cytokine and can act on B cells, T cells, and monocytes. In this study we demonstrate that IL-4Rs are expressed on human hepatocellular carcinoma (HCC) cells. We found that IL-4 suppresses hepatitis B surface Ag (HBsAg) mRNA and HBsAg production in the Hep3B cell line, which contains an integrated hepatitis B virus (HBV) genome and constitutively secretes HBsAg. When Hep3B cells are further transfected with the plasmid pHBV3.6 that contains >1 U of HBV genome, IL-4 could suppress the production of all HBV RNA and secreted HBsAg and hepatitis B virus e Ag. Furthermore, an endogenous DNA polymerase activity assay shows a decrease in HBV DNA after IL-4 treatment. Using luciferase reporter assays we have demonstrated that IL-4 could suppress the activity of the surface promoter II and the core promotor (CP). To delineate how IL-4 suppressed the transcription of HBV genes, we have examined the effect of IL-4 on the expression of transcription factors that are known to bind to the core upstream regulatory sequence, which colocalizes with enhancer II of the HBV genome. Our results demonstrate that IL-4 suppresses the expression of C/EBPα. Furthermore, overexpression of C/EBPα blocked 43 and 30% of the IL-4-mediated suppression of CP activity and IL-4-induced suppression of pregenomic RNA, respectively. Finally, we have demonstrated that mutations affecting the C/EBPα-binding sites on core upstream regulatory sequence/enhancer II completely abolish the IL-4-mediated suppression of CP activity. Thus, down-regulation of C/EBPα may be involved in the anti-HBV effect of IL-4 in Hep3B cells.
M Cell DNA Vaccination for CTL Immunity to HIV
Authors: Wang Xinhai , Hone David M. , Haddad Asmahan , Shata Mohamed T. , Pascual David W.
Published: April 2014

To facilitate invasion, reovirus has evolved to attach to M cells, a specialized epithelium residing within the follicle-associated epithelium that covers mucosal inductive tissues. Thus, we questioned adapting reovirus protein σ1 to ferry DNA vaccines to the mucosa to immunize against HIV. Three expression plasmids encoding HIV(Ba-L) gp160, cytoplasmic gp140, and secreted gp140 were tested in mice as protein σ1-poly-l-lysine-DNA complexes (formulated vaccine) via the intranasal route. Evaluation of cell-mediated immunity showed that the formulated gp160 DNA vaccine was more effective for stimulating envelope (Env)-specific CTL responses in lungs, lower respiratory lymph nodes (LN), cervical LN, submaxillary gland LN, and spleens. Three doses of vaccine were required for CTL responses, and intranasal naked DNA immunizations were ineffective. The greatest CTL activity was observed between weeks 8 and 10 for gp160-vaccinated mice, and activity remained detectable by week 16. These Env-specific CTL responses were perforin dependent in peripheral tissues, but mostly Fas dependent in the lungs. These Env-specific CTLs also produced IFN-γ. Mice vaccinated with the formulated gp160 DNA vaccine showed potent antiviral immunity against vaccinia virus-env replication in ovaries. Thus, compared with live vectors, protein σ1-mediated DNA delivery represents an alternative mucosal formulation for inducing cellular immunity against HIV-1.
Innate Inhibition of Adaptive Immunity:Mycobacterium tuberculosis-Induced IL-6 Inhibits Macrophage Responses to IFN-γ
Authors: Nagabhushanam Vijaya , Solache Alejandra , Ting Li-Min , Escaron Claire J. , Zhang Jennifer Y. , Ernst Joel D.
Published: April 2014

In humans and in mice, control of the intracellular pathogen, Mycobacterium tuberculosis (Mtb), requires IFN-γ. Although the adaptive immune response results in production of substantial amounts of IFN-γ in response to Mtb, the immune response is unable to eradicate the infection in most cases. We have previously reported evidence that Mtb inhibits macrophage responses to IFN-γ, suggesting that this may limit the ability of IFN-γ to stimulate macrophages to kill Mtb. We have also observed that uninfected macrophages, adjacent to infected macrophages in culture, exhibit decreased responses to IFN-γ. Here we report that IL-6 secreted by Mtb-infected macrophages inhibits the responses of uninfected macrophages to IFN-γ. IL-6 selectively inhibits a subset of IFN-γ-responsive genes at the level of transcriptional activation without inhibiting activation or function of STAT1. Inhibition of macrophage responses to IFN-γ by IL-6 requires new protein synthesis, but this effect is not attributable to suppressor of cytokine signaling 1 or 3. These results reveal a novel function for IL-6 and indicate that IL-6 secreted by Mtb-infected macrophages may contribute to the inability of the cellular immune response to eradicate infection.
Mice Lacking Myeloid Differentiation Factor 88 Display Profound Defects in Host Resistance and Immune Responses to Mycobacterium avium Infection Not Exhibited by Toll-Like Receptor 2 (TLR2)- and TLR4-Deficient Animals
Authors: Feng Carl G. , Scanga Charles A. , Collazo-Custodio Carmen M. , Cheever Allen W. , Hieny Sara , Caspar Patricia , Sher Alan
Published: April 2014

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2−/− and TLR4−/− animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88−/− mice failed to control acute and chronic M. avium growth and succumbed 9–14 wk postinfection. Infected TLR2−/− mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88−/− animals, while TLR4−/− mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88−/− mice revealed massive destruction of lung tissue not present in WT, TLR2−/−, or TLR4−/− mice. In addition, MyD88−/− and TLR2−/−, but not TLR4−/−, mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88−/− and TLR2−/− macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88−/− mice. Similarly, MyD88−/− mice displayed a profound defect in IFN-γ response that was not evident in TLR2−/− or TLR4−/− mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-γ responses.
Novel and Detrimental Effects of Lipopolysaccharide on In Vitro Generation of Immature Dendritic Cells: Involvement of Mitogen-Activated Protein Kinase p38
Authors: Xie Jin , Qian Jianfei , Wang Siqing , Freeman Muta E. , Epstein Joshua , Yi Qing
Published: April 2014

Dendritic cells (DCs) are recognized as major players in the regulation of immune responses to a variety of Ags, including bacterial agents. LPS, a Gram-negative bacterial cell wall component, has been shown to fully activate DCs both in vitro and in vivo. LPS-induced DC maturation involves activation of p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases, and NF-κB. Blocking p38 inhibits LPS-induced maturation of DCs. In this study we investigated the role of LPS in the in vitro generation of immature DCs. We report here that in contrast to the observed beneficial effects on DCs, the presence of LPS in monocyte culture retarded the generation of immature DCs. LPS not only impaired the morphology and reduced the yields of the cultured cells, but also inhibited the up-regulation of surface expression of CD1a, costimulatory and adhesion molecules. Furthermore, LPS up-regulated the secretion of IL-1β, IL-6, IL-8, IL-10, and TNF-α; reduced Ag presentation capacity; and inhibited phosphorylation of ERK, but activated p38, leading to a reduced NF-κB activity in treated cells. Neutralizing Ab against IL-10, but not other cytokines, partially blocked the effects of LPS. Inhibiting p38 (by inhibitor SB203580) restored the morphology, phenotype, and Ag presentation capacity of LPS-treated cells. SB203580 also inhibited LPS-induced production of IL-1β, IL-10, and TNF-α; enhanced IL-12 production; and recovered the activity of ERK and NF-κB. Thus, our study reveals that LPS has dual effects on DCs that are biologically important: activating existing DCs to initiate an immune response, and inhibiting the generation of new DCs to limit such a response.
DNA Vaccination Against Specific Pathogenic TCRs Reduces Proteinuria in Active Heymann Nephritis by Inducing Specific Autoantibodies
Authors: Wu Huiling , Walters Giles , Knight John F. , Alexander Stephen I.
Published: April 2014

We have previously identified potential pathogenic T cells within glomeruli that use TCR encoding Vβ5, Vβ7, and Vβ13 in combination with Jβ2.6 in Heymann nephritis (HN), a rat autoimmune disease model of human membranous nephritis. Vaccination of Lewis rats with naked DNA encoding these pathogenic TCRs significantly protected against HN. Proteinuria was reduced at 6, 8, 10, and 12 wk after immunization with Fx1A (p < 0.001). Glomerular infiltrates of macrophages and CD8+ T cells (p < 0.005) and glomerular IFN-γ mRNΑ expression (p < 0.01) were also significantly decreased. DNA vaccination (DV) causes a loss of clonality of T cells in the HN glomeruli. T lymphocytes with surface binding of Abs were found in DNA vaccinated rats. These CD3+/IgG+ T cells expressed Vβ5 and Vβ13 that the DV encoded. Furthermore, FACS shows that these CD3+/IgG+ cells were CD8+ T cells. Analysis of cytokine mRNA expression showed that IL-10 and IFN-γ mRNA were not detected in these CD3+/IgG+ T cells. These results suggest that TCR DNA vaccination produces specific autoantibodies bound to the TCRs encoded by the vaccine, resulting in blocking activation of the specific T cells. In this study, we have shown that treatment with TCR-based DV, targeting previously identified pathogenic Vβ families, protects against HN, and that the mechanism may involve the production of specific anti-TCR Abs.
IL-1 Induces Vesicular Secretion of IL-6 without Degranulation from Human Mast Cells
Authors: Kandere-Grzybowska Kristiana , Letourneau Richard , Kempuraj Duraisamy , Donelan Jill , Poplawski Sarah , Boucher William , Athanassiou Achilles , Theoharides Theoharis C.
Published: April 2014

FcεRI cross-linkage in mast cells results in release of granule-associated mediators, such as histamine and proteases, as well as the production of numerous cytokines, including IL-6. Mast cells have been increasingly implicated in inflammatory processes where explosive degranulation is not commonly observed. Here, we show that IL-1 stimulates secretion of IL-6 without release of the granule-associated protease tryptase in normal human umbilical cord blood-derived mast cells (hCBMCs). IL-6 secretion stimulated by IL-1 in hCBMCs is potentiated by priming with IL-4 and reflects the higher levels of IL-6 secreted from human leukemic mast cell line (HMC-1). Stimulating HMC-1 cells by both IL-1 and TNF-α results in synergistic secretion of IL-6. IL-6 is de novo synthesized, as its secretion is blocked by inhibitors of transcription or protein synthesis. IL-1 does not increase intracellular calcium ion levels in either hCBMCs or HMC-1 cells, and IL-6 stimulation proceeds in the absence of extracellular calcium ions. Ultrastructural Immunogold localization shows that IL-6 is excluded from the secretory granules and is compartmentalized in 40- to 80-nm vesicular structures. Selective secretion of IL-6 from mast cells appears distinct from degranulation and may contribute to the development of inflammation, where the importance of IL-6 has been recognized.
Transcriptional Regulation of Type I Diabetes by NF-κB
Authors: Lamhamedi-Cherradi Salah-Eddine , Zheng Shijun , Hilliard Brendan A. , Xu Lingyun , Sun Jing , Alsheadat Saaib , Liou Hsiou-Chi , Chen Youhai H.
Published: April 2014

Development of type I diabetes requires coordinated expression of myriad genes responsible for the initiation and progression of the disease. Expression of these genes are regulated by a small number of transcription factors including the Rel/NF-κB family. To determine the roles of the Rel/NF-κB family in type I diabetes, we studied multiple low-dose streptozotocin-induced diabetes in mice deficient in either c-Rel or NF-κB1. We found that mice deficient in each of these NF-κB subunits were resistant to streptozotocin-induced diabetes. However, the mechanisms of the disease resistance may differ in different cases. Deficiency in c-Rel selectively reduced Th1, but not Th2 responses, whereas NF-κB1 deficiency had little effect on T cell responses to anti-CD3 stimulation. Death of dendritic cells was accelerated in the absence of NF-κB1, whereas death of macrophages and granulocytes was affected primarily by c-Rel deficiency. Furthermore, Stat-1 expression was significantly reduced in macrophages deficient in NF-κB1, but not c-Rel. These results indicate that both c-Rel and NF-κB1are essential for the development of type I diabetes and that strategies targeting each of these subunits would be effective in preventing the disease.
Polyclonal CTL Responses Observed in Melanoma Patients Vaccinated with Dendritic Cells Pulsed with a MAGE-3.A1 Peptide
Authors: Godelaine Danièle , Carrasco Javier , Lucas Sophie , Karanikas Vaios , Schuler-Thurner Beatrice , Coulie Pierre G. , Schuler Gerold , Boon Thierry , Van Pel Aline
Published: April 2014

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3168–176 peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 × 10−6 to 9 × 10−4 of CD8+ blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.
Activation of Resting Human Primary T Cells with Chimeric Receptors: Costimulation from CD28, Inducible Costimulator, CD134, and CD137 in Series with Signals from the TCRζ Chain
Authors: Finney Helene M. , Akbar Arne N. , Lawson Alastair D. G.
Published: April 2014

Chimeric receptors that include CD28 signaling in series with TCRζ in the same receptor have been demonstrated to activate prestimulated human primary T cells more efficiently than a receptor providing TCRζ signaling alone. We examined whether this type of receptor can also activate resting human primary T cells, and whether molecules other than CD28 could be included in a single chimeric receptor in series with TCRζ to mediate the activation of resting human primary T cells. Human CD33-specific chimeric receptors were generated with CD28, inducible costimulator, CD134, or CD137 signaling regions in series with TCRζ signaling region and transfected by electroporation into resting human primary T cells. Their ability to mediate Ag-specific activation was analyzed in comparison with a receptor providing TCRζ signaling alone. Inclusion of any of the costimulatory signaling regions in series with TCRζ enhanced the level of specific Ag-induced IL-2, IFN-γ, TNF-α, and GM-CSF cytokine production and enabled resting primary T cells to survive and proliferate in response to Ag in the absence of any exogenous factors. Inclusion of CD28, inducible costimulator, or CD134 enhanced TCRζ-mediated, Ag-specific target cell lysis. Chimeric receptors providing B7 and TNFR family costimulatory signals in series with TCRζ in the same receptor can confer self-sufficient clonal expansion and enhanced effector function to resting human T cells. This type of chimeric receptor may now be used to discover the most potent combination of costimulatory signals that will improve current immunotherapeutic strategies.
NK Cell TRAIL Eliminates Immature Dendritic Cells In Vivo and Limits Dendritic Cell Vaccination Efficacy
Authors: Hayakawa Yoshihiro , Screpanti Valentina , Yagita Hideo , Grandien Alf , Ljunggren Hans-Gustaf , Smyth Mark J. , Chambers Benedict J.
Published: April 2014

Recent studies have implicated a possible role for NK cells in regulating dendritic cells (DC) in vitro. In the present study, we demonstrate that immature DC are rapidly eliminated by NK cells in vivo via a pathway dependent on the TNF-related apoptosis-inducing ligand (TRAIL). Elimination of NK cells and/or neutralization of TRAIL function during immunization with immature DC loaded with nonself or tumor Ags significantly enhanced T cell responses to these Ags and Ag-specific tumor immunity. These data suggested that NK cell TRAIL might regulate responses to vaccination by controlling the survival of Ag-loaded DC.
Cutting Edge: IL-7-Dependent Homeostatic Proliferation of CD8+ T Cells in Neonatal Mice Allows the Generation of Long-Lived Natural Memory T Cells
Authors: Schüler Thomas , Hämmerling Günter J. , Arnold Bernd
Published: April 2014

Healthy, nonimmunized C57BL/6 (B6) mice contain memory phenotype CD8+ T cells, which are assumed to be generated in response to environmental Ags. Since neonatal mice are functionally lymphopenic within the first days after birth, we investigated the alternative possibility that the memory CD8+ T cells of untreated B6 mice are the result of lymphopenia-induced proliferation during neonatal life. We show here that adoptively transferred CD8+ T cells proliferate in neonatal B6 mice, rapidly produce IFN-γ, and develop into memory cells which are maintained until adulthood. In contrast to CD4+ T cells, neonatal lymphopenia-induced proliferation of CD8+ T cells was IL-7 dependent. Thus, neonatal lymphopenia seems to allow CD8+ thymic emigrants to undergo lymphopenia-induced proliferation during early neonatal life to equip the immune system with a set of preactivated CD8+ T cells before any infection, which might contribute to the rapid initiation of immune responses in the adult.
A Repressor of GATA-Mediated Negative Feedback Mechanism of T Cell Activation
Authors: Miaw Shi-Chuen , Kang Bok Yun , White Ian Alexander , Ho I-Cheng
Published: April 2014

The NF-AT family is a group of potent transcription factors that are essential for T cell activation in vitro. However, NF-ATc2-deficient Th cells display hyperproliferation in response to stimulation, suggesting that NF-ATc2 functions as a negative regulator of Th cell activation/proliferation. In this study we show that the transcriptional repressor of GATA (ROG) is a direct target gene of NF-ATc2 and that NF-ATc2-deficient Th cells are unable to fully up-regulate ROG upon stimulation. Restoration of ROG expression in vivo partly corrects the hyperproliferation of NF-ATc2-deficient Th cells by attenuating TCR signals. Our data, therefore, depict a ROG-mediated negative feedback mechanism of T cell activation.
Role of TNF Receptor-Associated Factor 3 in the CD40 Signaling by Production of Reactive Oxygen Species through Association with p40phox, a Cytosolic Subunit of Nicotinamide Adenine Dinucleotide Phosphate Oxidase
Authors: Ha Yun Jung , Lee Jong Ran
Published: April 2014

To extend our previous report, which showed the production of the reactive oxygen species (ROS) after the CD40 ligation in the B cells, we further examined the possible mechanisms for ROS production and the involvement of CD40-induced ROS in p38 activation. Our research shows that the stimulation of WEHI 231 B lymphomas with anti-CD40 induced ROS production and p38 activation. An antioxidant N-acetyl-l-cysteine or an inhibitor for NADPH oxidase blocked both of these, but the inhibitors for 5-lipoxygenase did not. We also show that the treatment of cells with inhibitors for the phosphatidylinositol 3-kinase (PI3-K) interfered with the CD40-induced ROS production and p38 activation. In addition, when overexpressed with a dominant negative form of either Rac1 (N17Rac1) or the TNFR-associated factor (TRAF) 3, the WEHI 231 B cells did not show a full response to the CD40 stimulation to produce ROS. Molecular association studies further revealed that the TRAF3 association with p40phox, a cytosolic subunit of NADPH oxidase and p85 (a subunit of PI3-K), may possibly be responsible for the production of ROS by CD40 stimulation in WEHI 231 B cells. Collectively, these data suggest that the CD40-induced ROS production by NADPH oxidase in WEHI 231 requires the role of TRAF3, as well as activities of PI3-K and Rac1.
T Cell Clonal Conditioning: A Phase Occurring Early after Antigen Presentation but before Clonal Expansion Is Impacted by Toll-Like Receptor Stimulation
Authors: Maxwell Joseph R. , Rossi Robert J. , McSorley Stephen J. , Vella Anthony T.
Published: April 2014

After in vivo immunization, Ag-specific T cells disappear from circulation and become sequestered in lymphoid tissue where they encounter Ag presented by dendritic cells. In the same site and just after Ag presentation, they “disappear” a second time and we investigated this process. Using a mouse model of T cell deletion (without Toll-like receptor (TLR) stimulation) vs survival (with TLR stimulation), Ag-specific T cells indeed became undetectable by flow cytometry, however were readily detected by immunohistochemistry. Thus, whether or not the activated T cells were destined to delete or survive, they were difficult to extract from lymphoid tissue and did not disappear but in fact were abundantly present. Nevertheless, profound differences were observed during this time period when tolerizing conditions were compared with immunizing conditions. TLR stimulation induced an increase in CD25 expression, acquisition of surface MHC class II, and abnormally high increases in forward and side scatter of the peptide-specific T cells. Using a modified adoptive transfer approach, we demonstrated by flow cytometry that in the presence of TLR stimulation the Ag-specific T cells were tightly coupled to dendritic cells, explaining the unusual increases in size and granularity. Ultimately, these events induced the specific T cells to differentiate into memory cells. We postulate that this is a stage where T cells are either conditioned to survive or to delete depending upon the activation status of the innate immune system.
Expression of Preproinsulin-2 Gene Shapes the Immune Response to Preproinsulin in Normal Mice
Authors: Faideau Béatrice , Briand Jean-Paul , Lotton Chantal , Tardivel Isabelle , Halbout Philippe , Jami Jacques , Elliott John F. , Krief Patricia , Muller Sylviane , Boitard Christian , Carel Jean-Claude
Published: April 2014

Deciphering mechanisms involved in failure of self tolerance to preproinsulin-2 is a key issue in type 1 diabetes. We used nonautoimmune 129SV/Pas mice lacking preproinsulin-2 to study the immune response to preproinsulin-2. In these mice, a T cell response was detected after immunization with several preproinsulin-2 peptides and confirmed by generating hybridomas. Activation of some of these hybridomas by wild-type (wt) islet cells or recombinant murine proinsulin-2 demonstrated that two epitopes can be generated from the naturally expressed protein. Although T cells from wt mice responded to preproinsulin-2 peptides, we could not detect a response to the naturally processed epitopes in these mice. Moreover, after immunization with recombinant whole proinsulin-2, a T cell response was detected in preproinsulin-2-deficient but not in wt mice. This suggests that islet preproinsulin-2-autoreactive T cells are functionally eliminated in wt mice. We used a transplantation model to evaluate the relevance of reactivity to preproinsulin-2 in vivo. Wild-type preproinsulin-2-expressing islets transplanted in preproinsulin-2-deficient mice elicited a mononuclear cell infiltration and insulin Abs. Graft infiltration was further increased by immunization with preproinsulin-2 peptides. Preproinsulin-2 expression thus shapes the immune response and prevents self reactivity to the islet. Moreover, islet preproinsulin-2 primes an immune response to preproinsulin-2 in deficient mice.
Human Intestinal Fibroblasts Prevent Apoptosis in Human Intestinal Mast Cells by a Mechanism Independent of Stem Cell Factor, IL-3, IL-4, and Nerve Growth Factor
Authors: Sellge Gernot , Lorentz Axel , Gebhardt Thomas , Levi-Schaffer Francesca , Bektas Hueseyin , Manns Michael P. , Schuppan Detlef , Bischoff Stephan C.
Published: April 2014

In rodents, fibroblasts (FBs) mediate stem cell factor (SCF)-dependent growth of mast cells (MCs). In humans, SCF is mandatory for MC differentiation and survival. Other factors such as IL-3, IL-4, and nerve growth factor (NGF) act in synergism with SCF, thus enhancing proliferation and/or preventing apoptosis in MCs. In this study, we studied in vitro interactions between human MCs and human FBs, both isolated from the intestine and purified to homogeneity. In coculture with FBs, MCs survived for up to 3 wk, whereas purified MCs cultured alone died within a few days. TNF-α and IL-1β, which both did not affect MC survival directly, enhanced FB-dependent MC growth. We provide evidence that FB-derived MC growth factors are soluble, heat-sensitive molecules which down-regulate MC apoptosis without enhancing MC proliferation. However, only low amounts of SCF were measured in FB-conditioned medium (<0.2 ng/ml). Moreover, blocking of SCF/c-kit interaction by anti-SCF or anti-c-kit Abs and neutralization of IL-3, IL-4, and NGF did not affect MC survival in the coculture system. In conclusion, our data indicate that human FBs promote survival of human MCs by mechanisms independent of SCF, IL-3, IL-4, and NGF. Such interactions between MCs and FBs may explain why MCs accumulate at sites of inflammatory bowel disease and intestinal fibrosis.
CXCR1+CD4+ T Cells in Human Allergic Disease
Authors: Francis James N. , Jacobson Mikila R. , Lloyd Clare M. , Sabroe Ian , Durham Stephen R. , Till Stephen J.
Published: April 2014

Chemokine receptors play an important role in the migration of leukocytes to sites of allergic inflammation in humans. In this study, we have identified increased expression of the chemokine receptor CXCR1 on CD4+ T lymphocytes derived from patients with atopic disease compared with normal donors. Enhanced expression of CXCR1 by atopic donors was identified on freshly isolated peripheral blood cells and on expanded cell populations derived from nasal mucosal biopsies and from the periphery. Identification of CXCR1 expression on CD4 cells in the nasal mucosa was confirmed by double immunofluorescence. In addition, expression of CXCR1 was dramatically decreased in patients undergoing successful treatment of allergic rhinitis by specific immunotherapy. CXCR1 provided a functional receptor capable of regulating T cells in the context of allergic disease, since expression of CXC chemokine ligand 8 was up-regulated at the site of allergic inflammation and freshly isolated CXCR1+CD4+ cells from atopic donors showed an enhanced functional response to this ligand. CXCR1 expression on CD4+ T cells was increased in vitro in response to the pro-Th2 cytokine IL-4. Phenotypic analysis reveals that IFN-γ expression was lower in the CXCR1+CD4+ cells. The identification of CXCR1 as a marker of allergic rhinitis reveals a possible target for therapeutic intervention in atopic disease.
Dendritic Cell Maturation Controls Adhesion, Synapse Formation, and the Duration of the Interactions with Naive T Lymphocytes
Authors: Benvenuti Federica , Lagaudrière-Gesbert Cecile , Grandjean Isabelle , Jancic Carolina , Hivroz Claire , Trautmann Alain , Lantz Olivier , Amigorena Sebastian
Published: April 2014

The initiation of adaptive immune responses requires the direct interaction of dendritic cells (DCs) with naive T lymphocytes. It is well established that the maturation state of DCs has a critical impact on the outcome of the response. We show here that mature DCs form stable conjugates with naive T cells and induce the formation of organized immune synapses. Immature DCs, in contrast, form few stable conjugates with no organized immune synapses. A dynamic analysis revealed that mature DCs can form long-lasting interactions with naive T cells, even in the absence of Ag. Immature DCs, in contrast, established only short intermittent contacts, suggesting that the premature termination of the interaction prevents the formation of organized immune synapses and full T cell activation.